Le gkne vpu code pour une phosphoprotkine membranaire de 80 h 82 acides
...... The only exception is the SIVcpz, a chimpanzee isolate. ...... le troisieme
article de cette thhse (chapitre 4), 2 une analyse par mutagdnkse des rc5sidus et
des ...
Mutag6nkse de la proteine Vpu du virus de I'immunod6ficience humaine de type 1
Par Jacques Friborg jr. DCpartement de microbiologie et immunologie Facult6 de mddecine
Thtse prdsende 2 la Facult6 des h d e s supdrieures en w e de l'obtention du grade de Philosophia: Doctor (Ph.D.) en microbiologie et immunologie
Janvier, 1996
"Jacques Friborg jr., 1996
1*1
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Universite de Montr6al Facult6 des 6tudes sup6rieures
Cette thhe intitulbe:
Mutag6nbe de la proteine Vpu du virus de I'immunod6ficience humaine de type 1
prksentk par: Jacques Friborg jr. a CtC Cvalu6 par un jury composb des personnes suivantes:
Dr Daniel Lamarre, president-rapporteur
Dr Louise Poulin, membre du jury
TMse acceptde le: 1 2
.
c Cohen. directeur de recherche Dr ~ r i A.
Dr Marc Wainberg, examinateur externe
\=w
Le gkne vpu code pour une phosphoprotkine membranaire de 80 h 82 acides arninis unique au cycle rkplicatif du viurs de I'imrnunodificience humaine de serotype 1 (VM-1). Bien que la proteine Vpu ne soit pas esserttielle pour la replication in vitro,
elle pourrait jouer un r61e dans la r6gulation de l'expression virale. Au cours de I'infection par le VIH-1, le reliichement de la prog6nie virale peut etre reduit de 10 fois dans les souches vpu-. Il semble que Vpu soit impliquie dans I'assemblage et/ou le bourgeomement des particules virales de la surface des cellules infecdes. De plus, les effets pathogenes du virus en culture sont retard& lors de l'expression de Vpu. Enfin, de re'centes etudes ont montr6 que cette prodine pouvait dgstabiliser les complexes intracehlaires capable de se former entre le prkcurseur gp160 des glycoprotkines d'enveloppe virale et le recepteur CD4, en induisant la d6gradation spdcifique de ce dernier dans le riticulum endoplasmique. L'apport fonctionnel de Vpu dans la propagation du VIH-1,tant in v i t r ~ que
vivo, reste jusqu'2 ce jour knigmatique.
Les objectifs de ce projet de doctorat consistent B identifier le(s) domaine(s) actif(s) de Vpu irnplique(s) dam l'activid biologique de la prodine. Dans un but ultime, nous tenterons de caracte'riser les diverses fonctions de Vpu afin de mieux cerner son mode d'action au cours de I'infection par le VIH-1. La presence de Vpu pourrait en partie ttre responsable de la disshination plus rapide du VIH-1 face h celle observie avec le VIH-2. 11est donc important de dkfinir les aspects structuraux et fonctionnels de la protdine afin de developper une approche prbventive dans le contr6le de cette pande'mie. Pour delimiter des regions et/ou des sequences de Vpu essentielles B son activit6 biologique, nous avons utilis6 des techniques de mutag6n&sedirigke et &lification d'ADN g6nomique par PCR ("Polymerase Chain Reaction") afin d'introduire des mutations spkcifiques dam le gtne vpu. Ces genes muds ont par la suite tt6 ins&& dans un clone molkculaire infectieux du VIH-1,autrement isoginique, dans le but d'entamer des analyses de replication virale dam divers syst&mescellulaires in vitro.
Dans un premier temps, nos rbsultats dkmontrent que la phosphorylation de Vpu est essentielle B l'activid biologique de Ia proteine au cours de l'infection virale. En effet, Ies substitutions ponctuelles ou combides des dsidus serine en positions 52 et 56, identifies comme les sites de phosphorylation de Vpu, abolissent la capacitd de la proteine B retarder les effets cytopathiques du VIH-1 en culture. En revanche, ces mutations n'ont eu aucun impact majeur sur la propridd que poss&deVpu de faciliter le relfichement de virions matures. Les sites de phosphorylation de Vpu sont localisies dans une r6gion C-terminale hautement conservde qui pourrait renfermer I'un des domaines actifs de la proteine. Par ailleurs, l'introduction de mutations dam l'extrbrniti N-terminale de Vpu a permi de montrer que cette rkgion renferme non seulement le domaine d'ancrage de la proteine, mais prbsente bgalement des d6terminants n6cessaires
2 sa capacit6 de faciliter le reliichement de particules virales. Ainsi, nos observations
suggirent fortement que Vpu peut agir distincternent dam la cellule infectke afin d'optimiser la replication du VH-1. Finalement, nous avons proced6 A une etude exhaustive du phhomkne de degradation de la molt5cule CD4 observd lors de I'expression de Vpu afin de mieux cemer son made fonctionnel. L'ensemble de nos rbsultats demontrent clairement que Vpu est capable d'interagir avec la mol6cule CD4 dam le rCticulum endoplasmique.
Toutefois, cette liaison n'est pas suffisante pour engendrer la protdolyse de celle-ci. La phosphorylation de Vpu semble moduler la formation de complexes VpuKD4 mais pourrait egalement activer le(s) m&anisme(s) mol&ulaire(s) ntcessaire(s) au processus. D'autre part, notre analyse de mutagCn&sesur la queue intracytoplasmique de CD4 a rt5vel6 que cette rdgion prgsente des determinants confdrant ii la moltcule sa sensibilite au mode d'action de Vpu. En resume, l'integritb d'une structure secondaire en alphahClice predite de se former dam la queue intracytoplasmique de CD4 apparait importante dam la capacite de liaison de Vpu et au processus subsequent de degradation de la molecule.
TABLE DES ~ ~ A T I & E s Page PAGE TlTRE IDENTIFICATION DU .JURY SOMMAIRE TABLE DES MATI~RES LISTE DES TABLEAUX LISTE DES FIGURES LISTE DES AB&VIATIONS D~DICACE REMERCIEMENTS
CHAPITRE 1 REVUE DE LA LITT&ATURE.
Le virus de 1'immunodCficience humaine. Le VM: un rktrovirus pathoghe chez I'homrne. 1.1. ~ t i o l o ~du i e syndrome d'immunodeficience humaine. 1.2. Morphologie du VIH- 1. 1.3 Organisation gbnomique du VM-1. 1.4. Cycle rdplicatif du VM- 1.
La mol6cule CD4: principal rt5cepteu.r cellulaire du VIH-1. 2.1. Caractkristiques gen6rales de la rnol&ule CD4. 2.2. L'interaction entre la gp120 et la m o k u l e CD4: une liaison de haute afEdt6. 2.3. Rigulation de I'expression de la mol&uie CD4 par le VM-I.
Les glycoprot6ioes d'enveloppe du VIH. 3.1.
Structure et synthese des glycoprotkines de I'enveloppe virale.
Les protkines de structures du VIH-1. 4.1. Structure et synthtse des protdines Gag du VM-1. 4.2. La machinerie enzymatique du WH-1.
Assemblage et maturation du VM-1.
xvi
vii
6.
Les effets cytopathiques du VM-1.
7.
La proteine Vpu du VIH- 1. 7.1. Synthbe de la prodine Vpu. 7.2. Le r6le de Vpu dam le cycle replicatif du VM-I. 7.2.1. Vpu augmente le relfichement des particules virales. 7.2.2. Vpu diminue les effets cytopathiques du VM-1. 7.2.3. Vpu degrade sp6cifiquement la molCcule CD4.
8.
Le projet de recherche.
Article 1:
Functional analysis of the phosphorylation sites on the human immunodeficiency virus type 1 Vpu protein.
Anicle 2:
Structural and functional anaIysis of the membrane-spanning domain of the human immunodeficiency virus type 1 Vpu protein.
Article 3:
Degradation of CD4 induced by human immunodeficiency virus type 1 Vpu protein: a predicted alpha-helix structure in the proximal cytoplasmic region of CD4 contributes to Vpu sensitivity.
CHAPITRE 5 Article 4:
Structural requirements for the binding of the human immunodeficiency virus type 1 Vpu protein to the cytoplasmic domain of CD4.
viii
CHAPITRE 6 Discussion.
CKAPITRE 7
Conclusions et perspectives. CHAPITRE 8 Bibliographie.
157
LISTE DES TABLEAUX
Page
Tableau 1:
Fonctions et description des protdines viraies du VIH- 1.
Tableau 2:
Mdcanismes possibles ii l'origine des effets cytopathiques du VIH-I ou de ses protbines virales.
CHAPITRE 2 Tableau 1:
Effect of Vpu mutants on the level of Env glycoproteins at the cell surface.
LISTE DES FIGURES Page
Figure 1:
Reprksentation schdmatique des diffkrents stades de l'infection par le VIH- 1.
Figure 2:
Representation sch6rnatique du VM-1 et de son organisation g6nomique.
Figure 3:
Cycle rdplicatif du VM-1.
Figure 4:
Representation schtmatique de la mol6cule CD4 et de la tyrosine kinase p56fck.
Figure 5:
Maturation de la progenie virale.
Figure 6:
Analyse irrrmunobiochimique de la proteine Vpu au cours du cycle rbplicatif du VIH- 1.
Figure 7:
Reprdsentation sch6matique de la protiine Vpu du VIH-1.
Figure 8:
Schema reprksentant le relichement de particules virales i la surface des cellules infectees.
Figure 9:
La formation de syncytium au cours de l'infection par le VIH- 1 de cellules CD4+.
Figure 1:
Mutations introduced into the vpu dodecapeptide sequence of m-1.
Figure 2:
Replication of vpu mutant proviruses in MT4 cells.
Figure 3:
Viral protein expression of vpu mutant proviruses in HeLa cells.
Figure 4:
Effects of Vpu mutants on HIV-1cytotoxicity.
Figure 5:
Effect of Vpu mutants on syncytium formation.
Figure 1:
Mutagenesis of the predicted 8 1 amino acid Vpu protein.
Figwe 2:
In vitro translation of Vpu mutants in presence of canine pancreatic microsomal membranes.
Figure 3:
Expression of wild-type and mutated Vpu proteins.
Figure 4:
Effect of mutations in the N-terminal hydrophobic region of Vpu on the enhancement of virion release.
Figure 5:
Effect of mutations in the N-terminal hydrophobic region of Vpu on CD4 degradation.
Figure 6:
Effect of mutations in the N-terminal hydrophobic region of Vpu on HIV- 1-mediated cytotoxicity.
Figure 7:
Structural and functional analyses of the Vpu protein.
CHAPITRE 4 Figure 1:
Effect of deletion of the cytoplasmic domain of CD4 on Vpu-induced degradation.
Figure 2:
Sensitivity of CD4 cytoplasmic domain substitution mutants to Vpu-induced degradation.
127
Effect of substitution mutations in the proximal cytoplasmic region of CD4 on Vpu-induced degradation.
127
Effect of deletion and substitution mutations in the cytoplasmic domain of CD4 on the predicted alpha-helix conformation.
128
Figure 3:
Figure 4:
CHAPITRE 5 Figure 1:
The formation of Vpu/CD4 complexes in the ER: phosphoacceptor sites of Vpu modulate the binding to CD4 molecules.
Figure 2:
Effects of mutations introduced in the cytoplasmic tail of CD4 on the capacity to form Vpu/CD4 complexes.
Figure 3:
152
A putative a-helix in the proximal portion of the cytoplasmic
tail of CD4 is required for the stability of Vpu/CD4 complexes.
154
CHAPITRE 6 Figure 1:
Figure 1:
Reprbsentation schkmatique du transit intracellulaire de la protiine Vpu du VIH-1.
Representauon schhatique des domaines actifs de la protCine Vpu du VIH-1,
164
xiii
a
Alpha
AcM
Anticorps monoclonal
ADN
Acide dCsoxyribonucl~ique
Ag
ARN
Acide ribonuclkique
ARNm
Acide ribonucl6ique messager
ARNt
Acide ribonuclCique de transfert
Gsn ATP
Adknosine triphosphate
BFA
Brkfeldine A
C
Rkgion constante
CD
Marqueur de differenciation ou "Clusterof differentiaton
cm CMH
Complexe majeur &histocompatibilitd
CMM
"CanineMicrosomal Membranes": Membranes microsomales canines
CYS
D
DRB Glu
Acide glutamique
gp41
Glycoprot6ine transmembranaire de l'enveloppe v i d e
gp120
GlycoprotCine de surface de l'enveloppe virale
gp160
Pr6curseur polypeptidique de l'enveloppe virale
His
Histidine
k
Kappa
kDa
Constante de dissociation
xiv
LTR
"Long Terminal Repeat": Longue dquence r6pktitive
To
Pourcentage
'Pi
Orthophosphate inorganique
PH RE
Potentiel hydroghe R&iculum endoplasmique
Ser
Stkine
SIDA
Syndrome de l'immunod6ficiencehumaine
Thr
Thrkonine
v
Region hypervariable
VIH (1 ou 2)
Virus de 11immunodc5ficiencehumaine de s6rotype 1 ou 2
A mes parents et P mon friire qui m'ont encourag6 et support6 tout au long de mes travaw
xvi
Je voudrais exprimer ma tr&s grande reconnaissance A I'endroit du Dr Eric A. Cohen pour son excellente direction. Je tiens B le remercier particulitrement pour llintbr& et le dynamisme qu'il a su rt5flkter tout au long de ce projet de recherche et qui ont kt6 des plus motivateurs. Les prtcieux conseils scientifiques et les nombreux encouragements qu'il m'a prodigut5 ont 6tt trh apprkcibs ii maintes reprises. Tant au niveau de l'enseignement qu'au niveau humain, cette &ape de mon apprentissage a 616 des plus enrichissantes. Je le remercie dgalement pour m'avoir permis de participer B de nombreux congr2s nationaux et intemationaux et pour m'avoir encourag6 A poursuivre un stage post-doctoral l'institut medicale Howard Hugues de 11universitt5du Michigan. J'adresse rnes remerciements au Dr. Serge Montplaisir, directeur du dkpartement de rnicrobiologie et immunologie de la facult6 de medecine de 11universit6de Montrkal,
pour m'avoir conseiller dans mon choix de laboratoire d'acceuil et pour avoir accept6 ma candidature au programme de doctorat. Je remercie kgalement le Dr. Guy Lemay, professeur-chercheur au dkpartement de rnicrobiologie et immunologie de la facult6 de mddecine de 11universit6de Montrkal, pour son support tout au long de mon doctorat et sa contribution au cours de discussions d'ordre scientifique. Mes remerciements s'adressent aussi ii toute i'tquipe du Dr. Cohen dont la camaraderie a toujours 6t6 omniprksente tant dans les meilleurs que dans les plus dues moments. Je remercie en particulier Sylvie Beaulieu, Xiao-Jian Yao, Claude LavaMe et Azim Ladha avec qui j'ai entamt mon doctorat et dont la prdsence et I'amitid ddvoil6e ont 6t6 grandement apprdcides.
xvii
Je tiens B remercier Nash G. Daniel, mon protege au cours de nombreux stages d1&6et qui maintenant vole alldgrement de ces propres ailes. Je lui souhaite les plus belles dussites dam son projet de maltrise et une continuit6 qui saura le satisfaire. J'adresse des remerciements sinctres ii Carole Danis, Janique Forget, Dominique Bergeron, Gary Pignac-Kobinger et Mabrouck Taoufik qui ont subit mes alkas et qui ont bt6 des amis(es) proches au cows de mon doctorat. Je tiens aussi ii remercier d'excellents compagnons de travail mais avant tous des amis: Florent, Ramu, Serge, Robert, Jean-Philippe et Nie. Je dgsire souligner et remercier Johanne Mercier, Fran~oiseBoisvert, Nicole Rougeau et Angelo Tiganos pour leur amiti6, leur patience et leur assistance technique des plus apprdci6e dam Ie laboratoire, Je remercie dgalernent Serge Sdndchal pour son assistance technique en cytofluomktrie en flux. Je voudrais tdmoigner ma reconnaissance B toutes les personnes qui ont 6t6 associies de p r h ou de loin ii la r6alisation des publications obtenues au cours de mon doctorat et specialement aux organisrnes suivants pour leur soutien financier:
- Programme national de recherche et d6veloppement en matibe de s a t e (PNDRS) - L'universitk de Montrdal Je tiens findement B remercier et ii dkdier cette these a mes parents, Jacques Sr. et Marie-Claude, et ik mon petit fdre, Jean-Claude qui m'ont soutenu et encourage tout au long de mes dudes.
CHAPITRE 1
Revue de la littkrature
Le virus de I'immunodCficience hurnaine. Le virus de I'immunodkficience humaine (VTH),l'agent infectieux responsable du syndrome de I'immunodificience acquise (SIDA), est un rCtrovirus cytopathoghe appartenant 2 la sous-famille des Lentivirinae. Les lentivirus sont associees k des maladies d6g6niratives Cvolution lente affectant gknkralement les systiimes imunitaire et nerveux de 11h6te. Depuis de nombreuses annees, des virus tel le virus Visna-maedi du mouton ont CtC associCs 3 ce type &affections chez l'animal. On observe maintenant l'tmergence de rCtrovirus humain dont le prototype est le VIH. Aujourd'hui, la classification exhaustive des rktrovirus est surtout bas6 sur leurs morphologies en rnicroscopie 6lectronique et particuli2rement leurs modes de rkplication. Comme tous les rCtrovirus, le VIH est un virus enveloppt5, surmonti de spicules entourant la nuclkocapside virale. La particule virale infectieuse contient deux moI&cules d'ARN gCnomique monocathaire qui lors de I'infection pourront &re transcrites en un ADN bicatenaire afin de s'integrer dam le rnatkriel gCn6tique de la cellule. Par contre, outre les gknes gag,pol et env responsables de la formation de la particule virale, le VIH posskde une combinaison complexe de g h e s additionnels dits "auxiliaires" qui lui confere I'un des modes d'infection et de replication des plus Cnigmatiques. Le VIH jusqufk ce jour fait l'objet d'Ctudes des plus intensives. Afin de mieux comprendre la pathologie du SIDA, il est donc primordial de bien dbfinir et caracteriser les principaux acteurs de cette veritable pandimie.
1. Le VIH: un r6trovirus pathoghe chez I'homme. 1.1. ~ t i o l o ~du i e syndrome de I'immunod6ficience humaine. Au debut des annCes 80, le centre de contr6le des maladies (CDC: "Centers for Disease Control") note une augmentation de frequence inexpliquee de certaines affections rares. On rapporte plusieurs formes de pneumonies causees par le protozoaire Pneumocystis carinii. Dam d'autres cas, une forme particulii5re d'une tumeur de la peau
est diagnostiquee, le sarcome de Kaposi. Ces sympt8mes semblent etre le resultat d'un ddsordre commun, un &at d'immunosuppression sCv&rechez les sujets atteints. En 1982, le CDC reconnut l'existence d'une nouvelle maladie d6sormais connue sous le nom du syndrome de I'imrnunod~ficiencehurnaine (SIDA). Ce n'est que trois ans a p r h son apparition que l'agent etiologique du SIDA est is016 par l'iquipe du professeur Montagnier de l'institut Pasteur en France A partir de cellules ganglionnaires d'un sujet atteint (Barre-Sinoussi et al., 1983; Montagnier et al., 1984). Cet agent infectieux qui poss$de toutes les caractkristiques d'un r6trovirus fut nomme LAV ("Lymphadenopathyassociated virus"). Au cows de la meme annee, l'equipe de Robert Gallo aux ~ t a t s - ~ n i s fit la dkouverte d'un virus genetiquement apparent6 B un r6trovirus responsable d'une leuc6mie de type T (Gailo et al., 1984) qu'il dksigne HTLV-UI ("Human T-cell lyinphotropic virus type IT). Subs&pernment, plusieurs groupes (Gallo et al., 1984; Montagnier et al., 1984; Levy et al., 1984) d6montr5rent qu'un seul rktrovirus etait responsable du d6veloppement du SIDA. Ce virus resut l'appelation de virus de l'immunodkficience humaine de type 1 (VIH-1) par le cornit6 international de taxonomic des virus. Par la suite, un second virus apparent6 au W I - 1 , le VIH de serotype 2 (WI-
2), fut is016 chez des individus dlAfrique de l'ouest (Clavel et al., 1986). Bien que l'organisation genornique r6We l'existence d'une parent6 entre le VIH- 1 et le VM-2, I'homologie des proteines virales n'est de I'ordre que de 50 A 60%. Aujourd'hui, le SIDA a pris des proportions catastrophiques Zi travers le monde. L'organisation mondiale de
la s a t e (WHO: "World Health Organization") estime qu'environ 19 millions d'individus ont it6 contamint5s par le VDI-1 depuis Ie dibut de cette pandirnie. Jusqu'h ce jour, plus de 2 millions d'individus s6ropositifs ont developpk le SIDA, et la plupart en sont morts. Le VIH-1 se propage principalement par les voies genito-urinaires Iors de relations sexuelles, par transmission materno-fetale et par transmission sanguine. L'infection par le VIH-1 se caracterise initialement par une courte p&iode de rdplication virale intense (figure 1). Au cours des mois et des annCes qui vont suivre, le systirne immunitaire parvient A mdtriser la vir6mie. Durant cette phase asymptomatique de la mdadie qui peut durer entre deux A dix ans, il est difficile de detecter ou d'isoler le virus dam le sang des personnes atteintes. Cette longue periode de "latence clinique" n'est toutefois pas le reflet d'une inactivitk virale. La rkplication du VIH-1 est des plus dynamique tout au long de l'infection dam des tissues Iymphoi'des telles les ganglions lymphatiques et dam les macrophages (Pantdeo et al., 1993; Weiss et al., 1993). Recemment, il a 6t6 dernontrc5 qu'au cows de cette phase asyrnptomatique, le VIH-1 se replique continuellement de maniire active chez les individus atteints (Wei et al., 1995;
Ho et al., 1995). Ll semble exister un kquilibre constant entre l'infection de novo des populations cellulaires, la dplication virale et le renouvellement des cellules dktruites par le virus. Le VIH-1 proc*de donc B une destruction silencieuse et gradueile des principaux acteurs de l'immunid
Zi
mediation cellulaire, les lymphocytes T auxiliaires,
privant I'organisme de ses mecanismes de defense.
Bknificiant ainsi du deficit
immunitaire, de nouvelles souches virales, issues du taux eleve de r6plication et de la grande variabiIit6 genCtique du VM-1, pourront se propager sans que l'organisme puisse contraler leur disskmination. Une chute irr&ersible du nombre de lymphocytes T circulants et des troubles neurologiques deviendront alors des symptames apparants de 1'6volution de la maladie.
Infection aigiie
Phase asymptomafimafique
ARC
SIDA
Infection virale Figure 1. Reprbentation schhatique des diff&-ents stades de I'infection par le VIH-1. Avant la s6roconversion, on peut noter une courte piriode - de rkplication virale intense (-). Au cours des mois et des annkes qui vont suivre, la virernie est dduite A un faible niveau (phase I). Pendant cette pBriode, le nombre de lymphocytes CD4+ circulants (----.)chute progressivement. En revanche, le nombre de cellules CD8+ (----) est d e v i tout au long de la phase asymptomatique. On observe par la suite une seconde phase de replication v i d e intense (phase 2) et l'apparition des premiers symptdmes associt5s la maladie. Les celtules CD8+ (-.-----)qui ont un r6Ie jouer dans ta rkponse anti-virale vont disparaitre. C'est l'installation du dkficit immunitaire et la progression vers le SIDA. (modifike de la rdf6rence Levy, 1988)
Par ailleurs, on observe chez I'individu dipourvu d'un systsme immunitaire
efficace, la proliferation de nombreux autres micro-organismes, dont Pneurnocystis carinii et Mycobacterium avium, provoquant de nornbreuses infections opportunistes qui peuvent s'averer fatales (revue par Nelson et al., 1990). Il est clair que le VIH-1 a diveloppi de nombreux strataghes afin de survivre et de pouvoir se multiplier. Les mdcanismes par lesquels le VIH-1 induit une dkstruction progressive du sytkme immunitaire et dans certains cas, du syst&menerveux, demewent encore enigmatiques.
1.2. Morphologie du VIH-1. L'analyse en microscopic dlectronique du VM-1 mature laisse per~evoirun c6ne dense cylindrique recouvert d'une enveloppe lipidique acquise au cours du bourgeonnement 2 la membrane plasmique de la cellule infectde. Sa morphologie est celle d'un ritrovirus (famille retroviridae) membre de la sous-famille des lentivirus (lentivivirirrae). La particule virale enveloppee poss2de un diamktre d'environ 100 nm ii la surface de laquelle apparaissent de multiples (72 unitis trimdriques ou t&tram&iques) projections en fome de spicules, les glycoprotdines d'enveloppe virale (gp 120 et gp4 1) (figure 2). Le c6ne ou la capside virale est en grande partie composie de protiines structurales qui sont codties par le gene gag. La matrice interne de la capside est constitube de la proteine myristylde p17gag, tandis que la protdine p24gag, composante majeure de la capside, donne ii la particule sa fonne de cSne excentrique. La particule virale infectieuse posside deux moldcules identiques d'ARN monocatinaire d'environ 9.3 Kb qui sont etroitement associkes aux prodines de la nucldocapside (p9gag).
Figure 2. Reprkentation schkmatique du VIH-I et de son organisation gCnornique.
De plus, le matiriel nicessaire B la replication du VIH-1 lors d'infection subsequente sera entraher au cours des processus de maturation, soit: un complexe enzymatique form6 de la transcriptase inverse, d'une protease et d'une intkgrase, toutes d'origine v i d e (Gelderblom, 1991; Stevenson et al., 1992). O n retrouve kgdement un
ARN de transfert (ARNt) cellulaire codant pour une lysine, qui servira d'amorce au processus de ritrotranscription virale (Barat et al., 1989;Coffin, 1990). Findement on retrouve la proteine Vpr, la seule protiine accessoire du VM-1 incorporie
la particule
virale (Cohen et al., 1990). Son incorporation est mkdii par une interaction spicifique avec le domaine p6gag du prkcwseur polypeptidique Pr55gag (Paxton et al., 1993; Kondo et al., 1995; Checroune et al., 1995). Par ailleurs, au cours du bourgeonnement, la particule virak entrdinera avec e l k des protCines cellulaires prksentes sur la membrane plasmique de la cellule h8te. Ainsi, en fonction de la cellule infectee, on peut retrouver sur Ie V M - 1 des antiggnes (Ag) de classe I et de classe lI du complexe majeur d'histocompatibilitt5 (CMH) (Gelderblom et ai., 1987). Tandis que les glycoprotiines d'enveloppe v i d e representent 0.1% de la masse totale du virion, les moltcules HLA-
DR (HLA:"Human Leucocyte Antigens") peuvent constituer 4.4% des proteines totales associ6es au VM-1 (Henderson et al., 1987). O n peut Cgalement dktecter une variete de recepteurs cellulaires de surface tels les moICcules CD3 et CD4 (Meerloo et al., 1992).
1.3.
Organisation g6nomique du VIH-1.
L'organisation gdnornique du VIH-1,est sans contredit, la plus complexe de celles des rCtrovirus connus. A l'instar de ceux-ci, son materiel genitique est forme d'ARN monocathaire qui au cours de l'infection sera transcript en un ADN bicatenaire. On retrouve alors, bordant 2 chaque extrimitd de I'ADN, deux longues sequences ripdtitives, les LTR ("long Terminal Repeats"), qui contiennent tous les elements en cis nCcessaires 5 l'intigration et l'expression adequate du genome viral. Cependant, en plus
des genes de structures gag, pol et env comrnuns 3 tous rktrovirus, le gdnome du VIH- 1 prksente divers cadres de lecture ouverts (ORF: "Open Reading Frame") additionnels situks entre pol et e m . Cette region centrale renferrne les genes tat et rev auxquels ont associe des fonctions de r6gulation de l'expression des genes viraux, et les g5nes vif, vpr et vpu dits accessoires puisque exprimes tardivement dam le cycle viral et non essentiels & la replication v i d e dam les ligndes cellulaires CD4+ transformkes (tableau 1).
Findement, on retrouve il l'extr6rnitC 3' du gdnome, le gene nef dont la fonction prCcoce apparst ttre essentielle 3 la pathogthese du SIDA in vivo. Au cows du cycle rkplicatif, le VIH-1 aura donc recours B un epissage alternatif de 1'ARN genomique afin de lui permettre l'expression indipendante de ses nombreux gines. Le produit du gene tat ("Trans-activator") est un rdgulateur positif de l'expression de tous les ARN messagers ( a m ) viraux (revue par Cullen, 1993). La protkine Tat, exprimke t6t lors du cycle viral, se retrouve dans le noyau et dam le nuclkole de la cellule infectCe. Elle reconnait sur les ARNm viraux, une structure secondaire en forme d'kpingle 3 cheveux, appelde TAR ("Trans-acting responsive element"). En association avec des facteurs cellulaires, Tat se fixe sur une chaine naissante d'ARN par l'intermkdiaire de TAR afin d'en assurer I161ongationet de stirnuler Itinitiation de la transcription.
La protiine Rev ("Regulator of Virion protein
Expression") posside kgalement une fonction de regulation positive (revue par WongStaal et Haseltine, 1992). Elle permet aux ARNm viraux gag, pol et env d'ichapper aux mecanismes dlCpissage dam le noyau en se liant 8 une structure particuli&re,le RRE ("Rev Responsive Element"). Ces
ARN seront transport& vers le cytoplasme oii ils
seront traduits en protdines. Rev favorise ainsi la synth5se des protkines de structure formant les nouvelles particules virales et celle des protkines accessoires Vif, Vpr et Vpu. Les prodines Tat et Rev peuvent agir en trans afin de stimuler l'expression des autres genes et elles s'averent indispensables 3 l'infection productive du VM- 1.
Gat!
GagrPol
Int@ase; int&mtion de IfADN viral Env
m r s c u r polypeptidiquede l'envebppe
virak!
Glymprot&ne de d a c e de Iyenvekppe virale; bison ao e p l e u r C W Sous-unite trzu~~membrsaairt de I1envebppevide; pmprMd frrsiogbe Tat
Rev Nef Vif V P ~
V P ~
Augmente k rehhement des virions, d&rade les moMcules CD4, climinue les effets cytopathiques du vim
Malgrk certains aspects fonctionnels encore ne%uleux, le r61e de la protiine Nef dam le cycle riplicatif du VIH-1 est maintenant rnieux caracterisk. Les premieres itudes ont sugg5rk que Nef ("Negative Factor") pouvait agir c o m e rigulateur negatif rkprirnant la transcription des g h e s du VIH-1 (Terwilliger et al., 1986; Luciw et al.,
1987; Ahmad et Venkatesan, 1988). On retrouve ainsi dam la siquence U3 du LTR une skquence spkcifique, le NRE ("Negative Regulatory Region"), qui semblerait 2tre mediateur des effets de Nef. Les recentes etudes tendent plut8t
dCmontrer que Nef
augmenterait le taux de replication virale in vitro (Miiler et al., 1994; Chowers et al.,
1994; Spina et al., 1994). Les observations divergentes accumulkes jusqu'8 c e jour semblent Stre le rksultat d'un inorme polymorphisme entre les protkines Nef des diffkrents isolats du VM-1 (Tendliger et al., 199 1; Zazopoulos et Haseltine, 1993). Nef ne posssde aucun effet substantiel quantitatif ou qualitatif sur l'expression des gknes du VIH-1 dam la cellule infecte'e (Miller et al., 1994; Chowers et at., 1994). Toutefois, la proteine augmenterait Ia dissimination v i d e in vivo par un ou des mkcanismes encore ma1 dkfinis (Jamieson et at., 1994). Ainsi, Miller et al. (1995) ont ricemment dkmontrC que Nef pouvait agir en trans afin d'augmenter l'infectiviti du VIH-1 indkpendamment de l'expression de l'enveloppe virale et de ltentrCe du virus.
Par
ailleurs, l'un des phenotypes consistants avec l'expression de Nef est la capaciti que posskde la proteine d'induire l'internalisation et la degradation spkcifique des mol&xks
CD4 exprimdes B la surface de la cellule infectCe (Garcia et al., 199 1 ; Garcia et al., 1993; Aiken et al., 1994). Cette internalisation ferait suite 8 la dissociation par Nef de complexes pouvant se former entre CD4 et la tyrosine kinase p562ck (Salghetti et aL,
1995; Bandres et at., 1995). Ces phknomhes sembleraient inhiber l'activation et la prolifiration norrnale des lymphocytes T infect& (Niederman et al., 1992; Greenway el al., 1994; Greenway et al., 1995). Les effets de Nef sur l'infectivite du VM-1 et la
dkgradation des molecules CD4 sont indkpendants I'un de l'autre lors de la rkplication
virale (Goldsmith et al., 1995). Aujourdlhui, Nef est p e r p cornme essentiel ii la rkplication du VIH-1 et 2 la pathogCn2se du SIDA (revue par Cullen, 1994). La proteine Vif ("ViraI Infectivity Factor1') influence l'infectiviti des particules virales. Les h d e s fonctionnelles montrent que des virus Vif negatif sont 1000 fois moins infectieux que des virus Vif positif lorsque produit dam certains types cellulaires (Fisher et al., 1987; Strebel et al., 1987; Gabudza et al., 1992a). Rkcernment, la production in vitro de particules virales immatures a ktk associke avec l'absence d'expression de la protiine (Hoglund et al., 1994;Borman et al., 1995). Vif semble donc augmenter I'infectivitk v i d e en assurant la morphogknkse adkquate du virus dans certaines IignCes cellulaires.
Le produit du g h e vpr est la seule prot6ine accessoire incorporie dans la particule virale (Cohen et al., 1990; Paxton et al., 1993;LavallCe et al., 1994). EIle s'accumule dans le noyau de la cellule suivant I'infection par le VIH-1 en l1absencede toutcs autres protkines virales (Lu et al., 1993). Ces observations laissent prgsager un r6le prkcoce de Vpr au cows du cycle rkplicatif. Ainsi, Vpr peut s'associer au complexe de preintigration du V M - 1 et serait impliquC dam son transport dans le noyau de
cellules qui ne se divisent pas telles les macrophages (Heinzinger et al., 1994). Une ktude ricente a d'ailleurs dkmontr6 que I'expression de Vpr 6tait essentielle ii la r6plication du VIH-1dans les macrophages (Connor et al., 1995). Il est donc indressant de noter que Vpr poss5de un effet transactivateur mod&& sur le LTR du VIH-1, toutefois plus faible que Tat, mais kgalement sur divers promoteurs h6t6rologues (Cohen et al., 1990). Vpr semble egalernent capable d'induire en culture la replication du VM-1
chez des monocytes circulants du sang lorsque fourni de maniiire exogiine (Levy et al.,
1995). Par ailleurs, Vpr induit la differentiation de cellules musculaires humaines, les rhabdomyosarcomes (Levy et al., 1993)et inhibe la prolif6ration des lymphocytes T en altkrant la progression du cycle cellulaire (Rogel et ab, 1995). Les effets cytostatiques
de Vpr laisse entrevoir la possibilite que la protCine p u k e moduler la permissivite de certains types cellulaires au cours de l'infection in vivo en influenpnt leur cycle de division ou leur programme de diffkrenciation. Finalement, l'une des particularit& du VIH-1 est la presence du gene vpu qui ne se retrouve pas chez le VIH-2 et dont le r6le sera dCcrit en details dam les sections subsiquentes, En revanche, le VIH-2 possgde un cadre de lecture ouvert inexistant chez le VIH-1, le g h e vpx. Le produit du gkne vpx presente certaines homologies avec la protCine Vpr (Tristem et al., 1992, Westervelt et al., 1992). La conservation en acides amines entre ceux-ci permet d'envisager la possibilite que le g h e vpx, situe en arnont du g&ne vpr, provient originalement de la duplication de ce dernier (Tristem et al., 1990). De plus, tout comme Vpr, Vpx est incorpork dam le VIH-2 via une interaction avec le domaine p6gag du precurseur polypeptidique Pr55gag et semble contribuer avantageusement B la replication virale dam certaines lignies cellulaires (Yu et al., 1991; Wu et al., 1994). Ainsi, la conservation et la prisence de ces nombreux g k e s
chez le VIH de serotype 1 ou 2 aura un impact kvident sur la rkplication et l'infectivit6 virale, et dans la progression de la maladie.
1.4.
Cycle r6plicatif du VIH-1.
Le cycle rCplicatif du VM-1 ne differe que quelques peu de celui des autres retrovirus (figure 3). Dans ces grandes lignes, suite B l'entree du VIH-I dans la cellule cible, 1'ARN genomique viral est libere de la capside pour Etre transcrit en un ADN bicatenaire, qui sera integri au sein du matbriel ginetique de la cellule h6te. Lors de I'activation cellulaire, les g k e s virawc seront exprimks, commandant ainsi la synth5se des protkines nicessaires B la formation de nouvelles particules viraies. Suivant les Ctapes d'assemblage, les particules virdes matures seront libkrees par bourgeonnement i la membrane plasrnique et iront infecter d'autres cellules. Toutefois, le cycle rkpticatif du VIH-I prisente certaines particularitis puisqu'il met en jeu de multiples acteurs viraux et cellulaires.
Le VIH-1 r e c o ~ d sp&ifiquement t B la surface de la cellule cible, le recepteur CD4 (cellule CD4+). La glycoproteine mernbranaire CD4 est presente sur une grande varikte de cellules d'origine hkmatopo'i6tique. Elle joue entre autres un r61e essentiel dans l'elaboration de la rdponse immunitaire. Ainsi, les lymphocytes T matures et irnmatures, principaux rouages du syst5me immunitaire, constitue I'une des cibIes privalentes du VIH-I. Le rkcepteur CD4 est aussi apparant, quoique en plus faible concentration,
la surface des monocytes/macrophages et des cellules prksentatrices
d'antigknes telles les cellules dendritiques. Par ailleurs, certaines expkriences in vitro ont dkmontr6 que le VIH-1 est capable d'infecter des cellules n'exprimant pas la molicule CD4 telles les cellules nerveuses (Harouse et al., 1991) et les cellules fibroblastiques (Tateno et al,, 1989). Dans cet ordre d'idie, le galactocir~broside (GalC) (Bhat et al., 1991; Yahi et al., 1992) et la molCcule CD26 (Callebaut et a!.,
1993) ont Ct6 dicrits cornrne de potentiels co-rtkepteurs du VIH-1.
Lors d'infection de cellules CD4+, le VM-1 interagit avec sa future cible par I1intermCdiairede la glycoprotCine de surface de L'enveloppe virale, la gp 120. Cette interaction de haute affinitd entraine un changement de conformation de la gp120 conduisant ainsi B la fusion de la membrane cellulaire et de la membrane virale. Cette fusion est mCdi6e en grande partie par la sous-unite transmembranaire d e la glycoprot6ine d'enveloppe, la gp41. La liaison entre la gp120 et le rCcepteur CD4 entraine un changement de conformation dam la glycoproteine d'enveloppe necessaire B la presentation des domaines de fusion de la gp41. Cette Ctape permet par la suite la penetration de la capside viraie dam la cellule. Ce processus, contrairement 5 la plupart des virus enveloppis, ne s'effectue pas par endocytose et est ainsi indipendant du pH 5 la surface cellulaire (Stein et al., 1987). Au terme de plusieurs Ctapes rt5sumCs d a m la figure 3, I'ARN monocatinaire du VIH-1 relargui dam le cytoplasme est transcrit en un
ADN bicatenaire par la transcriptase inverse. Cet enzyme v i d e posdde 5 la fois une activiti ADN polym6rase et une activitk ribonuclease h i permettant d'effectuer la rktrotranscription du VIH-1. L'ADN nouvellement g6nM presentera alors B chacune de
ses extr6mids un LTR sur lequel on retrouve de multiples sequences susceptibles d'Ctre reconnues par des facteurs cellulaires et des proteines du VIH-1. Ces elements seront nkcessaires B I'intkgration et l'expression adequate du genome viral. L'ADN sera ainsi achemine vers le noyau cellulaire en un complexe de priintegration incluant une enzyme virale, l'intigrase, pour y &re insiri diatoirement au sein du matiriel ginetique de la cellule. Contrairement aux oncoritrovirus, ce processus de transport nuclkaire du complexe de preintc5gration du VIH-1 est ATP dependant et semble &re independant du cycle cellulaire (Bukrinsky et al., 1992). D'autres part, la rkplication de I'ADN cellulaire ne semble pas ttre un prerequis 2 l'intigration v i d e puisqu'elle n'est pas ntcessaire lors de reaction in vitro &integration (Fujiwara et Craigie, 1989). L'ADN viral prksent dans le noyau, se retrouve ainsi sous trois formes distinctes; soit linkaire, servant de precurseur B L'intCgration, et des formes circulaires possedant un ou deux LTR
(Haseltine, 1992). Une fois integre sous une forrne dite "provirale", le complexe de preintkgration du virus aura accompli sa fonction et toutes les &apes subskquentes de rkplication seront midiies par la rnachinerie transcriptionnelle dec. la c e h l e . L'expression des g h e s viraux sera donc optimisie grgce B des facteurs cellulaires et viraux qui conjointement participeront 2 l'activation des cellules infectees. Ainsi, Ies
LTR presentent des motifs spEcifiques pennettant la transcription du ginome viral en ARN par I'ARN polymkrase II de la cellule (Jones et al., 1986; NabeI et Baltimore, 1987; Folks et al., 1989; Lu et al., 1990; Kato et al., 199 1). On retrouve: (i) une sequence TATA classique servant de prornoteur, (ii) des sites de liaison pour le facteur de transcription ubiquitaire SPl, (iii) des sites de liaison pour le facteur cellulaire NF-
KB, et (iv) une sCquence NRE oh sont situks de nombreux sites de liaison pour des facteurs de transcription cellulaire. Griice 2 ces motifs le VIH-1 a d6veloppk plusieurs stratagimes d o ~ a nlieu t
une infection la fois persistante, lente et progressive (revue
par Laughlin et Pomerantz, 1994). L'expression des g h e s du VIH-1 de'butera avec la synthkse d'une copie complite
D'ARN 5 partir de 1'ADN proviral. Le taux d'initiation de transcription est sujet en grande partie B la nature et 11activitr5de divers facteurs cellulaires. Un grand nombre de ces proteines, produitent lors de l'activation de la cellule, vont se lier aux motifs situks pr6s du site d'initiation de la transcription, que l'on retrouve dam un des LTR du genome viral integre. 11 existe donc une Etroite relation entre l'activation des cellules et I'expression des g h e s viraux. La stimulation des cellules infectees par des agents mitogeniques, des antig5nes exoghes ou des cytokines est une condition prkalable B la replication du VIH-1 autant in vitro que in vivo (Zack et al., 1990). Suite 2 la stimulation des lymphocytes T infect&, on observers l'activation de facteurs de transcription cellulaire tel le NF-KB normdement associC
I-.
son inhibiteur, le facteur
La dissociation de IKB permettra le transport de NF-KBdans le noyau de la cellule
oh il se liera aux motifs presents dans le LTR de I'ADN proviral intEgr6. Cette
interaction spicifique induira l'expression basale des gknes viraux. Ainsi, le VIH- I n'entrera dans un cycle replicatif actif que lorsque les cellules infectees auront 6ti elles meme stimulies, favorisant ainsi l'evasion v i d e et une infection persistante. On doit cependant noter certaines restrictions 2 ce phenomhe puisque I'ADN proviral peut &re traduit dam des populations cellulaires matures telles les macrophages, les cellules dendritiques et les cellules microglides du cerveau (Watkins et al-,1990; Langhoff et al., 1991) chez lesquelles la division cellulaire est inexistante. Ces cellules sont
consid6rCes c o m e des r6servoirs de virus Ctant rkfractaires aux effets cytopathiques du
VIH- 1 et pouvant assumer la dplication de ce dernier. Suite & l'activation cellulaire, les LTR du VIH-1 deviennent des promoteurs actifs apte 5 la liaison de llARN polym6rase II cellulaire et contr6lant la synthke des ARNm viraux.
A ce stade de la replication, des moMcules complbtes D'ARNm
nouvellement synthdtisies vont s'accumuler dans le noyau de la cellule. Ils y subissent alors un ipissage alternatif donnant lieu
un groupe h6tirogene de transcripts multi-
epissks et desquelles sont synthitist5s les protCines Tat, Rev et Nef. La p r o t h e Tat favorisera la synthhe de toutes les nouvelles mol6cules d'ARNm viraux, tandis que la protCine Rev permettra le transport dam le cytoplasme des ARNm codant pour les genes de structures et leur disponibilit6 2 la machinerie de traduction cellulaire. Les ARNm qui n'ont subi aucun ou un seul ipissage vont Etre exprimis en protiines de structure soient les protkines de la capside virale (Gag) et les glycoprotkines d'enveloppe (Env), de mCme que les enzymes (GagPol) ndcessaires B la replication du VM- 1. Par la suite, les proteines virales vont subir des modifications post-traductionnelles incluant glycosylation, myristylation et phosphorylation. Les particules virales seront fmalement assemblt5es au n i v e z ~de la membrane plasrnique, encapsidant du meme coup deux moldcules d'ARN ghomique du VIH-1. Les virions matures nouvellement form& vont dors bourgeomer
travers la membrane ceUulaire e t vont entrainer avec elles une partie
de la bi-couche lipidique de la cellule ainsi que les glycoprotBines de l'enveloppe virale.
la gp41 et la gp120. Ces derniers pourront 2 leur tour infecter de nouvelles cellules et ainsi perpetuer le cycle replicatif du VIH-I. Plusieurs aspects de la replication du VIH-1 restent encore 6nigmatiques. Par quel(s) mhtnisme(s) le VIH-1 parvient-il ii infecter et se repliquer efficacement dans des lymphocytes T au repos ou encore dans des populations monocytaires? Les lymphocytes T CD4+ isolCs d'individus en phase asymptomatique representent majoritairement une population de cellules au repos contennant des forrnes dlADN proviral non-integre (Bukrinsky et al., 1991). De plus, I'interaction de la gp 120 ou d'anticorps anti-CD4 avec le recepteur CD4 semble inhiber I'activit6 de certains facteurs de transcription nucl6aire (NF-AT, NF-KB et AP-I) qui ont un r6le & jouer dans la regulation d'expression du g h e de l'interleukine-2 (Jabado et al., 1994). La liaison entre la gp120 et la molecule CD4 n'apparait donc pas suffisante pour pallier i I'activation des cellules infectees. Ainsi, il est clair que le VIH-1 entretient d'etroites interactions avec la cellule hate afin d'induire la stimulation ou la differentiation cellulaire, optimisant ainsi l'infection virale. De multiples facteurs cellulaires auront des rales prdponderants dans les diff6rentes &apes du cycle v i d e (revue par Gaynor, 1992).
Par ailleurs, des proteines du VIH-1 telles la p17gQg et Vpr participeront egalement i 116tablissementde l'infection productive dans des populations cellulaires inaptent B se diviser. Elles faciliteront via des interactions spkcifiques le transport du complexe de preintegration viral dans le noyau des macrophages et des cellules tpithiliales (Bukrinsky et al., 1993; Heinzinger et al., 1994). Le VIH- 1 peut donc procdder B l'infection de differentes populations cellulaires, dictant ainsi la progression du cycle
viral et 116volutionde la maladie.
2.
La mol6cule CD4: principal r6cepteur ceUulaire du VIH-1. Afin d'entreprendre son cycle d'infection, le VIH-1 reconnait B la surface des
cellules cibles, la mol~culeCD4 (Dalgleish et al., 1984; Klatzmann et al., 1984; Maddon et al., 1986; Clapham et al,,1987). Les travaux effectuks par Maddon et al., (1986) demontrent sans equivoque 1laffinit6 du virus pour les cellules exprimant 2 la surface la molkule CD4. Des anticorps monoclonawr (AcM) dirigis contre ce dernier inhibe l'infection virale. De plus, I'expression de CD4 dans des cellules autrement rdfractaires 5 L'infection par le VM-1 leur coserent la capacite de lier le virus (Maddon et al., 1986). Malgri certaines homologies avec la molicule CD4 humaine, 1'6quivalent
CD4 murin (L3T4) ne lie pas la gp 120 du VM-1 (Landau et al., 1988). L'interaction entre la glycoproteine d'enveloppe virale et le CD4 s'avkre donc des plus sp6cifique et de haute affinite.
La moEcule CD4 est une glycoproteine membranaire exprimke sous la forme d'un monomkre glycosylk d'environ 55 kDa.
Elle comporte quatre domaines
extracellulaires (Dl-D4), une region transmembranaire de 25 acides aminis et une queue intracytoplasmique de 38 acides aminks
son extremiti C-terminale (figure 4).
La mol6cule CD4 joue non seulement un r6le important dans 1'Claboration de la reponse irnmunitaire mais sert Cgalement de transducteur dam les phenomknes &activation cellulaire.
Extracellulaire
RWRFKK1 tlLW88LJtE Myrlstlfatlon
Cytoplasmique
Unique
SH3
SH2
Di-Leu
Klnase
Rcgulatoirc
Figure 4. Reprbentation schgmatique de la molCule CD4 et de la tyrosine kinase p5dCk. (modifiCe de la refdrence Geleziunas et nl., 1994)
Lors de la reponse imrnunitaire, la mol~culeCD4 n'interagit qu'avec les Ag de classe II du CMH prCsentCs par Ies cellules presentatrices d'antigknes (Bank et Chess, 1985). Cette interaction semble spdcifique puisque des AcM diriges contre l'un ou l'autre des intervenants inhibent leur association. De plus, des Ctudes de mutagknkse effectuies sur la mole'cule CD4 demontrent que des rdsidus pr6sents dam les domaines extracelluIaires D 1 it D3 sont essentiels B I'interaction avec les Ag de classe II (Clayton et al., 1989; Lamarre et al., 1989a). CD4 servirait ainsi de rnol6cule &adhesion afin de
stabiliser les complexes formCs entre les Ag de classe 11 sur la cellule cible et le rCcepteur de I'antigine des lymphocytes T (TCR; "T Cell Receptor") exprime 2 la surface de la cellule effectrice. En plus de son interaction avec les Ag de classe II, la molc5cule CD4 s'associe Cgalement en cis avec le TCR B la surface de la cellule afin d'amplifier les signaux conduisant B l'activation cellulaire. Cette interaction semble cependant faible et transitoire puisquleIle ne s'observe que dans 5 B 10% des lymphocytes T au repos (Anderson et al., 1988). Ainsi, la reconnaissance antighique
par les lymphocytes T entrafnera une redistribution des TCR et des mol&ules CD4, autrernent uniformdment reparties 2 la surface cellulaire, ii la zone de contact entre les celluies effectrices et les cellules prCsentatrices d'antigtnes (Kupfer el al., 1987; Rivas et al., 1988). La queue intracytoplasmique du CD4 semble jouer un r6le important dam
la formation des complexes CD4/TCR (Mittler et al., 1989). Par des techniques de
cytofluomc5trie en flux, les auteurs montrent que des moltcules CD4 tronquees, n'exprimant plus le domaine intracytoplasrnique, ne sont plus aptes 5 interagir avec le TCR. De plus, la formation de tels complexes semble avoir un effet synergique sur les &apes conduisant B l'activation cellulaire. L'absence de la queue intracytoplasmique sur la molecule CD4 inhibe 6galement l'activation des lymphocytes T (Sleckman et al., 1988). Il est clair que La molicule CD4 s1av5reun atout important dans les Ctapes de stimulation antigenique des lymphocytes T.
Outre sa fonction de molecule d'adhksion, le ricepteur CD4 sert 5 priori de transducteur dam les phinomhes d'activation cellulaire. Les travaux de Veillette et al., (1988) montrent la liaison non covalente de la rnoMcule CD4 B la tyrosine kinase p561ck (figure 4). Cette association spicifique est m&We par un motif cystCine-XX-cystiine (risidus en positions 20 et 23) dans la region N-terminale de p56[ck, "X"reprisentant un acide amini quelconque, et deux risidus cystkine situes en positions 420 et 422 de la queue intracytoplasmique de la molicule CD4 (Turner et al., 1990; Collins et al., 1992).
La tyrosine b a s e p561ck est un membre de la famille src implique dam la regulation des ph6nomhes d'activation et de diff6renciation cellulaire (Marth et al., 1988; Marth et al., 1989; Sefton, 1991). Ainsi, des mutants de la molicule CD4 incapable de lier la
p561ck perdent leur capacite d'induire la sicr6tion d'interleukine 2 en riponse B une stimulation antighique (GIaichenhaus et al., 1991). L'activation des lymphocytes T n'a donc lieu que si la molecule CD4 est associCe Zi la p561~k. Suite B la stimulation des lymphocytes T, on observe la dissociation des complexes ~ ~ 4 / p 5 6 et 1 ~I'internalisation & des molicules CD4 qui empechent une stimulation excessive des cellules. La molkule
CD4 par son interaction avec la p56lck est ainsi capable de transmettre des signaux d'activation cellulaire.
2.2. L'interaction entre la gp120 et la mol6cule CD4: une liaison de haute aEinit6.
L'utilisation de formes solubles de la molecule CD4 a permis de dklimiter les regions de la protkine responsables de l'interaction avec le VlH-1. Ainsi, des mol~cules
CD4 tronquies solubles, ne posskdant que les deux premiers domaines extracellulaires de la protkine, peuvent efficacement inhiber la fixation du VIH-1 ti la cellule CD4f (Traunecker et al., 1988; Berger et al., 1988). Des etudes de mutagin2se ont permis de restreindre le site d'interaction de la gp120 au domaine D l de Ia molt5cule CD4
(Peterson et Seed, 1988). Les acides amin& 1 B 83 sont suffisants pour permettre la liaison de la gpl20, tandis que les residus 40 B 55 sont indispensables (Arthos et al., 1989). La liaison specifique de la gp120 inhibe les interactions entre les mol&ules CD4 et son ligand, 1'Ag de classe II du CMH. Les etudes de mutagenhe effectuees sur la mol6cuIe CD4 demontrent bien que Ies sites de liaison B la gp 120 et aux Ag de classe II sont surperpos6s mais distincts les uns des autres (Clayton et al., 1989; Lamarre et al., 1989a). Seut le domaine D l est requis dam la liaison 2i la gpl20, tandis que les domaines D l B D3 semblent importants dans les interactions avec les Ag de classe II.
De plus, des formes mutantes de la mol~culeCD4 qui ont perdu la capacitk de lier la gp120, ont toutefois conserve celle d'interagir avec les Ag de classe II (Larnarre et al., 1989b). Les differentes etudes r6alisies jusqu18maintenant montrent bien que la gp 120 et les Ag de classe II ne peuvent lier simultankment la mol6cule CD4 (Fleury et al., 1991; Houlgatte er al., 1994).
Par ailleurs, la liaison de la gp120 B la mol6cule CD4 provoque egalement la phosphorylation et l'internalisation de la prodine (Fields, 1988). Suite ii une stimulation mitoginique ou antigenique, la mol6cule CD4 est norrnalement internalisee p i s degradee dans les lysosomes cellulaire (Acres et al., 1986). La stimulation entrdinera la phosphorylation de la mol6cule CD4 sur des residus sirine (en positions 408, 415 et/ou
43 1) presents au niveau de la queue intracytoplasmique, pre-requis au cherninement de la protkine vers les voies de dbgradation (Blue et al., 1987). U n phEnom2ne sembiable s'observe au cows de l'infection par le VIH-1. Toutefois, les etudes de mutagCn5se ont dEmontri que ni l'internalisation, ni la phosphorylation de CD4 ne sont requis dam la fixation et l'entree du VM-1 (Beddinger, 1988). La liaison de la gp120 engendre la dissociation du complexe CD4/p56lck entrainant ainsi l'internalisation de la molecule
CD4 et l'activation enzymatique de la p56fck (Soula et a/.,1992; Hivroz et al., 1993).
En rCduisant l'expression de la mol~culeCD4, le virus affecte non seulement la physiologie de la cellule infectke mais prkvient Cgalement une surinfection virale potentiellement nkfaste pour I'hbte (Garcia et al., 1991; Benson et al., 1993). De plus, les lymphocytes T infect& seront moins susceptibles de fusiomer avec des cellules non infectCes prolongeant la survie de la population cellulaire et l'infection par le VM-1 (Stevenson et al., 1988; Garcia et al., 1991; Benson et al., 1993).
2.3. R6guIation de I'expression de la mol&ule CD4 par le VIH-1. Suivant l'infection de cellules CD4+ par le VIH-1, on observe une diminution de l'expression de la mol6cule CD4 B la membrane plasmique (Klatzrnann et al., 1984; Stevenson er al., 1987; Clapharn et al., 1987). La disparition de la molCcule CD4 est dependante en grande partie du taux de rkplication virale. Ainsi, dans des IignCes cellulaires qui produisent du VIH-1 de manikre chronique, il est possible de rktablir l'expression de la mol6cule CD4 en inhibant le cycle rkplicatif du VIH- 1 (Shahabuddin et al., 1992). La r6gulation de l'expression de la mol6cule CD4 par le VIH- 1 s'av6re une
Ctape importante dans I'infection v i d e et les mCcanismes intracellulaires irnpliquks dam ce phinomhe, quoique nombreux, restent encore nebuleux. Plusieurs Ctudes ont montr6 l'implication du prCcursew polypeptidique gp 160 de I'enveloppe virale dans la diminution de I'expression de la molCcule CD4 B la surface de la celhle infectge (Kawarnura et al., 1989; Crise et al., 1990; Jabbar et Nayak, 1990; Butera er al., 1991). La gp 160 et la molCcule CD4 peuvent former des complexes dans le rCticulum endoplasmique (RE) de la cellule, empechant ainsi le transport et la maturation intracellulaire des deux molkcules (Crise et al., 1990; Jabbar et Nayak, 1990). Cependant, cette retention dam le RE ne rend ni la gp160, ni la molt5cule CD4 susceptibles aux voies de degradation de la cellule (Bour et al., 1991). La prodine Vpu semble plut8t mkdier un tel processus puisqu'elle d6stabilise les complexes gp 160lCD4
dans le RE en induisant spdcifiquement la degradation des mol6cules CD4 (Willey et al., 1992a; Willey et ale, 1992b). Les travaux r e c e m e n t effectuks par Bour et al. (1995) montrent d'ailleurs que Vpu peut lier la moldcule CD4 entrainant probablement sa dkgradation par un micanisme encore non klucidd. Des ktudes de mutagin2se ont dkmontri que la region transmembranaire et la queue intracytoplasmique de la moldcule
CD4 renferment des d6tenninants essentiels aux effets de Vpu (Lenburg et ai, 1993; Willey et al., 1994; Raja et al., 1994; Buoconore et al., 1994). Par ailleurs, la prodine Nef induit l'internalisation et la degradation des mole'cules CD4 exprimees 5 la surface des cellules (Garcia et al., 1993; Aiken et al., 1994; Anderson et al., 1994). Les travaux de Aiken et al. (1994) montrent que des skquences dam la queue intracytoplasmique de la mol6cule CD4 sont suffisantes pour induire son internalisation par Nef.
La
phosphorylation de la rnol6cule CD4 n'est pas requise dans ce phenomkne puisque la substitution des rdsidus skrine en positions 408,415 et 43 1 n'inhibe pas les effets de Nef (Garcia et ai., 1993). Toutefois, un motif leucine-leucine (risidus en positions 413 et 414), Cvocateur d'un signal d'endocytose et de ciblage vers les lysosomes cellulaires (Letourneur et Klausner, 1992), se revkle important dans l'internalisation et la ddgradation de CD4 par Nef (Aiken et al., 1994). La diminution d'expression de CD4 lors de l'infection v i d e semble &re aussi le resultat d'une baisse du t a u d'ARNm codant pour la molicule dans les lymphocytes T CD4+ (Hoxie et al., 1988; Salmon et al., 1988). Cependant, ce ph6nomCne ne s'observe pas dans des ligndes monocytaires en depit d'une faible expression de CD4 ii la surface des cellules (Geleziunas et al., 1991). Finalement, une etude montre que les moldcuies CD4 peuvent Etre entrainees lors du bourgeonnement viral par les virions nouvellement form& (Meerloo et al., 1992). Le VIH-1 a donc recours B une multitude de mecanismes
afin de moduler l'expression de la moldcule CD4 i la surface des cellules infectdes.
3.
Les glycoprot6ines d1enveIoppedu VIH-1. Les glycoprot6ines de l'enveloppe v i d e ont un r6le primordial B jouer dans la
pathoginhe du S I D A . Elles posskdent les ipitopes ndcessaires h I'interaction avec la cellule cible et les iliments responsables des phdnomiines de fusion membranaire pennettant 11entr6edu virus dans la cellule. Les glycoprot6ines d'enveloppe du VIH- 1, codee par le gkne env, sont tout d'abord synth6tisCes dam le RE sous la forme d'une polyprot6ine.
Celle-ci sera fortement glycosylie pour donner un prkcurseur
polypeptidique de 160 kDa (gp 160) (Allan et al., 1985). Le prkurseur gp 160 acquiert alors la capacite de liaison aux mol~culesCD4. Elle sera Cgalement capable de multimirisation (Earl et al., 1990). Au cours de son passage dam I'appareil de Golgi, la
gp160 sera scindC par la protCase cellulaire furine (Hallenberger et al., 1992) en deux sous-unites, la glycoproteine de surface gp120 et la glycoprodine transmembranaire gp41. Seulement 5 B 15% des polyprotiines gp160 seront achemindes vers la voie de
clivage, tandis que la majoriti sera digradde dans des lysosornes cellulaires (Willey et al., 1988). Les glycoproteines d'enveloppe seront findement transport~es3 la surface de la cellule oh, ancrdes 2 la membrane plasmique, elles pourront $we incorporkes aux particules virales naissantes.
3.1. Structure et synthke des glycoprot6ines de l'enveloppe virale. Le precurseur gp160 de 856 acides aminis de long subit au cours de sa maturation divers modifications ammenant
la formation des deux sous-unitis
fonctionnelles de l'enveloppe (Earl et al., 199 1). Lors de l'tlongation de la chafne polypeptidique, les 30 premiers acides amin& de la region N-terminale du prCcurseur servent de peptide signal. Cette sdquence est responsable de la translocation de la polyprot6ine naissante dans les microsomes du RE. Elle est par la suite 6lirninCe par une protease cellulaire avant mSme que soit accomplie la synthkse de la charne
polypeptidique. Cette nouvelle polyprotkine est ensuite glycosyEe par l'addition de mannoses sur certaines sbquences Asn-X-Ser ou Asn-X-Thr hautement conservees, ammenant 2 la formation du prdcurseur gp 160. Aprh Clagage de glucoses et de certains mannoses par des glucosidases et mannosidases cellulaires, la gp 160 est transport6e vers l'appareil de Golgi oii elle y subira d'autres modifications. Au cows de son transit dam le RE, la gp 160 est apte
former des dirn&reset possiblement des formes tktramCriques
essentieiles pour son transport dans la cellule. La gp160 sera alors excisee afin de dormer naissance B deux fragments capable d'interagir de fason non covalente: une portion N-terminale de 480 acides amin6s qui cornposera la gp120, et une portion Cterminale de 344 acides aminks servant d'ichine 2i la gp4 1. Ces sous-unitis seront elles memes glycosyl6es par l'addition de divers monosaccharides. Respectivement, 4 et 24 sites de glycosylation seront modifiks sur la gp41 et la gp120 afin de permettre le transport et la maturation adequate des glycoprotdines dlenveIoppe (Stein et Engleman, 1990; Lee et al., 1992; Li et al., 1993). Plusieurs Ctudes de mutagknise ont permis d'identifier des domaines fonctionnels sur la gp120 et la gp4l (Kowalski et al., 1987; Lasky et ai., 1987; Hwang
er al., 1991). Ces regions hautement conservCes parmi les differents isolats du VIH- 1 sont importantes dam les phCnorn6nes de liaison aux rnolCcules CD4, de fusion membranaire, d'interaction entre les sous-unit& de l'enveloppe et de multimkrisation. La gp 120, fortement glycosylee, comporte cinq domaines invariables (C LC5) et cinq domaines hypt;rvariables (V 1-V5) (Starcich et al,, 1986; Willey et al., 1986; Sullivan et al., 1993). Grace B ces domaines, le VIH-1 peut infecter une grande varied de cellules, mais egalement peut echapper au sysdme irnmunitaire de I'hbte.
Les sequences
hypervariables V1 et V2, situCes dam la portion N-terminale de la gp120. sont importantes dam les interactions gp 120-gp4 1. La r6gion hypervariable V3 est le principal determinant du tropisme cellulaire (Takeuchi et al., 1991; Shioda et a!. , 199 1). Cette region, qui forme une boucle entre les acides arnines 298 et 327, confere au VTW- 1
la capaciti d'infecter stlectivement des celules lymphocytaires ou monocytaires. Des itudes de mutagdnkse effectudes par Takeuchi et al. (1991) sur la rggion V3 ont dkmontrk que Ie simple changement d'un acide amin6 en position 3 11 pouvait modifier le tropisme cellulaire du VIH-1. La region V3 est Cgalement impliquee dans les mkcanismes de fusion membranaire. Des mutations dam la boucle peuvent r6duire ou abolir la formation de syncytium mkdid par la gp 120 (Freed et aL, 1991). Finalement, Ia rkgion V3 comporte le d6terminant antigenique prddominant du VIH-1 (Skinner et al., 1988).
Par ailleurs, plusieurs travaux ont ddmontrk que les rEgions hautement
conservees C2, C3 et C4, situees dans I1extr&nitCC-terminale de la gp120, etaient impliqukes dans les interactions du VIH-1 avec Ie recepteur CD4 (Lasky et al., 1987; Cordonnier et al., 1989; Olshevsky et al., 1990). Ces regions, servant de zone de fixation au CD4, semblent etre masquCes par les regions hypervariables. Ainsi, le VKH1 peut tchapper aux anticorps en modifiant les sequences de ces dernieres sans avoir 2 modifier son site de fixation. La portion interne de l'enveloppe virale, la gp41, posskde quatre domaines '
fonctionnels distincts requis dam l'infectivite du VIH- 1. La region N-terminale de la gp41 possgde entre autres les determinants responsables des processus de fusion membranaire (acides aminks 5 12 B 5 17) (Kowalski et al., 1991) et importants pour l'interaction avec la gp120 (acides amin& 528 B 628) (Kowalski, 1987; Helseth et al., 1991). Cette interaction de nature non covalente semble precaire et conduit B un relarguage spontant de la gp 120 de la surface des particules virales (WiUey et al., 1988; Moore et al., 1990). Par ailleurs, on retrouve des rksidus cystCine hautement conserv6s (en positions 598 et 604) qui sont essentiels au processus de clivage du precurseur
gpl6O (Dedera et al., 1992) et des sites de glycosylation importants dans la capacit6 fusioghne de la gp41 (Lee et al.. 1992). Le domaine transmembranaire de la gp4 1 (acides aminks 684 Zi 705) fortement hydrophobe sert d'ancrage B la glycoprotkine d'enveloppe 2 la surface du virus (Gabudza et al. 1991). Finalement, I'une des
caract6ristiques de la gp41 du VIH-1 est sa longue queue intracytoplasmique 2 l'extremitd C-terminale d'environ 150 acides aminks.
Eile semble jouer un r6le
important dam la structure gknerale de la glycoproteine et/ou dam Ies processus de changement de conformation de l'enveloppe Iors de la fusion rnembranaire (Lee et al. 1989; Spies et al., 1994). Cette region presente des determinants essentiels 5 l'oligomCrisation de I'enveloppe v i d e (acides aminks 68 ii 129) (Earl et Moss, 1993).
De plus, la gp4 1 semblerait interagir avec la protkine v i d e p l7gag afin de pennettre le transport et le bourgeonnernent polarise du VIH- 1 dans les cellules kpitheliales (Owens et al., 1991; Lodge et al., 1994).
4.
Les prot6ines de structures du VIH-1. Les produits du g5ne gag sont synthktisis dam le cytoplasme de la cellule par
des polyribosomes libres sous la forme d'un prkcurseur polypeptidique, Ie Pr55gag. Suite 2 Itattachement d'un acide myristilique il son extr6miti N-terminale, le precurseur Pr55gag est dirige vers la membrane plasmique oCi il est capable de multim&-isationpar des interactions non-covalentes.
On observe alors le ciivage du Pr55gQg en de
nombreuses proteines de structures (p17gag, p24gag, p9gQget pWg) par la protiase virale (Gottlinger et al., 1989; Kaplan et Swanstrom, 1991). La polyprotkine PrSSgag est la seule composante necessaire 2 l'assemblage de particules virales matures ou irnmatures (Smith at al., 1990; Mergener et al., 1992). Ainsi, 11int6grit6structurale et la maturation du pr&wseur sont importants dans la formation de particules virales (Hong et Bodanger, 1993).
4.1-
Structure et synthke des protCines Gag du VIH-1.
La proteine p17Wg produite B partir du clivage de la region N-terminale du precurseur Pr55gag forme la matrice interne de la particule virale. La myristylation post-traductionnelle qui s'effectue par I'attachement d'un acide gas, l'acide myristilique, sur une glycine en position 2 de la proteine pl7gag est essentielle B la formation de particules virales (DiMarzo et al., 1988). Elle permet le transport intracellulaire et l'attachement du precurseur Pr55gag Zi la membrane plasmique de la cellule (Gottlinger et ai., 1989; Wang et Barklis, 1993). La prodine p 17gW est &dement phosphorylke sur
des risidus sCrine (Mervis et al., 1988). Par ailieurs, elle presente des determinants essentiels au site d'assemblage et au bourgeonnement viral, puisque la delktion de sequences au niveau de la p 17gag entraine l'accumulation de particules virales dans le r6ticulum endoplasmique de la cellule (Facke et al., 1993). Plusieurs etudes de mutagCnise ont montrk l'importance de la p17gag dans l'incorporation des glycoprotiines d'enveloppe lors du bourgeonnement des particules virales (Yu et ai., 1992; Dorfman et al., 1994a). Elle semble interagir avec la partie intracytoplasrnique de la gp41 afin de retenir les glycoprotiines d'enveloppe au virion. Finalement, lors des &apes pr6coces de l'infection, la p17gaS contenue dans Ie virus serait impliquee dans le transport du complexe de preintegration virale dans le noyau de la cellule infect& (Bukrinski et al., 1993). La proteine posskde une rigion riche en lysines (acides amines 25 B 3 1) qui pourrait agir c o m e signal de localisation nucl6aire. La phosphorylation d'un r6sidu tyrosine en position 132 de la p l 7 P g semble egalement importante dans ce phknomhe (Gallay et al., 1995). La mutation ponctuelle de la tyrosine n'affecte pas la replication du VM-1 dans les cellules infectees, mais inhibe toutefois le transport du complexe de priintegration virale dam des macrophages diffdrenciis. La p r o t h e p24gag est la composante majeure de la particule virale. On estime ii environ 1200 copies, le nombre de p24Wg formant la capside virale (Layne ef al., 19%).
Elle est ainsi le principal determinant antighique du VIH-1 (Tatsurni et al., 1990). Les etudes rkalisees par Hong et Boulanger (1993) montrent l'importance de la p24gag non seulement d a m la structure des virions mais egalement lors de l'assemblage et le bourgeomement viral. Les mutations ponctuelles des risidus leucine en positions 136 et 190 de la region C-terminale de la p24Bag peuvent affecter l'assemblage intracellulaire des pr6curseurs polypeptidiques et le relschement des particules virales 5 la surface de la membrane plasmique. De plus, la portion C-terminde de la p248Qg semble comporter des determinants importants dam la morphogin2se des particules virales (Dorfman et al., 1994b). Findement, la rkgion N-terminale de la p24gQg presente des dkterminants
pennettant la liaison in vitro aux cyclophilines A et B (Luban et al., 1993; Thali et al., 1994).
Les cyclophilines sont des proteines cellulaires qui peuvent lier
l'imrnunosuppresseur cyclosporine A (Handschumacher et al., 1984). Ainsi, il est possible de detecter une grande quantiti de cyclophiline A dam les particules virales matures du VIH-1 (Thali et al., 1994).
Le clivage de I'extremitk C-terminale de la polyproteine m5gag dome naissance aux protCines p9gag et p6g%
La protiiine p9gag de la nucleocapside joue un r61e
important dans l'encapsidation de IfARN ginornique dans la particule virale nouvellement form6e (Gorelick et al., 1990). Deux motifs cystkine-histidine (acides aminks 15-28 et 36-59) presents dans la p9gag et hauternent conservCs parmi tous les retrovirus, s'avkrent essentiels pour l'encapsidation de 1'ARN gdnomique viral (Gorelick et al., 1990; Dorfman et al., 1993). Findement, l'un des aspects particuliers des produits
de clivage du Pr55gag est la presence de la protkine p6gag que l'on ne recomait pas chez la majorit6 des r6trovirus. Cette protdine riche en dsidus proline aurait un r6le B jouer dans le bourgeonnement viral (Gottlinger et al., 1991) et dans l'incorporation de la protCine Vpr dans la particule virale (Lu et al., 1993; Paxton et al., 1994; Kondo et al., 1995; Checroune et al., 1995).
4.2.
La rnachinerie enzymatique du VIEI-1.
L'une des particularitds du VIH-1 est le chevauchement des g h e s gag et pol. La synthkse des enzymes viraux requikrent un dtplacement de -1 nuclkotide entre les deux cadres de lecture ouverts du genome viral (Jacks et al., 1988). Au cours de la traduction de 1'ARN genomique, le dtplacement des polyribosomes survient au niveau d'une sequence homopolym6rique favorisant ainsi la synth2se des g h e s gag (Wilson er al., 1988). Ainsi, suite au changement de cadre de lecture, les produits du gene pol seront
synthttises B paair d'une polyproteine de fusion Gag/Pol, le prdcurseur ~rl60gag/p~l. Les proteines Gag et Pol sont produites dans un rapport de 20: 1 dans la cellule infectke. Tout comme la polyproteine PrSSgag, le prdcurseur Pr l6O@g/~olest mytistile suivant sa synthhe et migre jusqu'g la membrane plasmique de la cellule par des mecanismes encore mal ddfinis. 11 est alors capable de former des dim5res durant le bourgeomement du virus et de produire les enzymes essentiels A la replication v i d e , soient: la protdase (p lO@), la transcriptase inverse (p66@), et I'intt5grase (p32~0f). Au cours de l'assemblage des particules virales, la protiase est capable de
dimerisatioo meme si elle fait partie du precurseur ~ r l 6 0 g a d ~ oLes f . etudes de structure effectuees sur La protCase ont montre que la protCine n'ttait active que sous la forme de dim5res (Miller et al., 1989). La protCase se libkre du prkurseur ~rl60gag'pof par un phhomkne d'autocatalyse. Elle est par la suite responsable du phenomhe de clivage sdlectif amrnenant A la liwration des enzymes ainsi que des proteines de stuctures du
VIH-1 (Peng et al., 1989). La protease semble tgalement impliquer dans l'hydrolyse de prodines du cytosquelette de Ia membrane plasrnique, facilitant le bourgeonnement viral (Adams et aL, 1992). Elle est findement incorpor6e
un complexe enzymatique associt & la capside virale.
la particule virale naissante dans
Le clivage du precurseur ~rl60gag'~olentraine la liberation d'une proteine de 66 kDa, la transcriptase inverse, capable de former des dirn&res, Cet enzyme permet la transcription de I'ARN gCnornique du VIH-1 en un ADN bouble brim EUe poss2de
la
fois une activitk ADN polymtrase mais igalement une activit6 ribonucl6ase qui proc5de
A la degradation de llARNt lysine et I'ARN genornique de l'hybride ARN-ADN servant & la retrotranscription virale (Starnes et Cheng, 1989). La transcriptase inverse est un het6rodirnere former d'une protkine de 66 kDa ( ~ 6 6 ~ 0 1 et) d'un produit d e clivage ultkrieur, une sous-unit6 de 51 m a . On peut ainsi observer dans la particule virale mature la prksence d'une sous-unite de 51 kDa et une autre de 15 kDa ayant respectivement des activites polymCrase et ribonuclCase. Ces deux sous-unites ne sont toutefois actives que sous la forme d'un complexe hitQodim5re p66/p5 1 (Mizrahi et al., 1989). Findement, le VIH-1 possede une protkine de 3 1 kDa, l'integrase, provenant du clivage de la region C-terminale du prdcurseur ~rl60gag/po[. Cet enzyme est pr6sente en plusieurs copies dans la particule v i d e sous la forme d'un dimire. L'integrase est 2 la fois capable d'activitt endonuclkase spicifique sur I'ADN viral nouvellement transcript et non sp6cifique lui pennettant d'exciser I'ADN de 11h8te. Elle procedera par la suite B l'intkgration de L'ADN viral au genome de 11h6te. L1intCgrase est le seul facteur protkique necessaire A la reaction d'intdgration virale (Bushman et al., 1990).
5.
Assemblage et maturation du VIH-1. L'assemblage du VIH-1 dCbute par l'accumulation
des pricurseurs
polypeptidiques Pr55gag et ~ r l 6 0 ~ a g ' ~ Ao lla surface interne de la membrane cellulaire (figure 5). Tout comme les r6trovirus de type C, ces polyproteines interagissent 5 la membrane plasmique de la cellule infectde pour former la particule naissante. Des mutations empechant la myristylation post-traductionnelle des polyproteines inhibent la
replication du VIH-1 (Gottlinger et al., 1989; Wang et Barklis, 1993; Facke et al., 1993). L'association des polyprotkines Pr55gag par des dkterrninants prdsents dam la
region codante de la p24Wg semble donc induire les premi6res &apes du processus de bourgeonnement viral. Ces interactions donnent aux virions naissants leur structure courbke initiale A la surface de la cellule. Deux mole'cules completes d'ARN genornique viral seront par la suite encapsidkes dam la particule naissante. Des motifs cystkine-
H ~ ~ -en X~ une -C ou~dSe ) w copies A l'extrdmite Chistidine ( C ~ S - X ~ - C ~ S - X ~ -presents terminale des pr6curseurs Pr55gag (Gorelick et al., 1990; Dorfman et al., 1993), de
meme que des sequences (signal d'encapsidation Psi) uniques de la region 5' de YARN viral non-episse et qui ont la capacit6 de former quatre boucles independantes (Lever et
al., 1989; Aldovini et Young, 1990; Luban et Goff, 199 1; Clever et al., 1995) vont medids les phhom6nes d'encapsidation. Au cows du processus, une molEcule D'ARNt lysine ktroitement lide A I'ARN genomique est kgalement incorporee.
Les
glycoprotthes de I'enveloppe virale, transportdes et s'accumulant i la surface de la membrane cellulaire par des voies ind6pendantes de la biosynth6se des pricurseurs Pr55gag et ~r1608aB/P0f,seront entrainees lors du bourgeonnement de la particule naissante. Bien que I'incorporation des glycoproteines d'enveloppe soit essentielle
ii
l1infectivitCdu virus, la presence de ceiles-ci n'est pas requise dans la formation et le bourgeonnement de virions matures (Gheysen et al., 1989; Smith et al., 1990). La particule naissante acquiert membrane cellulaire.
? ce i
moment une partie de la bi-couche lipidique de la
A ce stade de l'assemblage viral, on observe alors 1'accumuIation
de virions ktroitement retenus A la surface de la cellule infectge. Ainsi, le relkhernent des particules virales semble &re un facteur limitant dam la maturation virale. En absence des sequences codantes de la p6Pg, la retention des particules virales assemblCes devient de plus en plus evidente (Gottlinger et al., 1991). La p6&% est Cgalement impliquie dam l'incorporation de Vpr, la seule proteine accessoire du VIH- 1 associke B la particule naissante.
Figure 5. Maturation de la proghie virale. (A) Mimgraphie aectronique de virions bourgeo~antsa la surface bun lymphocytes T. (B) Reprbentation sch&natique de l'assemblage de par&iculesvirales. (la figure 5B est de Fields et Knipe, 1990)
Les virions relaches B la membrane plasmique entameront quant 2 eux les derni5res &apes de la maturation.
La protkase active proc5dera au clivage des
polyprotkines Pr55gag et ~rl60gag/p*[afin de produire les protdines de structures et les enzymes de r6plication du virus, et de compl6ter ainsi le cycle rkplicatif du VIH- I. Ce n'est qu'8 ce moment de la morphogkn8se que l'on peut d6celer en microscopie klectronique la presence d'un c8ne dense cylindrique ou la capside virale, typique du virion mature. Plusieurs facteurs s'av8rent importants dans la maturation adequate des particules virales. L'utilisation d'inhibiteurs de la protdase conduit B la formation de virions noninfectieux renfermant une grande quantitk de pr&xmeurs polypeptidiques PrSSgag et ~r l60@g/~0[(Ashom et al., 1990). Ces particules virales seront plus larges en diamktre et dkpoumes d'un c8ne dense. L'inactivation de la protkase a m d n e non seulement Zi la formation de particules immatures, mais de plus, elle semble diminuer l'efficacitk de reliichement viral.
La protdine Vif semble &dement jouer un r6le dans la
morphogkntse des particules virales produites dans certaines lignies cellulaires (Hoglund et al., 1994; Borman et al., 1995). Ainsi, les analyses en microscopie dectronique effectukes par Hoglund et al. (1994) laissent entrevoir dans la majorit6 des virions n'exprimant pas Vif, une capside asymhique transparente. Ces particules noninfectieuses presenteront des quantites plus faibles d'enveloppe virale B leur surface et des proportions Pr55gaglp24gag a n o m d e m e n t klevt5es (Borman er al., 1995). L'encapsidation adiquate de I'ARN ginomique viral est egalement indispensable B l'assemblage et A la production de virions infectieux (Clavel et Orenstein, 1990). Par ailleurs, une diminution de la quantiti de cyclophilines A associ6es aux virions par I'usage d'agents immunosupresseurs telle la cyclosporine A semble attenuer l'infectivitd des virus naissants (Thali et al., 1994). 11 en convient qu'une meilleur connaissance des facteurs irnpliqu6s dam la morphogin2se du VIH-1 permettra la mise au point de nouvelles strat6gies antivirales destinies B inhiber cette 6tape du cycle &infection.
6.
Les effets cytopathiques du VIH-1. L'infection par le VIH-1 conduit g6ndralement A l'effondrement du systkme
immunitaire. On observe alors la destruction des principaux acteurs celiulaires, les lymphocytes T CD4+. Comment expliquer la disparition massive de ces ceIlules quand seulement un faible pourcentage d'entre eIles sont infectCes? Bien que les m6canismes conduisant & la mort cehlaire sont encore ma1 dkfinis, plusieurs processus mddiis par des interactions specifiques de virus 5 cellule et de cellule A cellule peuvent etre invoqukes dans la pathogenkse du SIDA. Afm de mieux comprendre les manifestations cytopathiques du VIH-1 chez l'individus, il est important d'en dkfinir les avenues vi tro. -
Les effets cytopathiques du VM- 1 en culture surviennent tardivement au cours de l'infection virale. 11s sont en grande partie attribuables aux interactions entre I'enveloppe v i d e et le rdcepteur CD4 (Sodroski, 1986). On constate lors de la replication du VIH-L en culture, la fusion entre une cellule infectee et environ 200 B 300 ceIlules CD4+ non infectdes ammenant B la formation de cellules geantes multinucl&5es de d&e de vie tres courte. La formation de syncytiurn contribue h la chute progressive de lymphocytes T en culture et pourrait fort bien influencer la progression de la maladie in vivo. Plusieurs etudes ont d'ailleurs r6vklC la presence, chez des individus en phase avansde de la maladie, d'isolats viraux capables de se reproduire et d'induire la formation de syncytium beaucoup plus activement en culture que ceux obtenus d'individus en phase asymptomatique (Tersmette et al., 1988; Tersmette et al., 1989). Toutefois, meme si la formation de syncytium se r&Ae c o m e l'un des m6canismes cytopathiques du VIH-1 en culture, son importance au cours de I'infection in vivo reste encore ii definir.
L'expression des glycoproteines d'enveloppe en absence de toutes autres proteines virales est suffisante pour induire la formation de syncytium dam des cellules
CD4+ (Lifson et al., 1986; Sodroski et al., 1986). Les etudes de mutagkn6se effectuees sur le gene env ont permis de dkfinir des regions dans les glycoprotkines d'enveloppe essentielles au processus. Le clivage du prkcurseur gp l6O est m e condition prkalable au phdnornene de fusion mernbranaire (McCune et al., 1988). Des mutations au niveau du site de chvage de la gpl6O rkduisent de mani6re significative la formation de syncytiurn en culture (Freed et al., 1989). De nombreuses etudes ont rkvi516 l'importance de la gp41 dam les phinomenes de fusion membranaire ainsi que dam la formation de syncytium (Freed et al., 1990; Kowalski et al., 1991; Dubay et al., 1992). Les travawc realisis par Kowalski et al. (1991) soulignent bien le r6le pivotant de la gp41 dans Ies effets cytopathiques du VLH-1. Les auteurs constatent une attenuation de la formation de syncytium et de la lyse individuelle des cellules infectkes par une mutation ponctuelle de l'acide amin6 517 de la gp41, sans toutefois altkrer la synthihe des glycoprot6ines d'enveloppe ainsi que la rkplication du virus. La presence de la gp120 est kgalement importante dam la capacitd fusiogene de la gp41 (Thali et al., 1992; Marcon et Sodroski, 1994). Des virus mutants renfermant une dilktion de la gp 120 ne sont plus aptent B induire la formation de syncytium (Marcon et Sodroski, 1994). Findement, on dknote une ktroite corrdation entre la concentration de gp120 exprim6 k la surface de la cellule infectke et sa capacitd de former des syncytia (Gabuzda et al., 1992b). L'interaction entre la gp120 et la gp41, l'expression de la g p 120 B la surface de la cellule infectee ainsi que la liaison au rkcepteur CD4 sont des Cvhements essentiels Zi la formation de syncytium. Le VM-1 provoque tgalement la lyse individuelle des cellules infectees. Au cows des dernicres annees, des etudes de rdplication in vitro ont dCmontr6 que le taux de synthhe protdique v i d e , Ie reliichement de particules virales ou encore I'accumulation d'ADN viral non-integri dans la cellule infectCe ne suffisait pas h induire la lyse
cellulaire (Kowalski et al., 1991; Bergeron et Sodroski, 1992). En revanche, les glycoprotiines de l'enveloppe v i d e auraient un r61e important A jouer dans ce processus, Au cours de son transit dans le RE, le pre'curseur gp160 acquiert la capacitk de liaison aux molCcules CD4. Ainsi, on denote la formation de complexes gp160KD4 dam ce compartiment de la cellule (Crise et al., 1990; Jabbar et al., 1990; Bour et al., 1991). Par ailleurs, suite au clivage de la gp160, le rneme phknomkne peut se produire entre la gpl20 et le rkcepteur CD4 (Koga et al., 1990). Ces complexes semblent etre toxiques pour la cellule. Un processus d'autofusion membranaire entre les differents compartiments de la cellule, m6diC par les complexes gp160/CD4, est p e r y cornme mkcanisme d'action. De plus, ces complexes peuvent bloquer les pores nuclkaires entratnant possiblement la mort de la cellule (Koga et al., 1991). Certaines cellules n'exprimant que faiblement le rdcepteur CD4 telles les cellules dendritiques et les macrophages sont rdfractaires awc effets cytopathiques du VM- 1 (Cheng-Mayer et al., 1990; Langhoff et al., 1991). La faible quantitk de complexes gp l6O/CD4 dktectable dam ces cellules pourrait en partie expliquer l e u risistance aux effets cytopathiques du VIH-1. Par ailleurs, I'expression de la gp41 peut Zi elle sede Stre toxique pour la cellule. L'ancrage de la gp41 peut alt6rer la permc5abilit6 ionique de la membrane plasmique (Miller et al., 1993; Chernomordik et al., 1994). On peut d'ailleurs observer lors de l'infection par le VIH- 1 une accumulation intracellulaire inhabituelle d'ions monovalents et divalents qui s'avkre nCfaste pour la cellule infectke (Cloyd et Lynn, 1991). La gp 120 semble induire un phhornhe semblable Iors d'infection in vitro de cellules nerveuses (Dreyer et al., 1990; Benos et al., 1994). Les glycoprotdines de l'enveloppe virale peuvent donc alt6rer l'intc5grite de la membrane plasmique qui non seulement affectera la fonction norrnale de la cellule mais entrainera ultdrieurement la mort de celle-ci.
D'autres mecanismes cytopathiques gCn6ralement pergus chez les individus atteints sont suggerks dans la lyse cellulaire (pour rksum6 voir tableau 2 ) . La destruction autoimmune des cellules infectkes soit par des lymphocytes T cytotoxiques (Walker er al., 1988) ou par une rdponse immunitaire anticorps-dipendante (OjoAmaize et a/., 1987) sont des processus omnipr6sents lors de l'infection in vivo. Findement, l'apoptose s'inscrit au rang des mecanismes cytopathiques du VIH- 1, s'observant souvent durant la pkriode intense de cytolyse (Meyaard et al., 1992; Banda et al., 1992).
Malgre une meilleur connaissance des effets cytopathiques du VIH- 1, il est bien souvent difficile, en raison d'ithique, de reconcilier les observations in vitro aux 6 v h e m e n t s encourues chez l'individus atteint.
Dans les meilleurs conditions
expdrimentales, des populations cellulaires isolees de sujets infect& ou sains seront cultivees et analys6es lors d'etudes de rdplication v i d e in vitro. Certains isolats viraux, particulikrement ceux isoler d'individus en phase avanc6e de la maladie, se r6v&lent en g6nCral plus lytiques que des souches virales rkupCr6es au cours de la phase asymptomatique (Schuitemaker et al., 1991). Hs pourront se rkpliquer tant dans les macrophages que dans les cellules T CD4+. A I'inverse, les souches isolkes d'individus sains apparaissent tr2s peu pathoghes et prksentent un tropisme pref6rentiel pour les macrophages (Schuitemaker et al., 1992). La variabiliti gCnktique du VIH- 1, le tropisme differentiel des isolats viraux et le stade de l'infection pourront, entre autres, influencer le degr6 de virulence in vivo. Ainsi, les phenom2nes d6finis ci-haut devront virtuellement expliquer la chute irrdversible des lymphocytes CD4+ ou encore les dksordres neurologiques resultant de l'infection virale.
7.
La prodine Vpu du VIH-1. L'une des caractkristiques du VIH-1 est la presence d'un cadre de lecture ouvert
codant pour une protdine d'environ 80 & 82 acides aminds, la proteine Vpu (Cohen et al., 1988; Matsuda et al., 1988; Strebel et al., 1988). Contrairement aux autres gknes du
VIH-I, vpu, situi entre le premier exon du g&netat et superposant la region 3' du g h e env, ne se retrouve pas chez le VIH-2. Les travaux effectuis par Cohen et al. (1988)
permettent de mettre en evidence in vitro Ia proteine Vpu de 16 kDa et riv5lent la presence d'anticorps dirigQ contre cette demi5re dam le serum d'individus atteints. Par ailleurs, des etudes ipiddmiologiques montrent que 30 B 40% des individus en phase precoce de l'infection prksentent des anticorps contre Vpu comparativement i 11% chez les individus en phase avancee de la maladie (Scheinder et al., 1990; Reiss et al., 1990). ~ t a n le t principal sujet de cette Wse, les diff6rents aspects structuraux et fonctionnels de la proteine Vpu seront abordds plus en detail dam ce chapitre.
7.1.
Synthke de la prot6ine Vpu. La synthbe de la proteine Vpu est unique dam le cycle rt5plicatif du V M - 1. Elle
s'effectue 2 partir d'un ARNm bicistronique exprimant kgalement les glycoprot6ines de l'enveloppe virale (Schwartz et al., 1990). La superposition des g&nes vpu et env laisse prdsager une regulation d'expression et fonctionnelle de ceux-ci. Toutefois, les travaux de Schwartz et al. ( 1990) montrent que la machinerie de traduction cellulaire reconnait tour B tour les deux codons d'initiation de vpu et de env. On n'ilirnine cependant pas la possibilitd que l'expression de Vpu puisse reduire celle des glycoprotCines d'enveloppe. La sCquence prt5dite du gkne vpu dome Lieu 3 la synthese d'une protdine de 8 1 acides amin& de long, poss5dant une rkgion N-terminale de 27 acides aminks fortement hydrophobe et une extrkmitt5 C-terminale A caracthe hydrophile.
gp160
-
gp120
'
p66
-
pss
-
p24
-
vpu
-
Figure 6. Analyse immunobiochimique de la prot6ine Vpu au cuurs du cycle replkatif du VIH-I. (A) Un s h m de patient sQopositif et un anticorps polyclo~~al de lapin dirigke contre la proteine Vpu permettmi d'immunoprdcipiter les diverses proteiaes du V IH-1 exprimhs dam les cellules infectks CI drro par 1111 virus vprc- (ligne 1 ) ou \pil+(ligne ?), et relarguks di111~ le milieu de culture (virus). (B) Des cellules infectees par le VIH-I sont fixkes sur rue lame microscopiqile. Le marquage fluorescent oblenu a I'aide de Itanticorps polyclonal anti-Vpu permet de visualiser I'accumulatiun des protkines Vpu (coloration jaunlre) au niveau de I'appareil de Golgi.
Bien qu'exprimee en grande quantitC dam la cellule, Vpu ne s'associe pas aux particules virales naissantes (figure 6A). Suivant sa synthhe, Vpu semble s'accumuler dans une region pCrinuclCaire du cytoplasme, correspondant 5 l'appareil de Golgi (Klimbait et al., 1990). Nos Ctudes immunohistochimiques confiirment ces observations et montrent une localisation v&iculaire dispersie dam le cytoplasme (figure dB). Les etudes de traduction in vitro en presence de membranes microsomales canines (CMM: "Canine Microsomal Membranes") effectuees par Strebel et al. (1989) ont permis de ddmontrer que Vpu est une proteine membranaire intrins5que. Les rdcentes analyses de structures montrent que la region N-terminale de Vpu s'ancre dans les membranes projetant son extrCmitk C-terminale vers Ie cytoplasme (Maldarelli et al., 1993). Cette agencemcnt est caract6ristique d'une protkine membranaire intrinskque de type I (figure 7). De plus, les auteurs montrent que Vpu est capable d'homo-oligom&isation
allant de
formes dimsres jusqu'h possiblement des complexes tdtram6riques. Par ailleurs, Vpu est phosphorylde par une enzyme cellulaire apparentie A la caskine kinase II (CKII) (Strebel et al., 1989; Schubert et al., 1992). La phosphorylation post-traductionnelle de Vpu
s'effectue sur deux r6sidus sirine en positions 52 et 56 (Ser 52 et Ser 56) situes dans une sequence de 12 acides a m i d s (acides aminCs 47 B 58) hautement conservts parmi les diffgrents isolats de VIH-1 (Schubert et al., 1994a). R&emment, des Ctudes de spectroscopie par resonance magngtique nucliaire (NMR: "Nuclear Magnetic Resonance") ont permis de mieux caractiriser la structure secondaire de Vpu (Wray er al., 1995) (figure 7). Ainsi, la region C-terminale semble former deux structures en
h6Iice a (acides amin& 42 3 50 et 57 B 69) liees entre elles par une portion flexible comportant les sites de phosphorylation de la prodine. La seconde hdlice a est suivie par une portion flexible qui presente le ddterminant antigenique predominant de Vpu (acides amines 68 ih 77) (Cohen et al., 1988; Schneider et al., 1990).
7.2,
Le r6le de Vpu d a m le cycle r6plicatif du VIH-1. Bien que non essentiel B la r6plication v i d e in vitro, les premieres etudes
fonctionnelles effectuees sur Vpu ont dkmontre que la proteine pouvait contribuer Zi la propagation du SIDA en augmentant l'efficacitd de transmission du VM-1 (Terwilliger er al., 1989; Strebei et al., 1989; Klimkait et al., 1990). Les travaux rt5alisks par
Terwilliger et al. (1989) montrent que I'expression de Vpu diminue les effets pathogimes du VIH-I in vitro d a m des lymphocytes CD4+ infect&.
Vpu semble retarder la
formation de syncytium par un mdcanisme encore non elucide. Par ailleurs, les cellules infectkes avec un virus vpu+ relachent environ 5 A 10 fois plus de particules virales comparativement 2 des cellules infectees avec un virus vpu-. La presence de Vpu pourrait en partie expliquer la pandkmie mondiale plus rapide du VIH-1 face h celle observde avec le VIH-2. Aujourd'hui, le r6le de Vpu dam le cycle replicatif du VIH- 1 est mieux dCfini. Nous passerons donc en revue au cours des sections suivantes les differentes fonctions attribudes B la protkine Vpu.
7.2.1. Vpu augmente le reliichement des particules virales, Vpu peut agir en trans afin d'augmenter le relichement de particules virales des cellules infectees (figure 8). Elle n'affecte ni la synth8se ni la maturation des protkines virales mais semble plut6t influencer l'assemblage et/ou I'exportation des virions naissants (Terwilliger et al., 1989; Strebel et al., 1989; Klirnkait et al., 1990). Au cours d'analyses en microscopie klectronique, Klimkait et al. (1990) observent un grand nombre de particules virales matures retenues h la surface des cellules infectees par un virus exprimant un mutant de deletion de Vpu. Les auteurs denotent kgalement dam ces cellules, la presence intracytoplasrnique de nombreuses vacuoles renfermant des virions matures et immatures. Par ailleurs, les effets d e Vpu sur le relilchement d e particules virales ne necessitent ni l'expression des glycoproteines d'enveloppe ni celle de la
molkule CD4 (Yao et al., 1992). Tout indique que Vpu joue un r6le dans le reliichement plutdt que dam la morphog&&e
des capsides virales. Cette fonction de
Vpu ne semble pas irnpliquer une interaction specifique avec les protiines Gag du VIH1. Griice B l'utilisation de chh&resde VM-1, Gottlinger et al. (1993) ont dirnontrk que
Vpu pouvait augmenter le reliichement des prottines Gag du VIH-2 et de r&rovirus aussi divergents que le virus visna de mouton et le virus de la 1eucCrnie murine. De plus, les auteurs montrent que la maturation des polyprott5ines Gag n'est pas ntcessaire au mode d'action de Vpu. La protdine est capable d'augmenter le reliichement de virus mutants de la protdase. Il devient de plus en plus plausible que Vpu puisse modifier une voie cellulaire commune au relgchement de tous r6trovirus 5 la surface des cellules infectkes. Les ktudes de mutaghkse effectutes par Schubert et Strebel (1994) ont dtmontrt5 que la phosphorylation de Vpu n'est pas requise dans la capacite de la prodine ii augrnenter ie reltchement des particules virales.
La substitution des sites de
phosphorylation (Ser 52 et Ser 56) par des rtsidus glycine ou asparagine n'affecte que partiellement le phenotype de Vpu. Par contre, la retention de Vpu dans le RE inhibe la fonction de reliichement de la protiine (Schubert et Strebel, 1994). Des cellules infectkes avec un virus vpu+ et subsiquement traitees avec la brefeldine A (BFA), un metabolite fongique qui possbde la propriktk de bloquer le transport v&iculaire du RE 1 l'appareil de Golgi (Misumi et al., 1986), vont alors libirer des quantitCs semblables de virions que des celIules infecties avec un virus vpu-. Ces observations suggerent que Vpu influence le rellchement des particules virales ii partir d'un compartiment cellulaire autre que le RE.
Figure 8. S c h h a repdsentaut le reuchement de particuks virales P la d a c e des cellules infect&. (A) Mimgraphie &ectroniquede virions retenus ii la surface dune cellule infect& par un virus vpu-. (B) Reprkentation schkmatique du bourgeomement viral au coun de I'infection par un virus vpuOU pu+.
7.2.2. V p u diminue les effets cytopathiques du VIH-1.
L'expression de Vpu est associie A une diminution des effets cytopathiques du
VIH-1 (Terwilliger et aL, 1989; Klimkait et al., 1990). Au cows de la riplication v i d e in vitro, on observe un retard dam l'apparition de syncytium en presence de Vpu. En microscopie optique, ces syncytium apparaissent moins nombreux et de plus petites tailles (figure 10). Les travaux effectuds par Yao et af. (1993) dkmontrent que Vpu affecte le taux de formation de syncytium sans toutefois d t i r e r la lyse cellulaire subsequente des cellules infecties par le VIH- 1. L'utilisation d'anticorps dirigks contre la gp120 et la rnolkcule CD4, qui peuvent inhiber la formation de syncytium, revkle clairement que le taux d e lyse cellulaire en culture est similaire en absence ou en presence de Vpu. Par des techniques de cytofluomt5trie en flux, les auteurs montrent tgalernent que les cellules infectkes par un virus vpu+ pr6sentent 3 fois moins de gp120 B leur surface comparativement
des cellules infectkes avec un virus v p u - .
Quoiqu'encore ma1 defini, Vpu semblerait interagir d a m le transit intracellulaire des glycoproteines d'enveloppe virale ou encore pourrait induire un relarguage accru de la
gp 120 dans le milieu extracellulaire. Cette diminution de gp120 B la membrane plasmique semble en partie expliquer les effets de Vpu sur la formation de syncytium.
Nos h d e s de mutag6niise ont demonwe que la phosphorylation de Vpu jouait un r6le dans la capacid de la protdine h diminuer les effets cytopathiques du VIH-1 in vitro (Friborg et al., 1995). M8me si des mutants de phosphorylation de Vpu sont toujours aptes B augmenter le relkhement de particules virales, ils ont cependant perdu la capaciti de retarder la formation de syncytiurn et de diminuer l'expression de la gp 120 ii la surface des cellules infectkes. Ainsi, la formation de syncytium beaucoup plus intense observee en absence de Vpu ne semble pas Etre le resultat de l'accumulation de particules virales 2 la surface des cellules infecties pouvant augmenter les probabilitks d'interactions de virus B cellule. Ces deux phdnomhes apparaissent independants l'un de I'autre et pourraient &tremedies par differents domaines actifs de Ia protiine.
7.2.3. Vpu digrade sp6cifiquement la mol6cule CD4. Finalement, Vpu posskde la capacitC de destabiliser les complexes gp 160KD4
qui peuvent se former dam le RE en induisant spicifiquement la degradation des molCcules CD4 (Willey et al., 1992a; Willey et al., 1992b). Le prkcurseur gp 160 ne sert que de facteur de retention puisque Vpu peut induire la degradation des molc5cules CD4 sequestrges dam le RE suite au traitement B la BFA. Ce phhomcne est rapide puisque la derni-vie de CD4 dans la cellule passe de 6 heures 3 environ 10 minutes en presence de Vpu. Ce phenornene est rapide puisque la demi-vie de CD4 dans Ia cellule passe de
6 heures B environ 10 minutes en prksence de Vpu. Par ailleurs, Chen et al. (1993) ont dCmontr6 lors d'ktudes de traduction in vitro en presence de CMM que la degradation de
CD4 par Vpu nkcessitait la presence des deux intervenants dans un meme compartiment membranaire. De plus, les auteurs montrent que la queue intracytoplasrnique de CD4 ainsi que la r6gion C-terminale de Vpu cornportent des dktenninants essentiels au processus de degradation.
De nombreuses itudes de mutaginese ont Cte rt5aliskes afin de definir les skquences ou regions de la mol6cule CD4 sensibles au mode d'action de Vpu (Vincent et al., 1993; Lenburg and Landau, 1993; Willey et al., 1994; Raja et al., 1994; Buoconore et al., 1994). Les travaux effectuis par Lenburg and Landau (1993) demontrent que des
siquences comprises entre les rksidus 418 et 424 de la queue intracytoplasmique de CD4 sont importantes dam la degradation de la molkcule.
Les auteurs rnontrent
toutefois que ni les sites de phosphorylation de CD4 (residus en positions 4 10 et 415) ni ie domaine d'interaction A la p561ck (residus en positions 420 et 422) ne sont impliquees dans le processus. Quant B eux, Vincent et al. (1993) dkfinissent par une s&ie de d616tion dam la queue intracytoplasmique de CD4, une region comprise entre les residus 414 et 419. En revanche, les itudes r6alisies tour B tour par Raja et al. (1994) et Buoconore et al. (1994) B l'aide de chirnkres de CD4 rkv2lent non seulement l'importance de la queue intracytoplasrnique dam les effets de Vpu, mais dgalement celle de la region transmembranaire de la rnol6cule.
La region C-terminale de Vpu presente des dkterrninants essentiels 2 la degradation de CD4. En effet, un mutant de d U t i o n (acides amin& 47-48 et 50 ii 52) ainsi que des protkines Vpu tronqukes, n'exprimant plus les cinq ou six derniers acides amin&, ont perdu la capacitk d'induire la degradation de CD4 (Chen er al., 1993). La phosphorylation de Vpu semble egalement importante puisque la substitution des Ser 52 et Ser 56 inhibe la fonction de la protkine (Schubert et Strebel, 1994). Recernment, des Ctudes de co-immunoprecipitation ont permis de montrer une interaction spkcifique entre Vpu et la molCcule CD4 dam un systkme d'expression cellulaire transitoire (Bour et aL, 1995). La ddgradation rapide de CD4 par la proteine Vpu de type sauvage rend difficile la detection de tels complexes. Cependant, cette liaison semble beaucoup plus stable en presence de mutants de phosphorylation de Vpu, qui ont perdus la capaciti de digrader la moMcule. Ces observations semble suggerer
que l'interaction entre Vpu et CD4 s'effectue t6t au cours de la synthsse des proteines et, pourrait possiblement &e un pre-requis au phenomkne de degradation. Les uavaux effectuis par Bour et al. (1995) ont dimontri que la queue intracytoplasmique de CD4 et la region C-tenninale de Vpu prksentait des determinants essentiels & l'interaction. Des chimikes de la moldcule CD8, comportant uniquement la region transmembranaire de CD4, ne peuvent interagir avec Vpu.
L'interaction entre Vpu et CD4 semble
s'effectuer par Ieur region C-terminale respective.
De plus, des mol6cules CD4
tronquees, n'exprimant plus les 22 derniers acides amines de la queue intracytoplasrnique de la mol6cule, ne sont plus aptes A former des complexes avec Vpu. Ces analyses ont permis de restreindre la region de liaison sur CD4 aux acides amin& 402 420, sequences igalement requises dans les processus de degradation par Vpu.
8.
Le projet de recherche. Sur le plan biologique, le VM-1 et le VIH-2 infectent Ies mSme cellules,
s'attaquant principalement aux populations lymphocytaires et monocytaires CD4+. Cependant, les domees tpidtmiologiques rivklent que le VIH- 1 s'est largement propage mondialement comparativement
son homologue. Bien que les modes de transmission
de ces deux virus et les sympt6mes observks chez Ies individus atteints soient similaires, I'apparition des premiers signes cliniques semble plus long avec le VIH-2 qu'avec le
VM- 1. Ces observations laissent supposer une propagation v i d e et une virulence plus faible du VIH-2 comparativement au VKH-1. Kl est clair que les diffkrences, les plus subtiles soient-elles, entre les d e w virus auront une importance dans la rkplication virale et la progression de la maladie. De nombreux facteurs viraux ou cellulaires encore non ddfinis devront expliquer ce phtnomhe et le gkne vpu semble un candidat approprii. La proteine Vpu ou une composante analogue n'est pas exprimte par le VIH-2. Or, la presence de Vpu, dont la sequence en acides arninCs est hautement conservee parrni les difftrents isolats du VIH-1,a tt6 associCe B une dCcharge virale bcaucoup plus intense et une diminution des effets cytopathiques au cours de l'infection in vitro. I1 est fort probable que ces propri6tCs conferont au VIH-1 un avantage dans sa replication et sa propagation.
Nous avons donc entrepris ce projet de recherche, afin de mieux
comprendre le r6le de Vpu dans le cycle rkplicatif du VIH-1. Ainsi, le but des travaux prtsentes dam cette thkse de doctorat ttait d'effectuer une analyse structure/fonction de Vpu afin de dCfinir le(s) domaine(s) actif(s) essentiel(s) au mode d'action de la proteine. Afin de rialiser de tels objectifs, nous avons donc entami des Ctudes de rnutag6ngse sur celle-ci. Notre approche itait de creer par des techniques de mutagCnhe dirigie, des mutations conservatives et non conservatives sur des sequences en acides aminds hautement conservts de Vpu. Pour analyser les effets des mutations sur les diverses fonctions de la prottine, les mutants
gc5nerks ont et6 par la suite reintroduits dam des vecteurs d'expression eucaryotes ou
dans un clone moliculaire infectieux vpu+ gardant ainsi un contexte viral isogknique. Au cours de nos travaux differents systemes cellulaires ont c5t6 employ& nous permettant ainsi d'Ctudier et de cornparer 2 phsieurs niveaux les propriitts fonctionnelles des diffkrents mutants avec la protiine Vpu de type sauvage. L1interpr6tationde nos r6sultats pr6sent6s dam Ies prochains cliapitres devrait contribuer 2 une meilleure comprkhension des effets biologiques de la protCine dans le cycle
riplicatif viral et sur la physiologie de la cellule. Tout en gardant une vision objective de nos risultats obtenus dans des systemes d'expression in vitro, nous tenterons de rnieux difinir Ie r6le de Vpu au cows de l'infection virale in vivo et dans la propagation du VIH- 1.
Article 1: Functional analysis of the phosphorylation sites on the human immunodeficiency virus type 1 Vpu protein.
Functional Analysis of the Phosphorylation Sites on the Human Immunodeficiency Virus Type 1 Vpu Protein Jacques Friborg. Azim Ladha, *Heinrich Gottlinger, *William A. Haseltine, and Eric A. Cohen Loboratoire de RC~rovirolo~ic Humaine. Ddparremenr de Microbiologie et Imrnunologie, Facultd de MPdecine, Universite de Montreal. Monrriul. Qlribec. Canada. and 'Division of Human Rerrovirology, Dana Farber Cancer Institute, Bosron , Massachusetts, U S A .
Summary: The human immunodeficiency virus type I (HIV-lkncoded vpu product is a small class 1 integral membrane protein that is phosphorylated by the ubiquitous casein kinase I1 (CKII) in HIV-I-infected cells. The Vpu protein facilitates the release of budding virions from the surface of infccted cells and delays the rate of syncytiurn formation. In this study, we investigated the role of phosphorylation in the biological activity of Vpu. Our results show that phosphoqlation of Vpu occurs on serine residues at positions S t and 56 located in a highly conserved dodecapeptide sequence. Mutation of either Ser 56, or both Ser 52 and Ser 56 impaired the ability of Vpu to delay the rate of syncytium formation while retaining virion release activity at levcls comparable to r p r - proviruses. Flow cytornetry analysis indicates that the relative amounts of envelope giycoprotein gp120 expressed at the surface of cells transfected with rhese 1 . p ~mutant proviruses was two- to threefold greater than that observed on cells transfected with a rpu' provirus. This increased expression of gplZO at the cell surface may explain the more rapid onset of syncytium formation ohcrved in cell transfected with vpu mutant proviruses. These results sugge3t that Vpu-facilitated virion release and delayed cytopathic effect are the consequence of two distinct functional activities of the protein. Key Words: HIV-Syncyriurn formation-Viral release-Vpu.
Human immunodeficiency virus type 1 (HIV-I)is characterized by a highly cornplcx genornic organization. In addition to the structurd (gag.enr.) and enzymatic ( p o f )gene products common t o all retroviruses, HIV-I encodes severdl reguiatory and accessory proteins (1.2). One of the accessory genes. vpu, is unique to HIV-I.The genomes of human and simian retroviruses most closely related to HIV- 1-HIV-2 and simian immunodeficiency virus (SIV)-do not encode analogous proteins come- -
Address correspondence and reprint requests to Dr. E. A. Cohen at Depanment of Microbiology and Immunology. Faculty of Medicine, University of Montreal. CP6128 Station A. Montreal. PQ, Canada ?I3C 317. Manuscript received March 2 1%; accepted June 6. 1994.
sponding to the vpu gene product (3-5). The only exception is the SIVcpz, a chimpanzee isolate. which was shown by sequence comparison to encode a vprr-like gene (6). The Vpu protein is encoded by a short open reading frame overlapping the 5' end of the envelope glycoprotein gene (env). Vpu and Env are made from a single rev-dependent bicistronic mRNA (7.8). This observation has been proposed to reflect a requirement for coordinate expression of Vpu and Env. Functional studies in CD4' T cell lines have shown that Vpu can act in trans to signiricantly increase the release of budding virions from infected cells since budding structures accumulate in intracellular vacuoles and at the surface of cells expressing vpu mutant proviruses (5,P-I I). It has also been
reponed that Vpu can facilitate the release of capsid proteins from retroviruses as divergent as HIV-2, Visna virus, and Moloney murine leukemia virus. raising the possibility that Vpu may act indirectly through modification of the cellular environment rather than by directly acting with viral proteins ( I?). Furthermore, Vpu delays the cytopathic effect of HIV-I by reducing the rate of syncytium formation in infected CD4' T cells (9-1 l). Infection of T cells with vpu defective (vpu') viruses results in a more rapid onset of syncytium formation in comparison to v p u - virus, even though significantly less progeny virus is produced. Apart from its effect o n virus-induced cytopathicity and on late stage of virion morphogenesis, Vpu was recently shown to regulate the formation of intracellular gp160-CD4 complexes in the endopiasmic reticulum by inducing a specific and rapid degradation of CD4 (13-16). The finding that VpufaciIitated virion release does not require Env or
ERAEFLFLYESEC ~
~
EiUDtGNESEG
6 VpU52-5s ~ 1 0 HXBHIO V P U ~ "
CD4 expression suggests that Vpu may have more than one target in infected ceHs and may serve several independent functions in the course of infection (17, IS). The exact functions and mechanisms of action of Vpu at the cellular and molecular ievels have yet to be established. Recent biochemical evidence indicates that Vpu is an otigomeric class I integral membrane protein phosphorylated at one or more serine residues by casein kinase I1 (CKII) (5.19.20). The predicted 8 Iamino acid sequence of Vpu suggests an amphipathic structure of the protein (Fig. IA). An Nterminal hydrophobic region of 27 amino acids presumably constitutes the membrane anchor domain. A C-terminal hydrophilic region that contains a I Z-amino acid sequence (dodecapeptide: residues 47-58) highly conserved among Vpu proteins from diverse HIV-1 isolates and SIVcpz (6) may represent an active site of the protein (Fig. IA). Two serine residues, Ser 52 and Ser 56, located in this
ERAEFNESEG
ERMDSGh'EgEG
HXBHlO V P U ~ ' ~HXBHIO V P U " ~
ERAEvm-EG HXBXlO V P U ~ " ~ ~
FIG. 1. Mutations introduced into the vpu dodecapeptide sequence of HIV-1. A: The schematic representation of the amphipathic structure of Vpu showing the Cterminal hydrophobic and N-terminal hydrophilic regions. The hydrophobicity plot was determined according to Kyte and Doolittle using the MacVector software (International Bioteehnologies, New Haven, CT. U.S.A.). B: The HXBH10-vpu' 81 amino acid (a-a.) sequence is indicated. with the highly conserved 12-a.a. sequence (dodecapeptide) of vpu framed. The mutated vpu dodecapeptide are listed below the wild-type sequence, and the mutated residues are underlined for each provirus.
loumaf of~cqrriredImmune Dejiricncy Syndromes and Human Re~rovirology. Vol. 8. No. 1 . 1995
region are potential phosphoacccptor sites for CKII (19.21). In this study, we performed a mutational anaIysis of the conserved dodecapeptide region of Vpu and investigated the importance of Vpu phosphorylation in the replication and the cytopathicity of HIV-Iin CD4+ T cell lines. MATERIALS AND METHODS Site-Directed Mutagencsis and Plasmid Construction HXBHlCLvpu' (gag4, pol*, vv'. vpr', tat+. rev', vpu', env ',ncf' ) aid KXBHl&vpu' (gag', pol', vf' ,vpr' ,tat', rev4, vpu*, env4, nef-) are isogenic infectious molecular clones of HIV-1 that differ only in their ability to express Vpu. As prrviously descri'bed, HXBH10-vpu' carries a point mutation in the initiation codon of vpu (9.17). Vpu muragenesis was performed on HXBHl&vpu' using a tw-step polymerase chain reaction (PCR)-based method. as d e s c n i elsewhere
Cclls and DNA Transfcctions MT4, a nV-I-uansformed human CD4' T ceU line. and Jurkat, a human CD4' T cell line. were msintained as previously d c j c r i i (23,24). MT4 (5 x 1Ob) and iurkat cells (10') were trsnSf& with 10 pg of plasmid DNA using a DEAE-dexaan traasfiion method (25). Trandccted cell cultures a-ere subsequcnlly washed once wirb RPMI 1640 medium and given a w m plete medium change with h h RPMl 1640 medium containing 10% of fetal a,t€ serum (FCS). JurLat cultures were resuspended daily in m .ppnoprirte volume of fresh RPMI IdlO containing 10% FCS so as to lMintain equivalent densities of txypan blueexcluding viabIe (lo6 teIls/ml) among cultures. Xnfcctim was monitored by determiaation of the percentage of cells in the cultures tbat arc positive by immunofluoresccnt labeling for virus-specific antigen p24 as d e s c n i below. Virusspecik reverse tRnscriptnse (KT) in the supernatant fluid was also mmsurcd u dtun'kd 0. To evaluate the cytopothic effect, the toul number ofviable cells and the percentage of tqpm blue-stPining cells in the cultures were monitored daily. The cultures were aim examined by l&ht microscopy for syncytium f d o n md scared by counting the number of syncytia per field. were maintained as previously d e ~ ~ n i b(12.17). d HeLa HeLa cells (lo6)were seeded into 80 ern2tissue d n u e flasks 24 b before transfection. The cells w m Plnsfectcd with HI ull of plasmid DNA using a caicium phosphate precipitation metbod
*
-
(22). Complementary oligonucleotide primers containing the desired mutations were used to generate the vpu mutated fragments by PCR. The nucleotidt sequences of the mutagenic oli0. gonucleotide pain having 3' ovedapping ends werc as follows: HXBH 1 0 - ~ p u ~ ' - ~ 5'-AAG ~: AGC AG AATTCCTTTGCTATGAGAGTGAAG-3' (sense) and 5'-CTTCACTCTCATAGCell Labeling and Immunopttcipitation CAAAGGMTTCXGCTCIT-3' (antisense); HXBH l s v p e . To assess viral protein expression, KT4 cells (109 were met5'-AGCAGMGACmGGCMTGAG-3' (sense) and S8-CTCabolically labeled with fuS]methiouine (100 pCilml) 48 h postA'ITGCCAAGGTCITCTGCT-3' (antisense); HXBH 10.vpum: trandection for 5 h. The W e d cells were lysed in RIPA buffcr 5'-AGCAGAAGACGGTGGCAATGAG-3' (sense) and 5'(10 mM Tris-HCI (pH7-41. 1 mM EDTA. 100 mM SaC1, 1% CTCATTGCCACCGTCTTCTGCT-3' (antisense); HXBHIOTriton X-100,0.1% sodium dodecyl sulfate [SDSI, 0.25% dtoxyvpuS: 5'-GGCAATGA GGGTGAAGGAGM-3' (sense) and cbolate, 0.2% phenyl-methylsulfonyl fluoride PMSF]) and im5'-'TTCXCCTTCACCCTCATrGCC-3' (antisense); HXBHlOmunoprecipitated as previously described (9) with botb a HIVvpum": 5'-AGCAGMGACGGTGGCMTGAGGGTGAAGI-positive human serum and an anti-Vpu semm difCcxed against GAGA-3' (sense) and 5 '-TCTCCfTC ACCCTCAlTGCCACthe c;uboxy terminus region of the Vpu protein (HAPWDVDDL) CGTCXTC];m-3' (antisense). A pair of oligonucleotide primin a 1:I ratio (3). Equal volumes of l a k l e d supernatant were e n located 23 bp upstream from the vpr initiation codon (sense coUccted and filtered on a 0.45-pm filter and virus particles were primer A: 5'4AGCCACCfITGCCrAGT-3') and 148 bp downlysed by adding 10 x RIPA buffer. HIV-I virio~associatedpros v c a m from the vpu termination codon (antisense primer B: 5'wins were immunoprccipitatedwith a HN-l-positive human seGGCTACACAGGCATGTGT-3') w e n used for DNA amplificarum. The immunoprccipitates were analyzed by elecuophoresis tion of the mutated fragments. Briefly, in the first PCR reaction, two DNA fragments werc generated simultaneously using sense on a 12.5% SDS-polyacrylamide gel a d a u t o r a d i w h y . To Malyze virion rcIasc. HeLa cells (lo6) werc metabofcally primer A with an antisense mutagenic oligonucIeotide aad mtisense primer B with the complementary sense muugdc o b labeled with ~ S l c y s t t i n (50 e pWd)48 h posftransfection for 12 onucleotide. Subsequently, the fragments generated were comh. V i i particles were pelleted from labeling medium through a bined in a second fusion PCR reaction in which the 3' overlop 20% sucrose cushion for 2 h at 27,000 rpm in a Beckman SW 41 ping a d s anneal to give a vpu mutated segment. The resulting rotor. Meted materials were lyscd in hcmml; sample buffer fusion product serves as a template and is further amplified by (125 Tris-Ha. [pH6.81,2% SDS, 4.8% 2-mcrsaptoethanol, PCR using primer A and B.The amplification product con^ 20% glycerol), and virPl proteins in the pellets were dihctly analyzed by electrophoresis on a 115% SDS-polyacrylamidc geI the mutation was digested at a Sal I site and Kpn I sites at positions 5820 and 6382 in the HXBHl&vput molecular clone. and 8 l I t o r r d i o ~ h (12). y The resulting 562-bp DNA fnsment was size fractionated by To dctrrminc the site@) of phosphorylation of the Vpu protein, Mf( cells (lo6) were incubated in phosphate-&ee RPMl 1640 electrophoresis through an agarose gel and cloned into HXBHIO~ ~ , for 30 min at 37°C to remove the cndogcnous phosphate vpu' as described previously (9,17) to yield H X B H ~ O - V ~ ~ medium B,H IO-V~U''~,HXBH 10-vpu5=, and H X B H ~ O - V ~ UH~X' ~ pool and subsequcntfylabeled with "PiCIU)pCi/ml) for 3 h at 62 H X B ~ l ( h p u - proviruses. All mutations w e n confirmed by h posttrmsfcction. ?be cells werc then washed with cold phosphate b a r d i n e (PBS) and lysed in RIPA buffer containing double-stranded DNA sequencing using a Sequenase kit (U.S. protuse inhiiitors (20 m M NaF. 10 m M EGTA, 5 m M MgCI, Bicxhemicals Cocp.. Cleveland, OH. U.S.A.).
Journal of Acquired Immune Deficiency Syndromes and Human Retroviriology, Vol. 8. No. I . I995
HIV-I VPU PROTEIN
13
and 10 mg PbfSF). Vpu proteins were immunopreci~itatedwith an &Vpu senrm and analyzed by electrophoresis on a 12.5% SDSgolyacrylarnidt gel and autoradiography. Densitometric Pnalyds of autoradiograms was performed with a Personal Densiromcter (MoIccuIar Dyaamics, Sunnyvale, CA, U.S.A.) using tsqftwarr version 3.22,
HXBH10-vpu', an infectious molecular clone of HIV-1 that encodes a finctional Vpu protein (9,17) to yield proviruses HXBH 10-vpusU-, HXBH 10vpunG, H ~ ~ H 1 0 - v p and u ~ .HXBH~O-vpuNM. In addition to these serine mutations, a mutant carrying nonconservative substitutions of amino acid residues Asp 51, Ser 52, Gly 53, and Asn 54 for, respectively, Phe, Leu, Cys, and Tyr within the highly conserved dodecapeptidt was generated and Mcamxemcnt of ell-assochtcd HIV-1 p24 was perfonncd pintroduced into the HXBHlO-vpu' to yield riodkaUy to evalua!e the spread of iafection in the d a m s . T r a m s f ' cells were fixed with methanol and aceme (1:l) fm ~ ~ B H 1 0 - v p This u ~ ~mutation ~. was designed to 30 min. IabcIcd wiIh an anti-p24 monoclonal m h i y (ABTno. affectthe overall structure and amino acid charge of 4 0 1 ; American BieTechnoIogies, Ossining, NY, U.SA.) and the dodecapeptide region. All mutations introduced stained with fluorescein isiothiocyaaatc ( F I T 0 coqiuwed goat ia this region of the vpu gene that overlaps the env anti-mouse IgG ( G i i , BRL, St. tawrcnce, MA, U.SA.). S u b open reading frame did not S e c t the 8-120amino se~uently,the percentage of imnrunofluorescent-positivecells acid sequence. The rationale bebind the cloning of was determined on a fluorescence microscope (Carl Zciss, Tharnwood. NY). the vpu mutants in the context of infectious HIV-1 Emdope glycoprotein gpf20 expmsed on the s u b of inmolecular clone was to assess the effcct of Vpu feaed cells was periodically rneaswcd by cytofluommeuic dphosphorylation and desired mutation in the dodeysis. JIPkU cells (2 x Id) were incubated with an anti-pl20 capeptide sequence on viral particle release and cymonoclonal antibody (ABT No. 1001; American Biotopathicity. Therefore, the stability of the Vpu muTechnologies) and stained with FITC-conjugated goat antimouse XgG as described elsewhere (I 1). Following the reaction tants as well as the preservation of the context of arirh the a n t t i e s , cells were tixed witb 1% padormaldehyde the env initiation codon and of the overlapping env in PBS. Positive ceUs were examined by irnmunofluomcence gene sequence were taken into account in designing and analyzed on a FACScan flow cytometcr (Becton Dickinmn, these mutations. Mountain View, CA, U S A ) , using FACSCAN software. Vpu Is Phosphorylated at Scrine Residues 52 and 56
RESULTS Construction of HW-1 vpu Mutant Provirus Vpu is phosphorylated at one or more scrine residues by a casein kinase II (CKIIkreIated protein (S,19). Two serine residues in the sequence of V p d e r 52 and Ser Skorrespond to the consensus sequence S e r m XX GlulAsp, which has been defined to be critical for phosphorylation by CKII (21). These potential phosphoryiation sites are located within a highly conserved dodecapeptide sequence of Vpu. To investigate the importance of this region and to assess the relevance of phosphorylation in Vpu biological activities, a set of five mutants was generated by a rnutagenesis PCRbasad method, as described in Methods (Fig. 1B). Individual substitutions of Ser 52 to a Leu or Gly and of Ser 56 to a Gly as well as a double substitution of Ser 52 and 56 for Gly residues were produced. Substitutionof Ser 52 for Leu is predicted to affect secondary structure according to the GOR method (28). In contrast, substitution of Ser 52 or/ and 56 with Gly is predicted to have minimal effect. These point mutations were introduced into
In an initial experiment, we determined the stability of the Vpu mutant proteins and investigated the nature of the Vpu phosphoacceptor sit&). Parallel cultures of human CD4+ T cell line MT4 (5 x lo6) were transfected with equal amounts (10 pg) of either HXBH10-vpu' or HXBHlO-vpu mutant DNAs. As a negative control, MT4 cells were transftctcd with 10 pg of HXBH10-vpu- (9,17), an infectious molecular clone isogenic to HXBH10-vpu ' except for the expression of Vpu. At 48 h posttransfection, viral protein expression was determined by labeling equal numbers (lo6)of viable cells with 100 )rCi/ml of [3s~]methioainefor 5 h. Both cell lysates and supernatants were immunoprccipitated with a HIV-I-positive human serum mixed with a rabbit anti-Vpu peptide serum in s 1:1 ratio (3). AU irnmunoprecipitated proteins were analyzed on SDSpolyacrylamide gels and autoradiography. As shown in Fig. 2A, expression of the vpu product was detected by the anti-Vpu serum in the labeled cell extracts of all HXBHlO-vpu mutants transfected cultures (lanes 3-7). A protein of apparent molecular mass of 16 kDa corresponding to Vpu
Journaf of Acquired Immune D&cicncy Syndromes and Human Rrtrovirotogy. Vol. 8. NO.1, I995
Ser 52 and 56 have been substituted. This result culture strongly indicates that both serine residues at posi(lane 2) but not from HXBHlO-vpu- culture (lane tion 52 and 56 in the dodecapeptide sequence are 1). The substitutions introduced in the vpu gene had phosphorylated and therefore represent the phosthe eect of increasing to varying extents the elecphoacceptor sites targeted by CKII. trophoretic mobility of the Vpu mutant proteins precipitated (land 3-7). This effect is most probably due to the charge modification introduced into the Effect of Mutations Within the Vpu Dodccapeptide Vpu mutant proteins, which are known to result in Rcgion on the Release of Viral Particles increased binding of negatively charged SDS molecules during dtctrophoresis. Densitometric scanThc relative amount of viral protein present in ning of the autoradiogram shown in Fig.2A as well MT4 cells transfected with the ~ W t r e n proviruses t as pulse chase experiments (data not shown) indiwas determined by immunoprccipitation of the lacate that the stability of the vpu product did not appear to be signXcantly altered by these substitubeled cell lysate and supernatant with a HIV-1positive human serum (Fig. 2A). The intracellular tions, because similar amounts of Vpu mutant p r e concentrations of the HIV-I Env glycoproteins teins were detected following transfection with (gpl60Aand gp120) as well as the capsid precursor wild-type or vpu mutant proviruses. Furthermore, our irnmunolocalization studies of Vpu with a rabbit p55 and the processed capsid protein p24 were comparable between the different cell cultures (lanes anti-Vpu senun indicated that the protein accumu1-7). In contrast, marked differences in the amount lates in the Golgi apparatus and is further transof capsid protein released into the cell culture suported to the plasma membrane through the endopernatants were detected. Densitometric scanning sornal pathway (X. J. Yao, F. Boisvert, S. ( h m n , of the autoradiogram reveaIed that at least three D. Bergtron, and E. A. Cohen, manuscript in preparatimes more viral capsid protein p24 and reverse tion, 1994). The immunolocalization of the Vpu mutranscriptase p66 were precipitated in the supernatant proteins in the transfectcd cells did not differ tant fluids of cells transfected wi* HXBH 10-vpu ' fkom the wild-type Vpu protein (data not shown). and HXBH10-vpu mutant DNAs than from cells To determine the nature of the Vpu protein amino acids that are phosphorylated, an equal number transfected with HXBH10-vpu- DNA (compare (lo6) of MT4 cells transfected with the different vpu lanes 2-7 to lane 1). The amount of virus particle released into the sumutant proviruses were metabolically labeled with 750 ~ C i / mof l 32Pi at 62 h posttransfection for 3 h. pematant of each MT4 culture was also determined Cells were subsequently lysed and immunoprecipiby measuring the virus-specified MA-dependent tated with an anti-Vpu serum. Immunoprecipitated DNA polymerase activity at different interval times Vpu proteins were analyzed on SDSpolyacrylfor 78 h posttransfection (Fig. 2C). Viral particle i g . arnide gels and autoradiography. As shown in F production was higher at all times posttransfection in the cell culture transfected with HXBH I 0-vpu ' 2B, a single 16-KDa phosphoprotein was immunoprecipitated by the anti-Vpu serum in the labeled or HXBH10-vpu mutant DNAs as compared to the cell extracts of the HXBH10-vpu+ transfected culcell culture transfected with HXBH10-vpu 'DNA. ture (lane 2) but not from the HXBH10-vpu- culAt 62 h posttransfection, the level of enzyme activture (lane l), indicating that the product detected ity present in the supernatant of cells transfected was the phosphorylated form of Vpu. The Vpu with HXBH10-vpu+ or HXBHIO-vpu mutants was phosphoprotein of HXBH10-vpu mutants carrying approximatively three- to fourfold greater than in individual substitution of Ser 52 ( ~ ~ B H 1 0 - v p u ~HXBHlO-vpu' ~ culture. and H X B H ~ O - V ~ U or~Ser * ~56 ) ( ~ ~ ~ ~ 1 0 - v p uInTsubsequent experiments, the Vpu-mediated rewas also detected in the labeled cell extract of translease of viral particles was further assessed and fected cultures (lanes 1-5). The H X B H ~ ~ - V ~ $ ~quantified ~, in the HeLa expression system. In this which encodes a Vpu protein with a nonconservacell system, the effect of Vpu on viral particIe retive substitution mutation at Ser 52, expressed also lease was shown to be more pronounced than in was precipitated from the HXBH10-vpu'
a nhnsnhawlatcd nrntein (data
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CELL LYSATE
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FIG. 2 Replication of vpu mutant proviruses in MT4 cells. A: Viral rotein expression of vpu mutant proviruses. MT4 cells ( 1 4 transfected with 10 (rg of HXBHIO-vpu- (lane 1). HXBHlOrpu (Iane 2). ~X%HlQ*rpu- (Iane 3). HXBHl0vpuUL (lane 4). HXBHlGvpu(Iane 5). HXBHlQvpu(lam 7) DNA proviruses were met(lane 6), HXBH~O-V~Uabolically labeled with -]methionins from 48 h posttransfection for 5 h. Cell Iysates (left panel) were imrnunoprecip itated with an anti-HIV-1 patient serum and an anti-Vpu serum. Viral proteins rsleasetd into supetmatants (right panel) wen immunoprecipitMwl with an antMIV-1 patient serum. Cell lysatets and clarified supam8tants were analyzed on 125% SDS-polyarcylamide gel. The mock transfected MT4 cells are shown in lane M of each panel. The positions of HIV-1 viral proteins am indicated. 6: Phosphorylation of the Vpu protein. MT4 cells (10') transfected with 10 bg of HXBH10-vpu' (lane 1). HXBH10-vpu ' (lane 2). HXBH10vpuPL (Iane 3). HXBHIOvpuM (Iane 4). HXBH10-vpu(lane 6) DNA proviruses were (lane 5). and HXBH10-vpumetabolically labeled with =Pi for 3 h. 62 h posttransfection. Cells were then washed with cold PBS and subsequently Iysed in RlPA buffer containing protease inhibitors. Labeled cell extracts were immunopmcipitated with an anti-Vpu serum and analyzed on 125% SDS-polyacrylamide gel. The mock transfected MT4 cells are shown in lane M. The position of the Vpu phosphoprotein is indicated. C: Effect of vpu mutants on release of viral particles in MT4 cells. MT4 cells HXBHlOwen transfected with 10 rp of HXBHlOrpu- (0). V ~ U HXBHIO-vpu6 as 01. H X B H I O - V ~ U ~(I).~ ~ HXBHIO-V~LP (A). H X B H l b p (A), and H x B H ~ W ~ U (+) DNA proviruses. The mock transfected MT4 cell culture is shown in line marked with X. At each posttransfectiontime indicated cell free supernatants were collected and virusspecified revene transcriptase activity was measured. +
(el,
-
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VPU
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(hours)
3. F m O R G ET AL.
16
beling with 50 pCilml of [35S]cysteine, viral particles released into identical volumes of supernatant fluid were pelleted through a 20% sucrose cushion. Labeled virion-associated proteins were then directly separated on SDS-polyacrylarnide gel and analyzed by autoradiography. Quaatitative measurements of the viral proteins associated to pclIeted virions shown in Fig. 3 indicate that at least I0 times more virus particles were released from HeLa cells transfected with the HXBHlO-vpu+ DNA (lane 1) as compared to HXBHlbvpu- (lane 3) msfected culture. As for cell culture transfeted with the E I X B H I O - V ~DNA U ~ ~(lane ~ ~ 2). at least a sixfold increase in virus particles release could be observed as compared to HXBHIO-vpu- (lane 3) transfected culture. Comparable effects on v h t release were also observed upon expression of Vpu proteins harboring single Ser substitutions (data not shown). Furthermore, the ratio of the amount of Env gp120 associated with viral particle was measured by quantifying, by densitometric scanning, the gp120 and the p24 bands from HXBHl&vpu+, HXBH 10-vpu , and HXBH 1 0 - v (Fig. ~ ~3).~ ~
-
The ratio of gp120 to p24 was similar (1:3) for all the pekted virus particles, indicating that the amount of Env glycoprotein incorporated into viral particle was not afftcted by the expression of Vpu. The reverse transaiptase activity detected in the supernatant of H ? C B ~ l & v p utransfected ~~ cells indicates that mutations in the dodecapeptide sequence still result in significant release of viral particles- In fact a substitution of seven amino acid residues (Glu 47, Arg 48, Glu 50, Asp 51, Ser 52, Gly 53, and Asn 54 for, respectively, Cys, Ala, Cys, Phe Leu, Cys, and Tyr) within the dodecapeptide sequence that significantly affected the global charge of this region (as demonstrated by the increased electrophoretic mobility) did not alter Vpu release fimction as determined by measurement of reverse transcriptase activity (data not shown). Furthermore, phosphorylation of the Vpu protein does not appear to be required for Vpu-mediated release of progeny virions, because substitution of phosphoacctptor sites at Ser 52 andlor Ser 56 did not abolish nor significantly alter the release of viral particles ~ ~ in HeLa and MT4 cells. Effects of Vpu Mutants on HW-1 Cytotoxicity
FIG. 3. Viral protein expression of vpu mutant proviruses in HeLa cells. HeLa cells were transfected with 30 pg of HXBH10-vpu' (lane I),~ ~ B ~ l ( l v(lane p u2).~ and ~ HXBH10-vpu' (lane 3) DNA proviruses. At 48 h posttransfection. cells were metabolically labeled with PSlcysteine for 12 h. Virions released into the supernatants were pelleted through 20% sucrose cushion and lysed in Laemmli sarnple buffer. Labeled peileted materials were directly analped on 11 .Soh SDS-polyacrylamide gel.
We and others have previously demonstrated that expression of vpu induced a delay in the appearance of cytotoxicity by syncytium formation (9-1 1). To examine the effect of Vpu dodecapeptide mutations on the rate of syncytium formation, cell killing kinetics studies were performed using the HXBHIOvpu mutants described above. Parallel cultures of Jurkat cells (10') were transfected with equal amounts (10 pg) of either HXBHIO-vpu-, HXBH 10-vpu ,or HXBH10-vpu mutant D N A s . In contrast to MT4 cells, Jurkat cells were previously shown to support long-term (up to 10 days) HIV-1 replication (9) and therefore represent a better system to study the effect of Vpu mutation on virusinduced cytopathic effects. Transfected cultures were monitored daily on day &12 for total viable cell number, percentage of dead cells, and appearance of syncytia. The spread of infection and virus release were determined periodically by measuring cell-associated p24 specific immunofluorescence and supernatant reverse transcriptase activity, respectively. A reproducible difference in the effect of virus replication on viable cell number was observed between cultures transfected with HXBH10-vpu+ DNA and cultures transfected with HXBH10-vpu'
Journal of Acquired l m m n e Deficiency Syndromes ond Human Renoviroiogy, Vol. 8. No. 1.1995
+
HW-I VPU PROTEIN
or HXBHlO-vpu mutants DNAs (Fig. 4A). In all cultures, the total number of viable cells rose norm a y until 5 days posttransfection. Through the next 4 days (to day 9), the rate of increase in viable cell number of HXBH10-vpu' transfected culture was almost nil. In contrast, the number of cells in the culture transf'ted with HXBHl&vpu+ DNA continued to increase until day 9 even though significantly morc progeny virus was produced as compared to HXBH10-vpu- transfccted culture (Fig.4B). At this point, the number of viable cells began to dtcreasc. The d t u r e s t r a n s f e d with H X B ~ l & v p and l ~ HXBHlO-vpuNM DNAs exhiiited overall a pattern of decrease in viable cell number similar to the culture transfected with HXBHlO-vpu' DNA, whereas HXBHl0-vpuand H X B ~ l 0 - v p uproviruses ~~ reproducibly induced a slower rate of cell killing than HXBHlOI vpu- provirus, as the numbers of viable cells began to decrease only 7 days posttransfection. The time scale of the effect of H X B H ~ O - ~ ~onUcell ~ ~via~ ~ bility was similar to HXBHlSvpu although cytotoxicity was morc pronounced. By day 10 posttransfection, there was no further increase in viable cell number in any culture. These differences in viable cell number observed between cultures cannot be attributed to virus production because cells transfectcd with HXBH10-vpu mutants DNAs that showed decreased cell viability earlier and faster than HXBH10-vpu transfectcd cells released comparable amounts of viruses into supernatant fluids +
20 syncytia per field. The Jurkat cell cultures transfected with either HXBH10-vpu- (A), HXBH10-vpu' (B), or ~ ~ B ~ 1 0 - v p u (C) - DNA provirus wem microphutographed 5 days posttransfection. The mock transfected Jurkat cell culture is shown (0). Spread of infection was monitored by periodically measuring cellassociated HIV-1 p24. The percentage of p24 immunofluor8s~nt-positiY8cells was determined by fluorescence microscopy and is shown in parentheses.
first appeared. However, the peak of syncytia was reached f day earlier (day 6) than in cultures transfectcd with a vpu+ provirus (day 7), suggesting a slightiy faster rate of syncytium formation. The magnitude of syncytium formation observed in the H X B H ~ G V transfected ~ U ~ ~ ~culture was consistently lower than in ~ X B ~ l 0 - v p u -and HXBHlOvpuSG transfected cultures. This observation may explain why HXBH 1O - ~ p u ~ ~ ~ ~cytotoxicduccd ity was less pronounced (Fig. 4A). four&
The percentage of single cells unable to exclude the vital trypan blue dye was also measured for each culture. As previously shown (11) for HXBH10-vpu-, the rate and extent of single cell killing, a process that occurs later than syncytium formation during viral infection, was not si@L cantly affected by the vpu mutation studied (data not shown). These results demonstrate that Vpu mutations altering the phosphorylation sites or the integrity of the dodecapeptide region affected to dif-
of Acqvirrd Inunntu Deficiency Syndromes und Human Rcrrovirobgy. Vol. 8, No. I . 1995
HW-I VPU PROTEIN
ferent extents the rate of HIV-induced syncytiurn formation but retained the capcity to facilitate virus export. Taken together, our data suggest that these two activities associated to Vpu expression may be the results of independent functions.
19
TABLE 1.
a e c r of Vpu muratus on rhe level of E h v glgcoproteins ar r h e cell swfacP
myt ~~
Vpu Dodccapcptide M u t a i h s Affect the Level of Env Glycoptoteins at the Cdl S u d h a
,
'
In view of the data obtained so far, the increased rate of syncytium formation observed with viruses lacking Vpu expression cannot be attniuteci solely to the impaired release of newly formed virions. Syncytium formation is the m t of cell-to-ccII fusion resulting h m the interaction of the envelope glycoprotein gpl2O expressed at the s u r f k c of infected cells and the C W receptor on uninfected cells (25,27)* It has been shown by flow cytometry analysis that the relative amounts of gp120 on the surface of cells infected with vpu' virus were markedly enhanced (two-to threefold) as compared to cells infected with vpu+ virus (11). To investigate whether Vpu dodecapeptide mutations had affected the concentration of gp120 at the cell surface, Jurcurs in infected cells expressing Vpu. Overall, these kat cells transfccted with the set of proviruses deresults suggest that the eariy appearance of syncytia scribed earlier were reacted with an anti-gpl20 in cell cultures oandectcd with H X B H ~ & V ~ U ~ ~ monoclonal antibody and stained with a FITCand H X B H I O - V ~ U ~proviruses ~ could be attrii conjugated goat anti-mouse IgG. The percentage of uted to the level of gp120 expressed at the cell surpositively stained cells as well as the relative face. Vpu expression appears to down-modulate the amount of envelope glycoproteins gpl20 at the cell levels of envelope glycoprotein at the cell surface surface were evaluated periodically by flow cytomby an undefined mechanism* Mutation at Ser 52 em. andlor Ser 56 may have exposed the Vpu domain As shown in Table 1, the mean fluorescence inresponsible for this function. tensity reveaIed an approximately threefold increase m the surface expression of gp120 in cells transfected with the HXBHlO-vpu- prowhen DISCUSSION compared to cells transfectfd with the H X B H 1 0 The vpu gene product of HIV-1 significantly invpu+ provirus. Similarly, by days 7 and 9, cells creases the release of budding virions from infected transfcctcd with provimses canying mutation in T cells. In the absence of Vpu, there is an the vpu sequence at Ser 56 ( H X B ~ 1 0 - v p u ~ ~ ~CD4+ . accumulation of mature particles associated with H X B H ~ O - V ~were U ~ ~expressing ~~) two times more plasma membrane of virus-producing cells, Apart gp120 on their cell surfact than cells transfe~td from tbis effect on viral release, Vpu delays the with the HXBHIO-vpu' provirus. The level of cytopathic effectof HIV-1 by decreasing the rate of gp120 detected at the cell surfaceof HXBH10-vpu' syncytium formation. Whether or not these events transfected cells was repeatedly higher than in art related has remained unclear because no do~ X B I i l & v p uor~ H X ~ ~ l l b v transfccttd pu~~ h f u n c t i o n corrcIation has been made so far. In Jurkat cells. In addition, early afttr transf'ction, the present work, we have carried out a mutational the percentage of positively stained cells was lower analysis of the conserved dodecapeptide region of in HXBH10-vpu transfected cells as compared to Vpu and investigated the importance of Vpu phosHXBH10-vpu mutant transfected cells. This d&ct phorylation in the replication and cytopathicity of is probably the result of the rliminished rate of synHIV-1 in CD4+ T cells. The results of the expericytium formation and thereby viral spread that oc+
Journal of Acquired Immune D&rcietwy Syndromes ad Human Rrrrovirobgy, Vol. 8. No. 1. 1995
20
J . FRIBORG ET AL.
shown that coexpression of vpu and env from the ments described here suggest for the first time that same viral transcript did not affect the synthesis or HIV-I Vpu-facilitated virion release and delayed processing of gp160 envelope precursor glycoprosyncytiun formation may be the consequence of teins (13,17). In the present study, the tot& amount two independent activities of the Vpu protein. of intracelldar gp 16Olgp120 immunoprecipitated Our first goal was to identify the phosphoaccep fkom transfectcd MT4 cells did not reveal sign& tor site(s) responsible for Vpu phosphorylation. cant differences in the presence or absence of Vpu Substitution mutation of two conserved serine res(Fig. 2A). Furthermore, although -HlO-vpu idues in t!x highly conserved dodecapeptide sedocs not produce Vpu due to a mutated ATG initiquence of Vpu, individually or in combination, ation codon, the vpu mutant proviruses analyzed in demonstrated that Ser 52 and Ser 56 were both this study did express a Vpu product and were stiU phosphorylated and represent the phosphoacceptor able to increase the rate of syncytium formation sites targeted by CKII (Fii. 2B),The effects of mu(Fig. 5). From these observations, we conclude that tations in the dodccapeptide sequence and the phosutilization of the env AUG initiation.codon during phoacceptor sites on the release of HIV particles translation of the bicistronic vpulcnv -A is unand on syncytium formation were next ernmined. likely to explain the effects of Vpu on s e e gp120 The data presented here show that neither phosexpression and syncytium formation. Alternative1y , phorylation of the Vpu protein nor maintenance of it was proposed that Vpu-facilitated virus release an intact dodecaptptide sequence appeared to be could reduce the concentration of surface envelope essential for Vpu-mediated release of progeny viriglycoproteins, thereby reducing the rate of synons since substitution of phosphoacceptor sites at cytium formation. From the phenotypic data o b Ser 52 and/or Ser 56 and nonconservative substituw e d with the proviruses carrying mutation in the tion mutations of the dodecapeptide sequence did vpu sequence at Ser 52 andor Ser 56, it is unlikely not abolish nor significantly alter the release of viral that the increase in the rate of syncytium fonnaparticles in M T 4 and HeLa cells (Figs. 2A,2B, and virus is solely the result tion observed with vpu' 3). In contrast, these mutations resulted in proviof impaired release, because H X B H ~ O - V or ~U~~ ruses that exhiiited an increased rate of syncytium H X B ~ 1 0 - v p uproviruses ~~~~ exhibited close to formation as compared to wild-type vpu proviruses. wild-type Vpu-mediated virus release function For reasons that are as yet unclear, mutations at Ser while 56 ( H X B ~ l & v p uand ~ ~ )Ser 52/56 ( H X B H I O - V ~ U ~ inducing syncytia at the same rate and magnitude as a vpu- v i m . The results obtained here by s)resulted in a more significant effect on the rate of cytofluorometric analysis show that the level of syncytium formation as compared to mutations at Ser 52 (HXBH 10-vpusZ o r HXBH 1~ - v p u ~ ~ - ~ gp120 ~ ) . at the surface of cells transfected with viruses lacking Vpu or harboring mutationg at Ser 52 and/or Overall, these results suggest that a functional doSer 56 was increased two- to threefold as compared main located within the highly conserved dodeto vpu+ provirus transfected cells (Table I). This capeptide region of Vpu modulates the rate of observation indicates that Vpu may affect the rate cytopathic effects of HIV-1 vpu+ viruses. Furtherof syncytium formation by directly or indirectly more, because Ser 52 and Ser 56 are phosphoaccepdown-modulating the level of envelope glycoprotor sites responsible for Vpu phosphorylation, we teins at the cell surf'. In support of these results, may postulate that phosphorylation of the protein is p r e w experiments involving cotransfection of required for its ability to modulate the rate of HIVvpu and env on two distinct expression plasmids in l-induced syncytium formationa HeLa-CD4 system resulted in the appearance of The relative syncytium-forming ability is proporlarge syncytia in absence of vpu (vpu initiation tionate to the level of envelope glycoproteins excodon mutation), whereas fewer and smaller syncypressed at the cell surface (29). We have previously tia were observed in the presence of vpu, suggesting reported that the concentration of envelope glycoprotein gp120 at the surface of cells infected with a modulation of gp120 surface expression (X. J. 1 viruses lacking vpu was increased two- to 'Yao, F. Boisvert, and E. A. Cohcn, unpublished threefold as compared to vpu+ vinu-infected cells results, 1994). These prcdata, obtained in a system in which no viral riplieation and release oc(11). It was proposed that the expression of Vpu may decrease the utilization of the env AUG initicurs, also rule out an effect of these mutations on the initiation of envelope glycoprotein translation. ation codon during translation of the bicistronic vpulenv mRNA. However, previous reports have The precise mechanism by which the HIV-1 vpu Journal of Acquired Intmune D&ciecncy Syndromes tznd Human RerroviroIogy. Vol. 8, No. I . 1995
HIV-I VPU PROTEIN
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down-modulates envelope glycoprotein expression at the cell s u r f k e remains unclear. The vast majority of unclcaved gp160 synthesized during HIV-1 replication is transported to lysosomes, where it is degraded (30). In CD4+ T cell lines, only 1&20% of gp160 is processed and reaches the plasma membrane. Furthermore, the formation of gpl60-CD4 complexes in the endoplasmic reticulum (ER) influences the trafficking and maturation of envelope glycoproteins (13,14,3 132). Vpu could have evolved to modulate envelope glycoprotein expression at the uIl surface by interacting with Env precursor gp160 during their intracellular sorting, thereby delaying syncytium formation as a result of the decreased expression of a 1 2 0 at the cell surface. Alternatively, in the presence of Vpu, envelope glycoproteins could transit at the cell surface and become quickly rcintenraliztd. Interestingly, our immunolOcaiization studies indicate that Vpu accumulates mainly in the Golgi apparatus but can also be detected in association with endosoma1 vesicles and the plasma membrane (X. J. Yao, F. Boisvert, S. Garzon, D. Bergeron, and ElA. Cohen, manuscript in preparation). In a transient expression system, Vpu was shown to indirectly increase the intracellular processing of Env gpl60 by d e s t a b h g gp160-CD4 complexes formed in the ER (13). This effect is the result of Vpu-induced degradation of CD4 molecules (14). However, in their studies, Willey et al. (13.14) did not investigate the levels of envelope glycoproteins and CD4 at the cell surface. Io this regard, a recent study has reported that W - l e n c o d e d Nef protein could interfere with envelope glycoproteins as well as CD4 cell surface expression (33). In fact, Nef can significantly decrease the cell surface levels of gp120 through a mechanism dependent on the intracellular binding of gp140 to CD4, resulting in a reduction of syncytium formation. In contrast to Vpu, Nef protein was shown to not alter the stability of CD4 molecules (34). A more recent mutational study of Vpu performed in an in vitro CD4 degsadation assay revealed that deletion of sequences within the highly conserved dodecapeptide region and the C-terminal portion of Vpu abolished the Vpu-induced degradation of CD4 (15). In order to detenaine whether the effects of Vpu on cell surface levels of Env glycoproteins and CD4 degradation are interrelated, we are currently testing the ability of the Vpu mutant proteins d e s c n i in this study to degrade CD4 molecuIes. Early studies have demonstrated that Vpu is a
91
membrane-associated protein (9. More recently, topographic studies revealed that Vpu is a class 1 integral membrane protein (20). The C-terminal hydrophilic region of Vpu that is directed toward the cytoplasmic fbcc contains two predicted a-helical sequences of opposite net charge linked by the highly conserved dodecapeptide sequence according to the Chou-Fasman aigorithm method (35) USing the hdacvector software (International Biotcchnologies. New Haven, CT,U.S.A.). It is generally assumed that protein phosphorylation will stab* different conformational states of the regulated molecules required for their biological activity. It has been shown that residues surrounding phosphorylation site(s) wiIl adopt aitematc side-chain conformations in the pbospbotylation state, enabling them to fonn intersubunit interactions (36). As Vpu may form hom9.oligomeric complexes (19,20), it is possible that the biological significance of Vpu phosphorylation at serine residues 52 and 56 is to induce the active conformational form of the protein. Interestingly, a structural similarity has been described between Vpu and the M2 transmembrane protein of the influenza virus (5.10). It has been suggested that the 97 amino acid M, phosphoprotein in a homotetrameric form can act as an ion channel capable of decreasing the pH of the intracellular endosomal compartment in influenza virus-infected cells (37-39). The M2protein is responsible for clevating the mildly acidic pH in the trans Golgi network or the Golgi complex (40). M2,by moddating the lurninal milieu of intracellular compartments through its function as an ion channel protein, can act at different stages of the influenza infection cycle (40,41). It is not known whether the HIV-1Vpu is functionally related to the M2 of influenza. Clearly, a better biochemical characterization and domain-function relationship will not only define the role of Vpu in HIV-1 replication cycle but will also unravel the general mechrvrism of action of the protein. -t: We want to thank Xiao Jian Yao, Tatyana Dorfman, I)ominique Bergcron, Karl H.Kalland and Michael Cody for helpful discussions and Serge Snechal for technical assistance with the flow cytomctry analysis. J.F. and A.L. are recipients of studentships from the Natioual Health Research and DeveIop ment R q p m (NHRDP), Health and W e b Canada. E.A.C. is E. recipient of a NHRDP AIDS career award. This work was suppoxtd by a NHRDPIMRC grant to E.A.C. and by a NM grant -267 to H.G., E.A.C. and WAR. are recipients of a NATO wllaborative research
grant-
Jounrol of Acquired Immune D&cieny Syndromes a d Hwnan Retrovirobgy. Vol. 8. No. 1. 1995
J- FRIBORG ET AL. rmmodefiaency virus type I Vpu is an oligomcric type I integral mcmbtpae protein. J Virol l993;67:SOSM 1. 21. Pinna LA-Casein khasc 2: "an eminence grise" in cellular 1. Cullen B R Use of akaryotic expression technology in the rtgdaion? Biochem Biophys Acta 1990;1054:26744. fun--onal urnlysis of c l o d gcoes. Methodi Eryrmol1987; 22. Ro SN, Huat HD, Horton RM, Pull-. JK, Pease LR. Sitc152:688-9. dihctcd by OVusing the p l y 2. Cullen B R Mecbznism of action of tegulatory proteins enmerase chain reaction- Gcnc 19%9;W514. cuied by complex retroviruses. Microbial Rev 1992Sk37S 23. T 'EF, Burghd R, Sm R The rm gene product of W. the human immPnod&deacy virus is rtquind for replica3. Coben EA, Terwilliger EF, Sodroski JG. Hasdtine, WA. tion J V W 1 9 8 8 S S S Identification ofa pmt& encoded by the vpu gent of HIV3.Hurb S, bymagi Y,Y m t o N. Infection of HTZV1. Nature 1988;3W=. IIULAV in HTLV-l+anyhg ccllr bsf-2 8ad m4 and a p 4. ~ Z , C b o u M J , ~ M . e t r Hmanimm~110l . d c f i c i e n t y ~ t y p e l h u . n . d d i ~ ~ r e q ~ m pliatioa in a plaque assay- Sciruce 1985$29:%3-6. s.Sodmski JG, ROSCXICA,FIIcltiac WA.T tranthe cerrtrrl region of the genome. Proc Nufl Acud Sci USA ~ Y l i n t i o n d t b c l c m g t e m b d ~ T iw~s3s:es68-n. lymphm@c vinucs in izrfectcd cells. Scicncc 1 W a : 5. Strebel K,KlimlPit T,Mutin MA- A novel m e of HfVll, vpu, and its 16-kiIoddfoapmduct. Science 1988241:1221-3381-56. Huct T, Qleynier R M e y m A, Rodants G, Wain26. Sodroski JG, Goh WC, Rosa C, (hmpkn K, EIoseltiac WA. Role of Tbe HTLV-IIULAV envelope in syncytium Hobson s. Geactic Orolaiptiou ofa cbimplrnzec ltrrtivinrs fbmaion and -city. Nature 1906;3=4704. ~elatcd to HIV-I. N I~SS:~SU. 7. Schwutt S,Felkr BK, Feay6 EM, PavlPkis GN. Env md 27. Lillroa JD, Feinberg MB, RCYCSGR. Induction of Vpu proteins of buaun immunodeficiency vinrs type 1 are dcpcadtnt d l fution by the m V / m LAV envelop glyproducedErom multiple bkhtmuic mRNAs. J VirollSC#);64: q x o t c h N a r m 1986323:7258. S%28. Gunicr J, OS#Utbrp~DJ, ROWn B-Aarlyris of the W8. Arrigo SJ, Cben ISY. Rev is nctess~ryfor tmmhlion but rrcy md implications of simple method for predicting the viT, vpr, md envlvpu not cytoplasmicaccamuIUion of -1 wcadary of gldbulpr proteins. J Mol Biol 1978; 2 RNAs. Genes Dcv 19913:808-19. 12&97-120. 9. T m d & r EF,Cohen EA, Lu YC, Sodroski JG,Hadtine 29. C;rbuzda DH, Lever A. Tcmdiigcr EF, Sodroski J. EEecu WA. Functid role of human immunodeficiency virus typc of deletions in the cyroplumic domain on biological func1 Vpu. Roc Natl Acad Sci USA 1989;86:5163-7. tions of humsn immunodeficiency virw type 1 eavclopc gly10. Klimkait T-S P e k l K. H MD,Martin MA, Orenstein coproteins. J Virol 1992;66:330615. JM,The hurmnimmunodtficiency virus type 1 spec& pro30. WSey R B o d k i n 0 3, Ports B. Biosymhtsis, cleavage and tein vpu is nquirrd for ciricicnt virus nrahrration aud re&&on of humrn immunodc6aency virus 1 envelope lease. J Virol19#),6):621-9. glycoprotcia gp160. Proc Nor! Acad Sci USA 1988;85: 11. Y.s, XJ, GYzon S, B o i e r t F, Haseltine WA,Cohen EA. 9 s w . Tbe effect of vpu on HIV-linduced syncytia formrtion. J 31- Buooocarr L, Rose JK. Rcvtntion of HIV-1 glycoprotein A c q w Immune &fie Syndr lB3;6: 13-1. musport by soluble CIX rctainod in the nedophmic retic12. HG, T.Cohm EA. Haseltine WA. Huulum. Nmwe 19#)34%6258man immunodcficiency virus type 1 Vpu protcin enhances 32. Crhe B, Buonocarr L, Ruse JK. CW is retained in the the production of capsids &om widely divergent retroviruseodoplprmic reticulum by the immunodeficiency v i m s typc es. Proc Acad Sci Natl USA 1993;90:7381-5. 1 dycovmtcin phcursor. J Virol 1990;64:558!5-93. 13, W e y RL, Maldarelli F, Martin MA, Svebel K. Hurrran 33- ~ c ' h k r k0 , Rhi&reY,H d JM,D a m s 0. Reduced cell immunodeficiency virus typc I Vpu protein induces m i d s&&cc q m s s i o n of processed humaa immunodeficiency degrada!ion of CD4. J Virol lB2$6:719>200. virus type 1envelope glycoprotein in the presence of Nef. 3 14. Willey RL, Mnldarclli F, Martin MA, Strebel K. Human Vwl 1993;m3274-80. immunodeficiency virus type 1 Vpu protein regdates the 34- Garcia JV, hiillcr AD. Serine phosphoryIation independent fdon of intraccUuIar gp160-CW complexes. J Vuol of &-srfacc CD4 by Ncf. Narure 1991; & on1=;66:226-U. 330-1 I. IS. Chen MY, Maldarclli F. Karczewski MK, Willey RL, 35. Chou PY,Fuman GD.Prodiction of protein conformation. S a t k l K. Human imnunodeficiency type 1 Vpu p r o w Biochemimy 1-4; l3:222-45. induces degndation of CD4 in vim: tbc cytoplasmic do36. Sprang SR, AchryP KR Goldsmith EJ, et al. Smaural of CDI conmbutts to vpu sensitivity. J Virol1993;67*S77changes in 3ycogcn phorpbarylase induced by pbosphory84. Irtion. Nature 1988;336:215-21. 16. Vincent MJ. RPja NU, Abdul Jabba M. Humln immunode37. S m e TU, &hadm G, ZIinbon MC,Hall-Smith M. Dougficiency virus typc 1 Vpu protein induces d-on of Irs AR, Hay AJ. Specific rPUcWal alteration of the M u chimeric envelope glywprotdns b e a m the cytoplasmic enz~ by m e . EMBO J 1m;9:3&e md aacbor domain ofCD4: role of the cytopl.smic domrin 76in Vpu-induced d-on in the endoplotmic reticulum. 3 38. Surgue RJ,m y AJ. Stmcbarl chaxacteristics'of the M2 p m V i d 1993;67553W9. tcin of iatluaua A vinutj: &deuce that it forms a tet17. Yao XJ, mttlingerH,Haseltint WA,Coben EA. Eaveiope d c &amill. Vi*dogy 1991;180:617-24. glycoprotein md CD4 independence of Vpu facilitated 39. Pinto LH, Holsiager U,L a m b RA. Snfluenza virus M2 prrz -1 -d expat. J Virol1992,66:5119-26. tdn bas ion chPnncl activity. CeU 1992;69517-28. 18. Gerrghty RJ,w iAT. Human immunoMcicacy vi40. Ruigmk RWH, Hint EM& Hay AT. 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REFERENCES
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l o u dof Acqnircd Immune Dl/&ncy
Syndromes and Human Retmviro/ogy. Vol. 8. No. 1. 1995
CHAPITRE 3 Article 2:
Structural and functional analysis of the membrane-spanning domain of the human immunodeficiency virus type I Vpu protein.
STRUCTURAL AND FUNCIlONAL ANALYSIS OF THE MEMBRANESPANNING DOMAIN OF THE HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 VPU PROTEIN
Jacques Friborg, Nash G. Daniel, Dominiquc Bcrgmn and &c A. Cohen*
Laboratoire de R&rovirologie Humaine, wartement de Microbiologie et Immunologic, FacuIt6 de Mdecine, Universite de MonmM, CP 6128, succursale centre-ville, MonQuebec, Canada H3C 3J7
*To whom correspondence should be addressed Tel: (5 14) 343-5967
Fax: (514) 343-5995 Electronic mail address:
[email protected]
ABSTRACT The human immunodeficiencyvirus type 1 (HIV-1) vpu gene product is a class I integral membrane phosphoprotcin capable of multhnmhtion. Expression of the Vpu
protein during HAL1 infection of UM+T cells delays cytopathic cffens by reducing the rate of syncytium formattion and disrupts HIV-1 gp160-CD4 complexes farmed in the endoplasmic reticulum by inducing a specific degradation of CD4. These Vpuassociated activities arc dependent on the phosphorylation state of the protein. In
addition, Vpu facilitates the release of budding virions from fIIV-1 infected cells by a mechanism which is c m n t l y unclear. To further dehcate structural requirements for Vpu membrane association and to better define the importance of the membranespanning domain in Vpu-associated activities, we have performed a mutational analysis of the hydrophobic N-terminal region of Vpu. Deletion and substitution mutations
introduced into the N-terminal region were initially tested for their ability to affect Vpu insertion in v i m into canine pancteatic microsomal membranes (CMM).The results of these experiments show that both the deletion of seven amino acids (residues Ile 8-AIa 14) and the substitution of residues Val 20, Trp 22 and Ser 23, had a deleterious effect
on Vpu insertion into CMM. Futhermore, these mutated Vpu proteihs exhibitad altered stability in HIV-1infected cells suggesting that membrane association may be critical for the stability of Vpu. Interestingly, two mutations that did not affect Vpu membrane
association, impaired the capacity of the protein to enhance virion release, while still being able to induce CD4 degradation and to delay syncytium f m t i o n during HIV-1 replication experiments. These findings suggest that the membrane-spanning domain retains not only information sufficient for the insemon and anchoring of Vpu in membranes but also contains determinants which contribute to Vpu-mediated enhancement of virion release.
INTRODUCTION
Human immunodeficiency virus type 1(HN-1) encales a Vpu protein, which is not found in the closely related HN-2 or in most simian immunodeficiency viruses (SIVs) (5,30,40).
Biochemical studies have shown that the vpu gene encodes an 81
amino acid intcgraI membrane protein capable of homo-multimerization (29,39). Vpu has the topology of a class I protein with a strongly hydrophobic N-tcmhal region of 27 amino acids (&a) and a C-terminal hydrophilic domain (29). Serine residues (Ser 52
-
and Ser 56), located in a C-tamigal stretch of 12 aa (residues 47 58) highly invariant
among Vpu proteins from diverse HIV-I isolates, have recently been shown to be phosphorylated by a casein kinast-II-dated protein (9,35,36). Numerous functional studies revealed that Vpu promotes the release of budding
virions from infected cells (22, 39, 43). This effect of Vpu is not restricted to HIV-1 since Vpu expression was shown to facilitate the release of viral capsids from HN-2and fiom retroviruses as divergent as Visna and Moloney muxine leukemia viruses in HeLa
cells (13). These observations raised the possibility that Vpu could act indirectly through modification of a cellular pathway rather than by interacting directly with virusspecific proteins. Vpu also has the capacity to delay HIV-1 cytopathic effects by
reducing the rate of syncytium formation during in vitro infection of CD4+ T cells (22, 43). This Vpu-mediated delay of syncytium formation is proposed to result from a
reduced accumulation of Env gp120 at the cell s d a c e during vpu+ virus infection (9, 51). Furthermore, Vpu was shown to decrease gp160-CD4 complexes fonaation during
HTV-1infection of CD4+ T cells by mediating a rapid and specific degradation of CD4 molecules in the endoplasmic reticulum (ER) (47, 48). Recently, Vpu was shown to
specifically bind CD4 molecules in the ER. This interaction appears to be an early event critical in triggering the process l e g to the Vpu-mediated degradation of CD4 (2).
Several lines of evidence suggest that Vpu has diff'ntial biological activities
during HIV-1 replication and may in fact have more than one target in infected cells. Indeed, Vpu-mediated enhancement of virion release was reparted to be independent, of the expression of HIV-1
Env glycoprotcins and CD4 receptor molecules (12, 52).
Furthermore,facilitation of virion release is efficieny inhibited when Vpu is retained in the ER by Brcfeldin A (BFA) treatment of infected cdls, while Vpu-mediated
degradation of CD4 is s t i l l prominent (34). Phosphorylation of Vpu was shown to be essential for its ability to induce degradation of CD4 molecules and to mediatt a delay in the rate of syncytium formation while it had only a partial efftct on Vpu-facilitated #
virion release (9, 34). Thus, biological activities of Vpu appear to be regulated by at least two different molecular mechanisms.
In this study, we have performed a mutational analysis of the hydrophobic N-
terminal region of Vpu to assess its importance in Vpu's biological activities. The results of our experiments show that deletion and substitution mutations i n d u c e d in
sequences which are highly conserved among Vpu proteins and were predicted to adopt an a-helical configuration, affected Vpu i n s d o n into canine pancreatic microsoma1 membranes (CMM)and firrther altered the stability of the mutated Vpu proteins in infected cells. In addition, we describe two mutations in the membrane-spanning domain of Vpu that had no effect on the capacity of the protein to inscrt into CMM but differentially altered Vpu biological activity during H N - 1 infection. These mutated
Vpu proteins were shown to induce CD4 degradation and to delay H N - I cytopathic effects but were otherwise impaired in their capacity to facilitate virion release. This result suggests that the membrane-spanning domain of Vpu contains determinants which contribute to the facilitation of virion release.
MATERIALS AND METHODS Site-directed mutagenesis and p-d
DNA constructions. HXBH10-vpu+
(LTR-gag+,pol+, v p , v p r , rm+, rev+, yu+, em+,4--LTR) and HXBH10-vpu- (LTR-
gag+, pol+, vif+, vpr-, z a P , rev+, vpu; en+, n&-LTR) are two isogenic infectious molecular clones of HIV-1that only differ in their ability to express Vpu (43.52). Vpu
mutagenesis was paformed on HXBHlO-vpu+ using a two-step PCR-bascd mutagcnesis as previously described (19).
The nmlcotide sequences of the mutagenic
oligonucleotide were as follows: HXBH10-vpu A7, sense: 5'-GCAACCT'ATACAT ATCGATAATAATAGCAATAG-3';
HXBH10-vpu
Ile6,
sense:
5'-
CmATACAAAAAGCAAGCITAGCA'ITAGTA-3'; HXBH10-vpu Alal 0, sense: 5'GCAATAGTAGAATTCGTAACAGCAATAATA-3'; HXBH10-vpu Ile 16, sense: 5' -
GTAGCAATATTAATAGGAACAGnGTGTW-3'; HXBH10-vpu Va120, sense: 5'-
ATAGCAATAGATGTGGATCCCATAGTAAT-3'; HXBH10-vpu Ile24, sense: 5'GAATATAGGAACGTAlTAACACAAAGAAAA-3'.
A pair of oligonucleotide
primers located 23 bp upstream from the vpr initiation codon (sense primer A: 5'AAGCCACCmGCCTAGT-3t) and 148 bp downstream from the vpu termination
codon (antisense primer B: 5'-GGCTACACAGGCATGTGT-3') were used for DNA
amplification of the resulting fragments.
The generated vpu mutants were digested at Sal I and K p n I sites located respectively at position 5331 and 5893 (+I = site of transcription initiation of the
HXBHLO molecular clone) and cloned into the in vitro expression plasmid pSP64 (Promega) to yield pSP64-vpu A7, pSP64-vpu ne6, pSP64-vpu AlalO, pSP64-vpu Kle16, pSP64-vpu Val20 and pSP64-vpu ne24. All mutant constructs were confitmed by the
didcoxyhucleotide chain termination sequencing method using a Sequenase kit (United States Biochemical Co., Cleveland, OH).
The SVCMV-CD4 construct was generated as described previously (50) by inserting a Xbo I - Smu I cDNA fiagmcnt encoding CD4 into the expression vector SVCMVtxPA. The CD4 cDNA was derived fiom the pT4B CD4 expressor, obtained
from Dr. Richard Axel through the AIDS Research and Reference Reagent Program (Division of AIDS, MAID, NIH) (28).
In vitro transcriptioneoupled translation. RNAs wcre synthesized by in v i m transcription of linearized pSP64-vpu plasmids DNA using SP6 RNA polymerase in the presence of mGpppG (5 mM) cap analogue (Phhmacia LKB Biotechnology) as described (5). Equimolar amounts of the transcription mixtures (equal amounts of radioactivity) containing the appropriate mRNAs were then translated in the presence of CMM in a rabbit reticuiocyte lysate system as described (5). Translation was carried out at 3 0'C
for 30 min in presence of 5 pl L-[fSS]methionine (1 pCi/pl, 1140 Ci/mmol) and
stopped by chilling on ice,
Cells and DNA transfation. MT4, an HTLV-I aransf'ed human CD4+T cell
line, Jurkat, a human CD4+T cell line and COS-7, an African green monkey kidney cell line transformed by an origin-defective mutant of simian virus 40 (SV40), were
maintained as described (14, 17, 44). MT4 (5 x 106) and Jurkat cells (10') were mmsfected with 10 pg of plasmid DNA using a DEAE-dextran transfedon method (38).
Transfected cell cuIturcs wcre subsequently given a complete medium change with fresh
RPMI 1640 medium containing 10% fetal calf serum (FCS). Jurkat cultures were resuspended daily in an appropriate volume of h h RFMI 1640 conraining 10% FCS so
as to maintain equivalent densities of viable cells (106 cell-
) in the cultures. COS-7
cells (1 x 106) were seeded into a 100-rnm petri dish and cultured overnight in
Dulbccco's modified Eagle's medium (DMEM) containing 10%FCS. Cells were subsequently cotransfected with a mixture of 5 pg of CD4 expressor (SVCMV-CD4)
and 20 pg of EW-1 plasmid DNA (molar ratio 1:3) using a calcium-phosphate method
(6)Virus-specific reverse transcriptase activity in the supernatant fluid was measured as previously described (37). Evaluation of the cytopathic effect was perf'omed daily by monitoring the totat number of viable cells by trypan bluewclusion. The cell cultures were also stand for syncytia by light microscopy.
Metabolic labeling and immunopredpitation. To assess Vpu membrane association, microsomaI membranes from in v i m translated reactions were sedimented by centrifugation of the translation mixtures for I h in an airfbge (Beckman I n s m e n t s
hc., Palo Alto, CA) at 20 psi of air pressure. Membrane pellets and supernatant fractions were collected, solubilized in a buffer containing 140 mM NaCl, 8 m M
NaHP04, 2 mM NaH2PO4, 1% Nonidet P-40,0.5% sodium deoxycholate and 0.058 sodium dodecyl sulfate (SDS), and immunoprccipitated with a rabbit anti-Vpu peptide serum directed against the C-terminal region of the Vpu protein (HAPWDVDDL) as previously described (5). The labeled immunoprecipitates were separated on a 12.58
SDS-polyacrylamide gel and visualized by autoradiography. For pulse-chase metabolic labeling, MT4 cells were smrved 48 h posttransfection
in methionine-free RPMI 1640 medium for 15 min.
Subsequently, cells were
metabolically labeled with 200 pCilrnl of [3%]methionine for 30 min. The labeling
media was then removed and the cells were washed once with phosphate-buffered saline (PBS). Equal aliquots of labeled cells were resuspended in 1 ml of warm RPMI 1640 containing 10 % FCS and incubated at 37'C. At each indicated time points, cells were lysed in RIPA buffer containing 10 mM Tris-HC1 (pH7.4), 1 m M EDTA, 100 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.25% sodium deoxycholate and 0.246 phenylmethylsulfonyl fluoride (PMSF),and Vpu proteins were innnunoprecipitated with the rabbit anti-Vpu peptide serum. The immunoprecipitates were analyzed as described above.
To assess Vpu-mediated enhancement of virion release, MT4 cells were
metabolically labeled with 100 pWml of [3SS]methioninc 48 h posmansfection for 5 h. The labeled cells were lysed in RIPA buffa and viral proteins wert immunoprccipitateci with both a HIV-1 positive human saum and a rabbit anti-Vpu serum in a 1:1ratio (44).
The immunoprecipitates were analyzed as described above. For each sample, viral particles were pelleted by ~Itracenaifugationof equal volume of supernatant fluid through a 20% sucrose cushion at 35000 rpm for 2 h at 4'C in a Beclrman SW 41 rotor.
Pelletcd materials were lysed in Laemmli sample buffk (125 mM Tris-HC1 [pH 6.8],2%
SDS,4.8% 2-mercaptoethanol and 20% glycerol) and virion-associated proteins were directly separated on a 12.5% SDS-polyacrylamide gel and visualized by autoradiography. To assess Vpu-induced CD4 degradation, COS-7 cells were starved in methionhe-fm DMEM for 15 min, 48 h posttransfection. Cells were then pulse-labeled
for 30 min by adding rnethionine-free DMEM containing 500 pCi/rnl of [3sS]methionine.
The labeling media was subsequently replaced with DMEM
containing 10% FCS and equal diquots of labeled cells were chased at 3732 At each
indicated time points, the radiolabeled cells were washed once with cold PBS and lysed in RIPA buffer. Immunoprecipitation of the cell lysates was performed with a mouse
anti-CD4 (OKT4) monoclonal antibody. Anti-CD4 serum was derived from ascitic
fluids of Balb/c mice that were injected with an OKT4 hybridoma obtained from the
American Type Culture Collection (ATCC), Rockville, MD. The immunoprecipitates were analyzed as described above. Quantitation of immunoprecipitated proteins was performed by densitomemc scanning of the autoradiographic signals on a Personal Densitomcter (Molecular
Dynamics) using the ImageQuant software version 3.22.
Immunofluorescence. Evaluation of cell-associated HIV-1 ~24808and Vpu protein by immunofluoresccnce as well as measurement of HIV-1Env glycoprotein
gp120 expressed at the surface of infected cells by flow cytometric analysis were performed as described previously (9.51). Anti-HN-1 p24gag and anti-HIV- 1 gp 120
monoclonal antibodies were obtained from American Bio-Techndogies Inc., (ABT; No
4001 and 1001), Ossining, NY. The fluorescein isothiocyanatc (FITC)-conjugated goat anti-mouse IgG was purchased fiom Gibco BRL Inc., S t Lawrence, MA.
RESULTS Construction of HIV-1 vpu mutants. In order to better characterize the
importance of the membrane-spanning domain of Vpu for the biological activities of the protein, a series of deletion and non conservative substitution mutations was introduced
into the 27 a.a. hydrophobic N-terminal region of Vpu by PCR-based mutagenesis i
. 1). The design of vpu mutants was b a d on the structuraI features of the protein
predicted by a computer-assisted analysis using the MacVcctor software program from International Biotechnologies Inc., New Haven, CT. Indeed, amino acid sequence alignment of Vpu from diverse HIV-1 isolates rtveded 88%of homology consewation between residues Ile 8 and Scr 23 (data not shown) which were similarly predicted to
adopt a-helical configuration according to the Robson-Gamier algorithm method (11). Membrane-spanning domains in integral membrane proteins most likely adopt a-helical configuration within the hydrophobic interior of the lipid bilayer which appear relevant
for proper protein anchoring (7,25,27). Thus, all substitution mutations were designed to alter the putative a-helical configuration in the N-terminal region of Vpu (Fig. 7).
The mutations affected three adjacent amino acids. Two mutants carrying nonconservative substitutions of amino acids Ile 6, Ile 8 and Val 9 for Lys, Ser and Leu (pSP64-vpu Ile6) respectively, and Ala 10, Leu 11 and Val 13 for Glu, Phe and Thr @SP64-vpu AlalO) respectively, were generated to affect the proximal pomon of the putative a-helical configuration (Fig. 1B). Two mutants carrying non-conservative substitutions of amino acids Ile 16, Ala 18 and Ilt 19 for Leu, Gly, Trp (pSP64-vpu Re16) respectively, and Val 20, Trp 22 and Ser 23 far Asp, Asp and R o (pSP64-vpu Val20) respectively, were designed to alter the distal portion of the putative a-helical
configuration (Fig. 1B). One additional mutant was produced by non-conservative substitutions of amino acids Ile 24, Val 25 and Ile 27 for Met, Ala and Lys (pSP64-vpu Ile24) respectively. Finally, a 7 a.a. deletion vpu mutant (pSP64-vpu A7) deleting Re 8
to Ala 14 was produced to completely disrupt the putative N-ttiminal a-helical motifs of the protein. Although the mutants were designed to disrupt the predicted structural
f e a ~ e of s the membrane-spanning domain of Vpu, they were shown to retain the hydrophobic profile of the wild-type (wt) Vpu N-terminal region by computer-assisted analysis (data not shown). Furthcrmort, all mutations introduced in Vpu sequences that overlap the env open reading fhne did not affect the gp 120 amino acid sequence.
In vitro translation of Vpu mutants in the presence of canine pancreatic microsoma1 membranes: characterization of the hydrophobic N-terminal region of Vpu. In vim translation in the presence of CMM has previously proved to be useful in
defining the function of hydrophobic domains from a variety of viral proteins, including Vpu, and in elucidating their topology in biological membranes (16, 23, 29, 39).
Therefore, run-off RNA transcripts from the expression plasmid SP64-vpu+ encoding the wt vpu gene were translated in rabbit reticulocyte lysatc in the presence of
CMM. As
shown in Fig. SA, a 16 kDa translation product corresponding to Vpu was immunoprecipitated from the membrane pellet (lane 1). In contrast, the presence of Vpu in the supernatant fraction was barely detectable (lane 2). Since Vpu lacks a cleavable
signal sequence (39), an electrophoretic mobility shift should not be expected from microsoma1 vesicles containing the protein. Treatment of CMM with sodium carbonate
at alkaline pH has been shown to release secreted and pcripherai membrane proteins from microsoma1 membrane preparations (10). Thus, a translation mixture containing Vpu was treated with sodium carbonate at pH 11.0 and then centrifuged to separate
peripheral fiom membrane-bound components. As shown in Fig. 2A (lanes 3 and 4) Vpu was still predominantly detected in the pellet indicating that the protein is specifically membraneeassociated. Moreover, Vpu was found in the supernatant fraction when CMM were disrupted by the addition of detergents Nonidet P-40 (NP-40)(Fig. 2A; compare lanes 5 and 6) or Triton X-100 (Fig. 2A; compare lanes 7 and 8) after the
translation reaction, indicating that Vpu was membrane-bound. As previously reported (29-39)-these data dearly demonstrate that this in vim, system can be useful for testing
Vpu membrane association. Therefore, the effect of mutations introduced in the
hydrophobic N-terminal region of Vpu on the protein membrane association was next evaluated. As shown in Fig. 2B.equivalent levels of wt Vpu and mutated Vpu proteins were synthesized. Tbree mutations (Vpu A7, Vpu AlalO and Vpu Val2U) had the effect
of increasing Vpu's electrophoretic mobility (Fig. 2B; lanes 3-4, 7-8 and 11-12). However,the changes in cle~~~~phoretic mobility did not always corrtlatc with a loss of
membrane association (Fig. 2B; compare lanes 3-4, 7-8 and 11-12). Densitometric
scanning of the autoradiogram shown in panel 2B revealed that 90% and 80% of the vpu product from pSP64-vpu A7 and pSP64-vpu VaI20, respectively, were immunoprecipitated h m supernatant hctions suggesting that these mutated Vpu lost their ability to efficiently insert into CMM (Fig. 2C). All other Vpu mutants retained the ability to associate to membranes since they were predominantly detected in membrane-
containing pellet fractions (Fig. 2B; lanes 5, 7, 9 and 13). It is noteworthy that a vpu mutant encoding a 45 aa Vpu auncated at the C-terminus (Fig. 7) was translated in this system and shown to insert into CMM at a level comparable to that of wt Vpu (data not shown). Expression of wild-type and mutated Vpu proteins during HIV-1 replication, To assess the importance of the membrane-spanning domain of Vpu in the biological activities of the protein during HTV-1 replication, the mutations described
above were introduced into the infectious HXBH10-vpu + molecular clone (43). In an initial set of experiments, we analyzed the effect of the mutations on stability and intracellular localization of the Vpu products. For this purpose, the vpu mutant proviruse,~were transfected into MT4 cells. Forty-eight hours posmansfection, the mutated Vpu proteins were analyzed by pulse-chase labeling and immunoprecipitated as
described in Materials and Methods. As shown in Fig. 3A, all vpu gene products were
recognized by the rabbit anti-Vpu serum, albcit to different extent, Moreover, as observed in the in Vim system (Fig 2B),an incrcascd electrophoretic mobility of the mutated Vpu proteins expressed from HXBH10-vpu A7, HXBH10-vpu AlalO and HXBHlO-vpu Val20 proviruses was noticed in MT4 cells.
Quantitation of the
radioactive signals by densitomeuic scanning of the autoradiogram revealed that mutated Vpu proteins (Vpu Ile6, Vpu AlalO and Vpu fle16) which retain membrane association capacity were stable in MT4 cells. While the half-life (t 112)of wt Vpu was estimated herein to be approximately 5.5 h, the calculated t
JQ
of these mutated Vpu proteins
fluctuate between 5 to 5.5 h (Fig. 2B). In contrast, the stability of Vpu mutants impaired
in their capacity to insm into (SMM (Vpu A7 and Vpu Val.20) was significantly altered (t 1~ =
0.5 h) when compared to wt Vpu. Moreover, the lower level of Vpu Ma10
detected at t = 0 cannot be attributed to rapid degradation of the protein since this mutant
was as stable as wt Vpu during pulse-chase experiments. The increased electrophoretic mobility of Vpu AlalO rather suggests that the substitution mutations induced a conformational change which decreased the immunoprecipitation efficiency of the rabbit anti-Vpu serum.
Immunolocalization studies have previously showed that Vpu accumulates mainly in a perinuclear region (22) which corresponds to the Golgi complex (Yao and Cohen, manuscript in preparation). Therefore, we next evaluated the inaaceHular localization of the mutated Vpu proteins in transfccted MT4 cells by an indirect immunofluorescence technique using the rabbit anti-Vpu peptide serum (51). As shown
in Fig. 3C,specificfluorescence labeling of the wt Vpu (panel c) can readily be seen in a perinuclear region of the cytoplasm as an intense stain. In addition, some Vpu staining appear dispersed throughout the cytoplasm, spreading toward the plasma membrane. A similar pattern of perinuclear staining can be seen with the HXBH10-vpu Ile16 (panel
g), HXBH10-vpu Ile6 @anel e) and HXBH10-vpu AlalO (panel f) transfected cells,
although the labeling intensity of the la& was reduced. In contrast*no specific fluorescence labeling was observed in the HXBH10-vpu- (panel b), or in the HXBH10vpu A7 (panel d) and HXBH10-vpu Val20 (panel h) aansfectai cells suggesting that
membrane association and proper sorting may be ncccssary for the stability of Vpu.
Mutations in the hydrophobic N-termiaaI region affect Vpu-mediated enhancement of virion release. To analyze the effect of the mutations on Vpumediated enhancement of virion release, the amount of virus particles released into the
supernatant of each MT4 uansftcted cell culture was evaluated by initially measuring
the virus-sptcific reverse transcriptase (RT) activity at different intcrval of times (Fig. 4A). As previously reported (9-22, 39,43,52), RT activities were higher at a l l times in
the supernatant of HXBHIO-vpu+ transfected cell culture when compared to the cell culture transfected with HXBHIO-vpu- provirus which lacks expression of a Vpu
protein. At 62 h posttransfection, a sevenfold increase in the level of RT activity was measured. Proviruses expressing mutated Vpu proteins impaired in their capacity to insert into CMM (HXBHlO-vpu A7 and HXBH10-vpu V W ) (Fig. 2B),exhibited viral release phenotypes comparable to the HXBH10-vpu- provirus. Interestingly, at 48 h posttransfection the HXBH 10-vpu Ile6 provirus exhibited a partial Vpu-mediated enhancement of virion release (27% of wt Vpu) whereas HXBH10-vpu Ala 10 revealed a dramatic reduction in Vpu-mediated viral release. Subsequently, the HXBHlO-vpu Ile6
reached wt Vpu activity while only a slight increase in the level of RT in the H X B H I O vpu AlalO transfected cell culture was observed at 78 h posttransfection. Finally, HXBH10-vpu Ile16 and H X B H l h p u Ile24 (data not shown) cell cultures constantly
revealed levels of RT activity comparable to the cell culntre transfccted with H X B H I O vpu+ provirus. Similar results were obtained (i) in several independent experiments
ruling out the possibility of differential transfection efficiency between the cell cultures,
and (ii)were further reproduce in the HeLa expression system (data not shown) in which
the effect of Vpu on viral particle release was shown to be more pronounced than in Q)4+ T cell lines (9,13,52).
The Vpu-mediated enhancement of vmon release from the transfmed cells was further assessed by irnmunoprecipitsh'on of viral particles from the cuIture supernatants
as described in M a w s and Methods. Fig. 4B (left panel) shows that the Vpu product
was immunoprecipitated from lysates of cells transfected with HXBH10-vpu+, HXBH10-vpu A7, HXBH10-vpu IIc6, HXBHlOIvpu AlalO, HXBH10-vpu Ile16 and
HXBH10-vpu Val20 (lane 2-7, respectively) but not fiom lysate of cells transfected with the HXBHl0-vpu- provirus (lane 1). Furthermore, the intracellular concentrations of
Env glycoproteins (gp 160 and gp 120) as well as the Gag polyprotein p558ag and the processed capsid protein p24gag were comparable between the different cell cultures (lane 1-7, respectively). To evaluate the facilitation of virion release mediated by the expressed Vpu mutants, quantitative measurements of radioactive signals corresponding to viral proteins p248a8, pS58aB and p66RTassociated to pelleted virions were made by densitomemc scanning of the autoradiogram (Fig. 4B, right panel). As measured by densitometry, the amounts of major capsid protein p24W associated to pelleted fractions were increased by 4.2, 2.5 and 4.0 fold with HXBHIO-vpu+ (lane 2), HXBH10-vpu Ile6 (lane 4) and HXBH10-vpu Xlel6 (lane 6) respectively when compared to HXBHlO-vpu-. In contrast, as already shown with RT values, similar amount of viral particle-associated
proteins were detected in the supematants of MT4 cell cultures transfected with HXBH10-vpu- (lane I), HXBH10-vpu A7 (lane 3), HXBH10-vpu AlalO (lane 5) or HXBH10-vpu Val20 (lane 7). Despite the saiking differences in virion release, the amount of intracellular viral proteins were indistinguishable between the MT4 transfected cell cultures (Fig. 4B, Ieft panel). Several reports have shown that the expression of Vpu has no effect on the synthesis and processing of viral proteins (9, 13, 34), indicating that the results obtained with the latter mutated Vpu proteins are the
consequence of impaired Vpu-mediated enhancement of virion release.
These
observations were further confirmed in experiments in which p24Pg irnmunofluorescence staining was performed on transfcctcd MT4 cells following cycloheximide treatment (21). At 62 h postuansfection, an obvious accumulation of p 2 W staining at the surface of HXBH10-vpu-, HXBH10-vpu A7, HXBH10-vpu AlalO and HXBH10-vpu Val20 -transfccted cells was obsemed 6 h after cycloheximide
treatment (data not shown). This cell surface ~24808accumulation was not observed in cells transfected with HXBH10-vpu+ , HXBH10-vpu Ile6 or HXBH 10-vpu Xle 16. Electron microscopy studies have previously shown that the accumulation of p248r18 at the plasma membrane of cells infected with
vprr viruses correlates with v i r a l particles
closely tethered to the cell membrane (51). Taken together, these data suggest that the membrane-spanning domain of Vpu contains determinants which contribute to the facilitation of virion release.
Effect of the hydrophobic N-terminal region mutations on Vpu-induced CD4 degradation. Previous studies have demonstrated that motifs in the C-terminal domain of Vpu are essential for the ability of the protein to degrade CD4 molecules in the ER (4, 34). Interestingly, Schubert and Strebel (1994) have shown that phosphorylation of Vpu
is absolutely required for the Vpu-induced degradation of CD4 but has only a partial effect on the Vpu-mediated enhancement of virion release.
Thus, to assess the
importance of the hydrophobic N-terminal region of Vpu on CD4 degradation, we tested the induced degradation of CD4 molecules in the presence of gpl60 using the different
Vpu mutants. COS-7 cells were cotransfected with the CD4 expression plasrnid
SVCMV-CD4 and the different v p u mutant provirusts.
Forty-eight hours
posttransfection, cells were metabolically labeled for 30 min and chased for different intend of times. Newly synthesized Q)4 were immunoprecipitatcd fiom cell lysates with an anti-CD4monoclonal antibody and visualized by autoradiography. The results
shown in Fig. 5 are npresentarive of several independent experiments. Quaotitation of the radioactive signals by densitometcic scanning of the autoradiogram revealed that the
relative levels of CD4 were significantly reduced in the presence of Vpu (HXBH10vpu+) when compared to those detected in i s absence (HXBH10-vpu-) (Fig- 5). At least t h e e times less CD4 were imrnamopncipitatcd in the presence of Vpu throughout the
pulse-chase period. Since the t
min
ln of CD4 in the presence of Vpu is approximately 10
(a), the reduced level detected at the earliest time point indicates that CD4
molecules have already undergone Vpu-induced degradation during the pulse-labeling time as previously described by others (26, 45)- Similarly, mutated Vpu proteins expressed h m HXBH10-vpu b 6 or HXBH10-vpu AlalO induced CD4 degradation: a
2.5 to 3.0 fold CD4 decrease was measured throughout the 180 min chase when compared to the amount of CD4 detected in the absence of Vpu. Moreover, the relative
leveIs of CD4 detected in ceIls aansfectcd with the HXBH10-vpu A7 provirus were comparable throughout the pulse-chase experiment to those measured in the absence of Vpu. Thus, in contrast to their impaired capacity to enhance virion release, Vpu ne6 and
Vpu AlalO retained the ability to induce CD4 degradation in the ER.
Effect of the hydrophobic N-terminal region mutations on Vpu-mediated delay of HIV-1cytotoxicity. To evaluate the effect of Vpu N-terminalregion mutations
on HIV-induced cytopathic effects* replication kinetic studies were performed in the Q)4+Jurkat
cells. Parallel cultures of Jurkat cells were transfected with either
HXBH10-vpu', fIXBH10-vpu+ or HXBHlO-vpu mutant proviruscs. Cell cultures were
then monitored daily for up to 10 days postuanSfection for total viable cell number and appearance of syncytia as previously described (9,44,51). As previously reported (9.44, 51), the vpcc+ virus exhibited delayed cytopathic effect as compared to the vprr virus. As shown in Fig. 6A, there was a marked difference in the total number of viable cells between cultures transfected with
HXBH10-vpu- and HXBH10-vpu+ proviruses. The cell culture transfected with HXBH10-vpu A7 exhibited an overall cell viability panem similar to the cell culture
msfected with HXBHlhpu-. In contrast, cells transfecccd with HXBHlO-vpu Xle6 or HXBHIO-vpu AlalO continued to proliferate throughout the 10 days of replication kinetic, but, at a lower rate as compared to HXBHlO-vpu+ transfectcd cell culture. Funhermore, a difference in the rime-course of syncytium formation was obsemed between HXBHlOqu- and HXBH10-vpu+ transfed cell culturts (Fig. 6B). While, syncytia first appeared in HXBH10-vpu' culture 3 days posttransfection and reached a
maximum level by day 5 or 6, formation of syncytia was seen in HXBH10-vpu+ culture on the 4th day posttransfection and only reached a peak on day 7 and 8. The timecourse of syncytium formation o b m e d in the cell culture uansfected with HXBH10-vpu A7 was indistinguishable from the one seen in the HXBHIO-vpu- uansfected cell culture. In
contrast, the time-course of syncytium formation observed in cell cultures uansfected with HXBH10-vpu Ile6 or HXBH10-vpu AlalO was similar to that obsewed with
HXBHlO-vpu+ culture.
W e have previously suggested that Vpu-mediated delay of syncytium formation was the result of a two- to three-fold reduced accumulation of
Env gp120 at the surface
of cells infected with vpu+ viruses as compared to cells infected with vpu- viruses (9,37,
51). Thus, the level of Env gp120 expressed at the smface of transfected Jurkat cells was determined by flow cytometry. After staining, positive cells were selected and the
relative amount of cell surface Env gp120 was analyzed by FACScan as previously
described (51). As shown in Fig. 6C, a three-fold demcase in the Env gp120 mean fluorescence intensity was measured 7 days postuansfection in cells transfccted with
HXBHlkpu+ ( I ) provirus when compared to cells transfccted with HXBH10-vpu- (c) provirus. Cells transfected with the HXBH10-vpu A7 (d) expressed at the surface
similar level of Env gp120 when compared to HXBH10-vpu- transfected cells. In
contrast, cells transfecfcd with either HXBH10-vpu Ile6 (e) or HXBH10-vpu Ma10 ( f )
showed a reduced accumulation of Env gp120 at their surface, similar to that observed with HXBH10-vpu+ transfected cells.
DISCUSSION It has previously been suggested that Vpu has independent biological activities which show differential sensitivities to Vpu phosphorylation and requirt the presence of the protein at different sites within the cell (9,34). While previous mutagenesis analyses
of Vpu have attributed to the C-terminal domain a functional role in Vpu-induced CD4
degradation and -delayed H N - 1 cytopathic tfftcts (2, 4, 9, 3 4 , the importance of the Vpu membrane-spanning domain has not been addressed In order to gain turther insight into the mode of Vpu action, we thus investigated the structural requirements of the hydrophobic N-terminal region of Vpu for membrane association of the protein and its functional relevance in Vpu-associated activities.
Vpu is an integral membrane protein with the hydrophobic N-terminal region
spanning the lipid bilayer once and the protein's C-te~minalregion exposed to the cytoplasm (29,40). The results of our mutagenesis analysis of the N-terminal region of Vpu support and extend this model. Indeed, computer homology alignment from 15 different HIV-1 isolates revealed a high level of amino acid conservation (88%) between
residues 8 and 23 within the N-terminal hydrophobic region of the protein (data not shown). These sequences were funher shown to retain an a-helical configuration as predicted by a computer-assisted analysis. Both the deletion of seven amino acids encompassing residues Re 8 and Ala 14 (Vpu A7) which completely disrupts the putative N-terminal a-helical configuration of the protein and the substitution of residues Val 20,
Trp 22 and Ser 23 (Vpu V W ) , had a deleterious effect on the ability of Vpu to insert in vim into CMM (Fig. 2A). At this point, we cannot rule out the possibility that these
deletion and substitution mutations may have affected the o v d conformation of the Vpu products s h e increased electrophoretic mobility was observed using denaturating conditions. However, substitution mutations exhibiting similar electrophoretic mobility
changes (Vpu AlalO) or C-terminus auncarion (Vpu 45stop) (Fig. 7) did not result in a
loss of membrane association. These data indicate that the hydrophobic N-terminal region contains determinants sufficient for the insertion and anchoring of Vpu in
membranes, Fnnn a structural point of view, Vpu is a low molecular weight (16 kDa) integral membrane protein that lacks a cleavable signal sequence (39). The majority of proteins with an uncleaved signal sequence uses a signal-anchor (S-A) domain that saves to both
mrget the protein to the ER and act as a stoptransfer to stably anchor the protein into membranes (1 5, 18). Class I integral membrane proteins with S-A domain are poorly characterized The most common feature of class I membrane proteins of the S-A-type
is the absence or low number of positively charged amino acid residues at the N-terminal
side of the.hydrophobic segment (18). The proximal partion of the N-terminal sequence of Vpu illuseate such a feame (Fig. IA). Furthermore, it has been shown that the
presence of Trp residue at the interface of the N-terminal and C-terminal regions of class
I membrane proteins may llfillthe stop-transfer function and stabilize the a-helix with respect to the lipid bilayer (25). Interestingly, Trp at position 22 in the Vpu amino acid sequence is consewed in all *qu+ HIV-1isolates. The result of our in v i m experiment show that Vpu lost its ability to efficiently insert into CMM when this residue was
substituted in Vpu Val20 pig. 2A). Thus, this finding suggests that the distal portion of the N-terminalregion of Vpu may contain the S-A domain of Vpu. Additional structural analyses will be needed to characterize and delineate the signal and stop-transfer
functions of the hydrophobic region of Vpu. In this regard, Vpu may represent a good model to investigate the requirements for membrane targeting and insertion of single-
spanning class I S-A proteins* Conversely to the other mutated Vpu proteins which retain the ability to integrate into CMM (Fig. 2B-C),both the deletion of seven amino acids in Vpu A7 and
substitution mutations introduced in Vpu Val20 were shown to affect the stability and the localization of Vpu products in HIV-1infected cells (Fig. 3A-B). Most viral integral
membrane proteins, like Vpu, possess a single membrane-spanning domain composed of at least 20 hydrophobic residues which often adopt an a-heIical configuration within the hydrophobic interior of the Iipid bilayu (7). Studies have shown that mutations in the membrane-spanning domain or within the signal sequence can lead to the misfolding of membrane proteins in cells (1,33). These misfdded proteins arc retained in the ER and
are eventually degraded through mechanisms still poorly understood (8). Thus, it is conceivable that the abve mutations that prevented Vpu membrane association, resulted in misfolding and degradation of the mutated Vpu proteins. These results suggest that
membrane association is important for the stability of Vpu. In addition, as previously reparted by Chen et al. (1993) and as seen with Vpu A 7 and Vpu Val20 during HIV-1
replication experiments (Fig. 7), membrane association appear to be critical for a l l Vpuassociated activities.
The molecular basis of Vpu function has yet to be defined. Several studies have shown that Vpu induces a specific degradation of CD4 molecules in the ER (3,32,46, 47,48) and both the cytoplasmic and transmembrane domdns of CD4 were reported to
be required for this process (3,32). Recently, we have shown that a putative a-helical
structure in the proximal cytoplasmic region of CD4 contributes to Vpu-induced degradation (50). Funhermore, Vpu was shown to specifically bind CD4 molecules in the ER leading to the Vpu-induced CD4 degradation (2). The two molecules were
proposed to interact through their respective C-terminal cytoplasmic domains. In mutagencsis analyses of the C-terminal region of Vpu, Schubert and Strebel (1994) have
shown that phosphorylation of scrine residues (Ser 52 and Ser 56) was required for Vpuinduced CD4 degradation. In contrast, Vpu-enhancement of particle release was only partially dependent on Vpu phosphorylation (9,34). Using phosphorylation mutants that we have previously described (9) as well as C-terminal truncation mutants (Fig. 7), we
have c o n f i i e d these data and have shown that phospharylation mutants of Vpu lost the ability to delay syncytium formation during HTV-1 infection. These observations
suggested that Vpu contains two independent functional domains. The results of this study provide, now, evidence that the hydrophobic N-terminal region of Vpu contains
determinants critical to Vpu-mediated enhancement of virion release Wrn infected cells. In HIV-1 replication experiments, substitution mutations of amino acids Ile 6, Ile 8 and Val 9 in Vpu Ile6 and Ala 10, Leu 11 and VaI 13 in Vpu Ah10 impaired to different extent the capacity of Vpu to enhance virion release, while still being able to delay the cytopathic effectsof the HIV-1and to induce CD4 degradation (Fig. 7). In contrast to Vpu A7 and Vpu Va120,these mutations did not affect the ability of the protein to insen in vitro into CMM (Fig. 2AB). Funhermore, the m o v e r rate of Vpu ne6 or Vpu
AlalO were shown to be similar in T cells when compared to wt Vpu (Fig. 3A-B). In recent reports, the hydrophilic C-terminal domain of Vpu has been shown to form an ahelix-m-a-helix structure harboring the phosphorylation sites of the protein (35, 49).
This structural feature of wt Vpu was also prcdictcd in our computer-assisted analysis and was further retained in Vpu Ile6 and Vpu AlalO (data not shown). These
observations are consistent with the ability of these mutated Vpu proteins to induce CD4 degradation in the ER and to delay HIV-1 cytopathic effects (Fig. 7). Thus, the overall data indicate that the impaired Vpu-mediated enhancement of virion release observed with Vpu Ile6 and Vpu AlalO are most likely the result of specific alterations in the
membrane-spanning domain of the protein. This notion is especially apparent with Vpu Ile6 of which expression was indistinguishable from wt Vpu but had impaired activity in tenns of release function in MT4 and HeLa cells. Moreover, these results provide
additional evidence for two functionally distinct domains within Vpu. Apart from their anchoring role, membrane-spanning domains have been
implicated in sorting of membrane proteins (1,42). In this study, we cannot exclude the possibility that the mutations introduced in Vpu k 6 and Vpu AlalO may have affected their proper sorting. The exact inuacellular localization and secretory pathway of Vpu is still unclear. The protein accumulates mainly within the perinuclear region (22) which
corresponds to the GoIgi complex (Yao and Cohen, manuscript in preparation) and further retains a widespread vesiculpr pattern- Cell sulfact expression of Vpu has yet to be demonstrated. Recently, it was shown that retention of Vpu in the ER of infecred
cells by BFA treatment could efficiently inhibit Vpu-mediated enhancement of viral
release, indicating that this function requires the presence of the protein in different ceilular compartments (34). Thus, the phenotypes obtained with Vpu Ile6 and Vpu AlalO might have multed from failure to nach tbe proper cellular compartment fTom
which Vpu controls particle release.
However, this stems unlikely since our
immmo1ocalization analysis of both mutated Vpu proteins revealed similar pattern of perinuclear staining as compared to wt Vpu (Fig. 3 0 ,
.
The data accumulated so far is consistent with the notion that Vpu modifies
fundamental cellular pathways which may indirectIy contribute to the enhanced viral capsid release from infected cells (13, 22, 39,43, 51). Most notable, it was proposed that with respect to hydrophobicity and domain structure, Vpu exhibits homoIogies with the influenza virus M2 protein (22, 34). M2, a small integral membrane phosphoprotein
of 97 a.a., was recently shown to form a homo-tctrameric ion channel capable of modulating the pH of endosomal compartments in the mm-Golgi complex of infected cells (20,24,3 1,41). It has been suggested that a putative a-helical configuration in the M2 19-residue membrane-spanning domain forms the port region of the channel (20).
Mutations in this region of M2 were shown to alter ion channel activity of the protein by affecting the structure of the pore. There is no experimental evidence to date to suggest
that Vpu has an ion channel activity. Nevcnheless, it is tempting to speculate that the
integration of Vpu, mediated by its membrane-spanning domain, may promote the formation of complex structures which may dircctly o r indirectly influence the physical
properties of membranes. In chis regard, our preliminary studies performed on cell lines that stably express wt Vpu have revealed that expression of the protein can indeed affect
membrane fluidity (unpublished data). More detailed biochemical and structural
analyses will be needed to better characterize the effect of Vpu on membrane integrity and its relevance in the biological activities of the protein,
ACKNOWLEDGEMENTS W e want to thank Andrew Mouland, FIortnt Checroune, Ramu Subbramanian and Xiao-Jim Yao for helpful discussions, and Serge SCn6chal for technical assistance with the flow cytomemc analysis. We an grateful to Richard Axel for the pTAB plasmid
which was obtained through the AIDS Research and Reference Reagent Program,
Division of AIDS, NIAlD, NIH. J. F. and N.G. D. are recipients of studentships from the National Health Research and Development Program (NHRDP). Health and Welfare
Canada. E. A. C. is a recipient of a NHRDP AIDS career award This work was supported by grants from NHRDP and MRC to EAC.
LEGENDS
Fig. 1. Mutagen& of the predicted 81 amino add Vpu protein. (A) Ampbipathic profile of the Vpu protein exhibiting a strongly hydrophobic N-termid region and a Cterminal hydrophilic portion. The hydrophilicity plot was determined according to Kyte and Doolittle using the MacVector software. Tht amino acid sequence of the HXBHlO Vpu protein is indicated N-terminal sequences of Vpu predicted to adopt a-helical
configuration (a)arc underlined. A 12 aa. sequence highly invariant among Vpu proteins from diverse HIV isaIate is boxed- (B) Mutations were introduced into the predicted 27 a.a N-terminal hydrophobic region of Vpu by PCR-basedmumgenesis (see Materials and Methods). The deleted sequence (A) and altered amino acids are indicated in bold. The putative a-helical configuration is indicated by dashed boxes,
Fig. 2. In vitro translation of Vpu mutants in presence of canine pancreatic microsomal membranes.
(A) Run-off RNA transcripts from pSP64-vpu+ were
translated in a rabbit reticulocyte lysate in the presence of canine pancreatic microsomal membranes (CMM). FoUowing sedimentation by centrifugation, microsomal membranes pellets @) and supernatant fractions (s) werc collected, immunoprecipitated with an antiVpu serum and analyzed through 12.5% SDS-polyacrylamide gel. Treatments of CMM with 0.1 M sodium carbonate at pH 11.0 (lanes 3-4) or by addition of NP-40 (lanes 5-6)
or Triton X-100 (lanes 7-8) were also performed. (B) Run-off RNA transcripts from
pSP64-vpu+ (lanes 1-2), pSP64-vpu A7 (lanes 34), pSP64-vpu Ile6 (lanes 5-6). pSP64vpu AlalO (lanes 7-8), pSP64-vpu Ile16 (Ianes 9-10), pSP6evpu Val20 (lanes 11-12) and
pSP64-vpu De24 (lanes 13-14) werc translated in rabbit reticulocyte lysate in the presence of CMM. Vpu products in membrane pellets @) and supernatant hctions (s)
were immunoprecipitated with an anti-Vpu strum, separated on a 12.5% SDSpolyacrylarnide gel and visualized by autoradiography.
(C) Quantitation of
immunoprecipitated proteins was performed by densitomemc scanning of the
autoradiogram shown in panel B and expressed as percentage of immunoprecipitatedVpu associated with the membrane pellet fraction over total immunopt.eCipitatedVpu.
Fig. 3. Expression of wild-type and mutated Vpu proteins.
(A) MT4 cells were
uansfected with 10 pg of either HXBH10-vpu+ or HXBH10-vpu mutant proviruses. Forty-eight hours posttransfection, cells were pulse-labeled for 30 min with [3%]methioninc and chased fm the indicated rims. Lakled cells w a c l y s d and Vpu products were immunoprccipitated with a rabbit anti-Vpu peptide serum. immunoprecipitates were separated on a 12.5% SDS-polyacrylamide gel and visualized by autoradiography.
(B) Quantitation of immunoprccipitated Vpu products was
performed by densitomctric scanning of the autoradiogram shown in panel A. The percentage of Vpu products recovered from lysates of MT4 cells transfccted with either HXBHlO-vpu+ (a),HXBH10-vpu A7
(a),HXBH10-vpu Ile6 (H),
HXBH10-vpu AlalO
(A), HXBH10-vpu nel6 (A)or HXBHXO-vpu VaI20 (+) relative to the amount present at the start of the chase
(Z
= 0) was expressed as a function of time. (C) MT4 cells
transfected with either HXBH 10-vpu-(b), KXBH10-vpu+ (c), HXBH10-vpu A7 (d), HXBH10-vpu ne6 (e), HXBH10-vpu AlalO (f), HXBH10-vpu ne16 (g) or HXBH10-vpu Val20 (h), were Iayered on slides and fixed with ethanoVacetone (75%/25%) solution.
The ceIls were subsequently reacted with a rabbit anti-Vpu peptide serum and stained with FITCconjugated goat anti-rabbit IgG. The mock-transfected MT4 cells are shown
(a).
Fig. 4. Effmt of mutations in the N-terminal hydrophobic region of Vpu on the enhancement of virion release. (A) Parallel cultures of MT4 cells (5 x 106) were
transfccted with 10 pg of either HXBH10-vpu' (o), HXBHlO-vpu+ (a),HXBH10-vpu
A7 (0). HXBH10-vpu Ile6 (H), HXBH10-vpu AlalO (A), HXBH10-vpu ne16 (A)or HXBHlevpu Val20 (+) provirus. At each posttransfection time indicated, cell free supernatants were collected and virus-specific reverse transcriptase (RT) activity was
measured, RT activity in mock-transfecttd supernatant is indicated by (x). (B) Fortyeight hours posttransfection, viral expression and production was detcnnincd by labeling cqual number (106)of viable MT4 cells with [3%]rnethionine
for 5 h. Labeled d s were
lysed and cell-associated viral proteins were immunoprccipitated with both an HIV-1 positive human serum and an anti-Vpa serum. Immunoprecipitates were separated on a 12.5% SDS-polyacrylamide gel and v i s d i z c d by autoradiography. For each sample,
equal amount of labcling medium was collected and viral particles wtrt peUetcd through a 20% sucrose cushion by UltracentrifUgation. Pelletd materials were lysed in laemmli sample buffer. Virion-associated proteins were directly separated on a 12.5%SDSpolyacrylamide gel and visualized by autoradiography.
Fig. 5. Effect of mutations in the N-terminal hydrophobic region of Vpu on CD4
degradation. COS-7 cells were comsfected with a mixture of 5 pg of CD4 expression plasmid SVCMV-CD4and 20 pg of either HXBHlO-vpu-. HXBH10-vpu+ or HXBH10vpu mutant proviruses. Forty-eight hours posrtransfction, cells were pulse-labeled for 30 min with :f%]methionine and chased for the indicated times. Labeled cells were
lysed and CD4 was immunoprecipitated with an anti-CD4 (OKT4) monoclonal antibody, separated on a 12.5% SDS-polyacxylamide gel and visualized by autoradiography. Fig. 6. Effect of mutations in the N-tennind hydrophobic region of Vpu on HIV-1-
mediated cytotoxicity. (A) Total viable cell counts in Jurkat ceU cultures transfected with 10 pg of either HXBH10-vpu' (o),HXBH10-vpu+ (a), HXBH10-vpu A7
((OX
HXBHl@vpu Ile6 (I or) HXBH10-vpu AlalO (A) provirus, 6r mock-transfected (x) were determined daily. (B) Transfected Jurkat cell cultures wcrc periodically monitored for
syncytium formation by counting the number of syncyria as follows: +, 1 to 2 syncytia per ( 1 0 0 ~ field ) in a culture of 1 x 106 cells/ml;
++, 3 to 5 syncytia per field; t++, 5 to
10 syncytia per field; ++++, 10 to 20 syncytia per field; + - ~ t t>20 , syncytia per field.
The data represent an average of 5 fields. (C) Effect of Vpu mutants on the level of Env
gplu) expressed at the surface of infected cells. Jurkat cells transfccted with 10 pg of either HXBH10-vpu+ @), HXBH10-vpu- (c), HXBHlGvpu A7 (d), HXBHlO-vpu Ile6
(e) or HXBHlO-vpuAlalo (0provirus wcre collected on ice 7 days posaransfection and washed twice with cold PBS. Cells w e n then incubated at 4'C with anti-gpl2O
monoclonal antibody for 1 h and stained with FITCumjugated goat anti-mouse IgG for
30 min. The relative fluorescence intensity of Env glycopteins gpl2O expressed at the cell surface was evaluated by flow cytofluometry (FACS). The mock-aansfected Jurkat
cells are indicated (a). Fig. 7. Structural and functional analyses of the Vpu protein. A series of deletion, substitution and mncation mutations wcre introduced into the vpu gene by PCR-based mutagenesis. These mutations targeted the N-terminal hydrophobic region (hydrophobic
mutants) and the C-terminal region (phosphorylation mutant and truncated mutants) of the 81 a.a. Vpu protein.
The effect of the mutation on the putative a-helical
configuration according to the Robson-Gamier algorithm method are shown by the length of dashed box. Mutational substitutions of serine residues (S) which were
identified as the phosphoacceptor sites of Vpu are indicated (*). The vpu mutants were
introduced into the infectious HXBH10-vpu+ mo~ecula.clone and analyzed for all known biological activities of Vpu. The phenotypes of the Vpu mutant proteins
obtained were compared to the wild-type Vpu protein. Data not determined are indicated (ND).
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viruses in infected cells. Science 225: 381-385. Strebel, K, Klimkait, T, Maldarelli, F., and M. A. Martin. 1989. Molecular and biochemical analyses of human immunodeficiency virus type 1 vpu protein. J. Virol. 63: 3784-379 1.
Strebel, K., Klimkait, T, and M. A Martin. 1988. A novel gene of HIV-1, vpu, and its 16-kilodalton product. Science 241: 1221-1223.
Surgrue, Re J., and A. J. Hay. 1991. Structural characteristics of the M2 protein of influenza A viruses: evidence that it forms a tctrameric channel. Virology
12Uk 617-624. Swift, A. M., and C. E. Macbamer. 1991. A golgi retention signal in a membrane-spanningdomain of Coronavirus El protein. J. Cell. BioL 112: 19-30. Terwilliger, E. F.9 Cohen, EoA., Lu, Ye-C., Sodroski, J. G., and W. A. Haseltine. 1989. Functional role of human immunodeficiency virus type 1 upu.
Roc- Natl. Acad. Sci. USA 86: 5 163-5 167.
Terwilliger, Eo, Burghoff, R, and R Sia. 1988. The an Gene product of The
Human Immunodeficiency Virus is Required for replication. 3. Virol. 62= 655658.
Vincent, M. J., Raja, N. U., and M. A. Jabbar.
1993.
Human
immunodeficiency virus type 1 Vpu protein induces degradation of chimeric
envelope glycoprotcins bearing the cytoplasmic and anchor domains of CD4: role of the cytoplasmic domain in Vpu-induced degradation in the endoplasmic
reticulum. J. Virol, 67= 5538-5549. Willey, Ro, Buckler-White, L. 11,and K. Strebel. 1994. Sequences present in the cytoplasmic domain of CD4 are necessary and sufficient to confa sensitivity to
the human immunodeficiency virus type 1 Vpu protein. J. Virol.
=1207-1212.
Willey, R L., Maldarelli, Fo, Martin, M. Am,and K. Strebel. 1992. Human
immunodeficiency virus type 1 Vpu protein induces rapid degradation of CD4. J. V i d . 66: 7 193-7200. Willey, R. L., Maldarelli, Fo, Martin,
M. A, and K. Strebel. 1992. Human
immunodeficiency virus type 1 Vpu protein regdates the formation of intracellular
gp 160-CD4complexes. J. Virol. 66: ZZG234.
Wray, V., Federau, T, Henklein, P., Kalbunde, S., Kunert, O., Schomburg,
D, and U. Schubert. 1995. Solution structure of the hydrophilic region of HIV-1 encoded virus protein U (Vpu) by CD and H NMR spectroscopy. Int. J. Peptide Protein Res. 45: 35-43.
Yao, X.-J., Friborg, Jo, Checroune, F.9 Gratton, S., Boimert, F., SOkaly, It. P., and Eo A. Cohen.
1995.
Degradation of CD4 induced by human
immunodeficiency virus type 1 Vpu protein: a predicted alpha-helix structure in the proximal cytoplasmic region of CD4 contributes to Vpu sensitivity. Virology
51.
Yao, X. J, Ganon, S, Boimert, F., Haseltine, W.A*,and E. A. Cohen. 1993.
The effect of vpu on HIV-1- induced syncytia formation. J- AIDS dt 135-141. 52.
Yao, X J, Gtlinger, H , Haseltine, W. A, and E. A. Coben. 1992. Envelope &coprotein and CD4 independence of vpu facilitated HIV-1 capsid export. J. Virol. 66: 5 119-5126.
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Article 3:
Degradation of CD4 induced by human immunodeficicency virus type 1 Vpu protein: a predicted alpha-helix structure in the proximal cytoplasmic region of CD4 contributes to Vpu sensitivity.
Degradation of CD4 Induced by Human Immunodeficiency Virus Type 1 Vpu Protein: A Predicted Alpha-Helix Structure in the Proximal Cytoplasmic Region of CD4 Contributes to Vpu Sensitivity XIAO-]IAN YAO.' JACQUES FRIBORG.' FLORENT CHECROUNE.' SOPHIE GRATT0N.W FRANCOISE BOISVERT.' RAFICK P. SEKALY.~and ERIC A COHEN'.' ':.?Somrare de Rerrovirologie Humaine. D&anernent de Microbiologie er Irnmunologie. Facuh&de MtMecine. Uniwmir8 de Monrr&a/. CP 6128. Srarron A. Monrreal. Quebec. Canada. H3C 317: tL8boraroire dlmmunologie. Instirut de Recherches Cliniques de Mont&8/. 1 rO Awe. des Pins Ouesr. Monrr&al. Qu&bec. Canada. H2W 1R7: and SDiM'sion of &~eninenral Medicine, McGill University. Mofl~&l. Quebec. Canada. H3G rAc Received Febnraly 16. 1995: accepted
March
74. 1995
m e HIV-lencoded Vpu protein induces a rapid and specific degradation of C04 molecules in the endopfasmic reticuium (ER). In this study. Vpu-induced degradation of CD4 in the ER was investigated by quantitative immunoprecipimtionof CD4 following cotransfemion of COS-7 cells with CD4 and Vpu expressors i n the presence of brefeldin A a drug that blocks protein transpon from the ER to the Golgi complex 19 order to precisely define me sequence(s)or structural element(s) in the CD4 cytoplasmic domain necessary for Vpu-induced degradation. a panel of deletion and s u b ~ o mutants n in the cytoplasmic domain of CD4 was generated and anatyzed. In agreement with previous reports. our deletion analysis indicates that a region encompassing amino acids 411 to 419 (KRUSEKKT) in the cytoplasmic domain of CD4 was required to confer Vpu sensitivity. However. Six specific substitution mutations within this region did not confer C04 resistance to Vpu. suggesting that neither the amino acid sequence nor the charge of the amino acids in this region wss critical to Vpuinduced CD4 degradation. A dileucine motif that is imporcamfor intemaiiirion of CD4 and Nef-induced CD4 down-regulation two substitution m u t a m (CDQEMKL and was also not required for Vpu-induced CD4 degradation. Interestingly. C04MK407.11 PP)located in a more proximal cytoplasmic region of CD4 abolished Vpu-induced CD4 degradation. Computerassisted analysis of the substitution and delm-on mutants conferring CD4 resistance to Vpu-induced degradation indicated that these mutations disrupted a putative alpha-helix formed in the proximal cytoplasmic region of C04. Taken together. these studies strongly suggest that a struCtura1 element in the proximal cytopfasmic region of CD4 contributes to Vpu sensitivity. c 1995 A a d e m i c Press. ~ n e
INTRODUCTION
CD4 is an integral membrane glycoprotein found mainly or;the surface of T-helper cells and monocytes (Maddon er aL. 1985). The cytoplasmic domain of CD4 interacts w~thp56'Ck. a member of the src family of cytosolic tyrosine kinase. and this complex pIays an irnponant role in T-cell activation (Glaichenhaus et aL, 1991: Shaw er a/., 1989; Veiltette eta/., 1988). Previous studies have shown that a region at the C-terminal end oi CD4 encompassing Lys418 to He433 is involved in the binding with p56"* (Shaw et a/., 1989). The proximal membrane region of that cytoplasmic domain, in which a putative alpha-helix structure has been predicted, is necessary for CD4 internalization and lysosomal targeting (Shin et a/.,1991). In addition to its involvement in T-cell activation. the CD4 molecule is also the primary receptor for the human ~mmunodeficiencyvirus (HfV) as the result of the highaffinity binding of the viral surface envelope glycoprotein
' To
wnom correspondence and reprint requests should be ad-
cressec. Fax: (514) 343-5985.
gp120 to the external domain of CD4 (Dagleish et a/.. 1984). Several studies have shown that HIV down-regulates the cell surface expression of CD4 during infection. This down-regulation appears to be caused by several viral proteins. Previous in virro studies have shown that the binding of HIV envelope glycoprotein precursor gp160 to CD4 molecules can retain CD4 in the endoptasmic reticulum (ER) and can efficiently block the transpon of CD4 to the plasma membrane (Crise er a/., 1990: Jabbar and Nayak. 1990). Recent studies have shown that the HlV-1 regulatory protein Nef could mediate the downregulation of surface CD4 expression in infecred cells. This Nef-induced down-regulation of CD4 does not appear to be the result of an effect at the level of transcription Or protein synthesis (Garcia and Miller, 1991). Indeed, a recent study has shown that Nef acts by promoting CD4 endocytosis and degradation in iysosomes (Aiken eta/., 1994). In addition, several reports have now shown that the HIV-1 Vpu protein, in conjunction with gp160, mediates a rspid degradation of CD4 in the ER and as a result may cause reduced levels of cell surface CD4 (Willey er a/., 1992a.b). 0042-6822'95 56.00 Coovrbgni C 1995 n y
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YAO ET A L
The Vpu protein is encoded by an open reading frame which is located in the central region of the HIV-1 ge-
nome and panially overlaps the 5' end of the envelope reading frame (Cohen et at., 1988). Biochemical studies have shown that Vpu is a class 1 integral membrane protein in which the N-terminal hydrophobic region of 27 ammo acids (aa) presumably constitutes the membrane anchor domain. The C-terminal hydrophilic region contains a highly conserved 12-aa sequence (residues 47 to 58) facing the cytoplasmic side and may represent an active site of this protein (Strebel eta/., 1989; Maldarelli et al.. 1993).This protein is post-translationally phosphorylated by casein kinase I1and is able to form homooligomers (Strebel et at.. 1989: Schuben and Strebel. 1994; Frrborg eta/., 1995). Phenotypic studies indicate that Vpu has several functions during HIV-1 infection of CD4' cells CTerwilliger et a/.. 1989: Willey et at., 1992a; Yao et a/.. 1992. 1993). Vpu can disrupt HIV-1 gp160-CD4 complexes in the ER by inducing COLs degradation (Willey et 21.. 1992a.b). Vpu also facilitates the release of virions from infected cells in a manner which is independent of ;he presence of HIV envelope glycoproteins and CD4, suggesting that the Vpu protein has a t least two independent functions (Yao et at.. 1992). Moreover. recent studies suggest that different activities of Vpu are regulated by the phosphorylation state of the protein (Schuben and Strebel, 1994; Friborg et at.. 1995). Vpu-induced CD4 degradation has been shown to reourre the retention of CD4 in the ER either via the trapping of CD4 by gpl60 or by the treatment of cells with brefeldin A (BFA) Nilley et a/.. 1992b). a drug that blocks the transpon of proteins from the ER to the Golgi complex Several sTudies using CD4 and the CD8-C04 hybrid have indicated that the cytoplasmic domain of CD4 is the target cf Vpu-induced degradation and thaz this effect is independent of the phosphorylation siare of CD4 (Chen et at., 1993: Lenburg and Landau, 1993; Vincent et at.. 1993: Willey et a/.. 1994). Deletion studies have revealed that the segment LSEKKT (amino acids 414 to 419) identified by Vincent er 81. (1993) and the overlapping segment EKiCTCQC (amino acids 418 to 424) identified by Lenburg and Landau (1993) located in the cytoplasmic domain of C34 represent sequences responsive to Vpu. However. recent studies using HIV gp160-CD4 or Vesicular Stomatitis virus glycoprotein-CD4 chimera constructions revealed that both the transmembrane and the cytoplasmic domains of CD4 are required for the sensitivity of CD4 to Vpu-induced degradation (Buonocore er a/., 1994; Raja eta/.. 1994). These apparent conflicting results may be explained by the presence of related sequences or structures in the transmembrane domains of CD4 and CD8 that are involved in Vpu-induced degradation. In the present study, we investigated the sequenceb) or structural elernent(s) of the CD4 cytoplasmic domain which contributes to Vpu-induced degradation. Our muta-
tional analysis indicates that a computer-predicted alpha-helix structure located in the proximal cytoplasmic region of CD4 contributes to Vpu sensitivity.
MATERIALS AND METHODS DNA constructions SVCMVCD4 was constructed by insening a XbalSmaI cDNA fragment encoding CD4 into the corresponding sites of the expression vector SVCMVexpa which contains a SP64 polylinker (Promega) flanked by a cytomegalovirus (CMV) immediate-early gene promoter (Jeang et a/.. 1987) at the 5' end and a simian virus 40 (SV40) poly(A) signal and small T antigen intron at the 3' end. The CD4 cDNA was derived from the pT4B expressor (Maddon et a/., 1985). The Vpu expressor SVCMWPU' and the negative control plasmid SVCMWPU' used in this study were described previously (Yao et a/., 1992). CD4 deletion mutants SVCMVCD4A7, SVCMVCD4Al4, SVCMVCD4A23. and SVCMVCD4P32 were constructed by PCR using the 5' primer (5'-CTGGAGCTCCAGGATAGT-3') which includes a natural Sad site (nucleotide position 598; +1 = site of transcription initiation) in the CD4 cDNA and the 3' primers in which the CD4 amino acids Gln427, Cys420, Lys411. or Arg402 were. respectively, replaced by a stop codon followed by a Sacl site. The FCR fragments were then digested with Sad and cloned into the corresponding site in SVCMVCD4. CD4 substitution mutants were generated using a twostep PCR-based method that was described previously (Ho er a/., 1989). A 5' primer identical to the one described above was used for the construction of all mutants. The 3' primer (5'-GAAACTAGAGCTCPPICACTCAA3') was derived from the CD4 3' noncoding region and included a Sacl site, Complementary oligonucleotide primers containing the desired mutations were used to generate the CD4-mutated fragment by PCR. The nucleotide sequences of the mutagenic oligonucleotide pairs were synthesized as follows: CD4LL413.4AA: sense, 5'-GATCAAGAGAGCCGCTAGCGAGAAGAAG-3', antisense. 5' -CTTCTTCTCGCTAGCGGCTCTCTTGATC3'; CD4KK417.8lG: sense, 5'-TCAGTGAGATTGGTACCTGCCAG-3'. antisense. 5'-CTGGCAGGTACCAATCTCACTGA-3'; CD4EM405.7CD: sense, 5'-GGCGCCAAGCTTGTCGGGATTCTCAGATCA-3'. antisense. 5'-TGATCTGAGAATCCCGACAAGCTGGCGCC-3'; CD4Kt411.3TP: sense, 5'-TCTCAGATCACGCGTCCCCTCAGTGAG-3'. antisense, 5'-CTCACTGAGGGGACGCGTGATCTGAGA3'; CD4M407P: sense. 5'-AAGCAGAGAGGCClTCTCAGATC-3'. antisense. 5'-GATCTGAGAAGGCCTCTCTGCTT3'; CD4K411P: sense. 5'-TCTCAGATCCCGAGACTCCT3'. a ntisense. 5'-AGGAGTCTCGGGATCTGAGA-3'. The CD4EMKL and CD4MK407.11PP mutants were created by hybridizing the firsr PCR reaction fragments from CD4-
c
DEGRADATION
EM405.7CD and CD4KL411.3TP or CD4M407P and CD4K41 1P. respectively. A fragment containing the two sets
of mutations was generated in a second PCR reaction using the 5' and 3' primers described above. All of the PCR fragments generated after the second reaction were digested with Sac1 and cloned into the SVCMVCD4 expressor to yield CD4LL413.4AA; CD4KK417,aIG; CD4EM405,7CD; CD4Kt411,3TP; CD4M407P; CD4K4'11P: CD4EMKL and CD4MK407.11 PP expression plasmids. CD4CC420.2AA and CD43S were constructed by replacing the Sac1 fragment of SVCMVCD4 with the PCR-generated fragment from pMNCCD4CC420.2AA and pMNCCD43S. The pMNCCD43S mutant was generated by two-step PCR. First. the serine residue at position 431 was mutated to an alanine by using a 5' and 3' primer complementary to MNC vector sequences. as described before Oremblay et a/.. 1994). and a pair of complementary oligos coding for the desired mutation: sense, 5'-AAGACATGTGCCCCCATTTGA-3': antisense, 5'-TCAAATGGGGGCACATGTCTT-3'. Second. Ser408 and Ser415 were mutated to alanines using the CD4 cDNA mutated at position 431 and complementary oligos coding for the new substitutions: sense, 5'CGGATGGCTCAGATCAAGAGACTCCTCGCTGAGAAG-3'. antisense. 5'-CTTCTCAGCGAGGAGTCTC-TTGATCTGAGCCATCCG-3'. The fulllength mutated cDNA was subcloned into the HindlllBarnHl sites of the MNC vector. pMNCCD4CC420.2AA has been previously described (Tremblay et a/., 1994).
Metabolic labeling and radioimmunoprecipitation Forty-eight hours post-transfeaion. transfected cells were pretreated with 10 mM %FA for 30 min and metabolically labeled with 200 pCi rsS]methionine for 5 hr in the presence of BFA Radiolabeled cells were then washed with PBS and lysed on ice in radioimmunoprecipitationlysis buffer (140 mM NaCL. 8 m M NaHP04.2 m M NaH2P04,1% Nonidet P-QO.0.5% sodium deoxycholate. 0.05% SDS). Total cell lysates were immunoprecipitated with OKT4 monoclonal antibodies. In some experiments, cell lysates were subsequently immunoprecipitated wm a rabbit anti-Vpu serum. lmmunoprecipitates were then analyzed on a 10 or 12.5% SDS-polyacrylamide gel followed by autoradiography- Densitometric analysis of autoradiograms was performed with a Molecular Dynamics Personal densitometer using ImageQuant software version 3.22 Protein secondary structure analysis by computer The secondary structure of the CD4 protein was analyzed by using MacVectorsequence analysis software from Internationat Biotechnologies Inc. (New Haven. CT) and ANTtfEPRO sofware from the Instim de Biologie et Chimie des Proteines (France) (Geourjon and Deleage, 1993). The secondary structure prediction of proteins by these softwares is based on the Robson-Gamier and Chou-Fasman rnethods (Chou and Fasman. 1978; Gamier er al.. 1978). RESULTS
Cell lines and transfections COS-7. an African green monkey kidney cell line transformed by an origin-defective mutant of SV40, was cultured in Dulbecco's modified Eagle medium (DMEM) (GIBCO Laboratories). For transfection. one million COS7 cells were seeded into a 100-mm petri dish and cuftured overnight in DMEM containing 1O%fetal calf serum. Cells were then cotransfected with a mixture of 5 p g CD4 expressor and 12.5 p g Vpu expressor (SVCMWPU' or SVCMWPU-, molar ratio 1:3) by the calcium-phosphate method. Antibodies and chemical compound The anti-CD4 (OKT4) monoclonal antibodies were derived from ascitic fluids of Balbk mice that were injected wiTh an 0KT4 hybridorna. The OKT4 hybridomz was obtained from the American Type Culture Collection (ATCC, Rockville. MD). Rabbit anti-Vpu serum was raised by immunization of rabbits with a synthetic peptide corresponding to amino acids 73 to 81 of the BH10 Vpu protein (Cohen er a/., 1988). BFA was obtained from Sigma Chemical Co. and slored as a stock solution of 10 m M in ethanol at -20°.
Effect of truncations of the cytoplasmic domain of CD4 on Vpu-induced degradation Previous studies have shown that Vpu stimulates the degradation of C04 molecules in HeLa cells as well as in vitro in rabbit reticulocyte lysate in the presence of canine microsomal membranes (Willey et a/., 1992a; Chen er at-. 1993). To identify sequence@) or structural element(s) within the CD4 cytoplasmic domain which contributes to Vpu-induced degradation. we constructed four truncated mutants at the carboxyl Terminus of CD4 by PCR (Fig. 1A). Stop codons were introduced at positions 402. 41 1. 420. and 427 of CD4 to generate the truncated CD4 expression plasmids CD4A32, CD4023. CD4A14. and CD4A7, respectively. Each CD4 mutant was analyzed in COS-7 cells in the presence or absence of Vpu as described under Materials and Methods. Preliminary results clearly showed that Vpu-induced degradation of CD4 could be observed in COS cells. Quantitation of the rate of CD4 decay in these cells by pulsechase labeling indicated that the half-life of CD4 was less than 30 min in the presence of Vpu, whereas similar amounts of CD4 were maintained for up to 5 hr in the absence of Vpu (data not shown). The data in Fig. 10 show that mutants harboring deletion of, respectively. the last 7 and 14 aa from the C-terminus of CD4 were
activities in T cells. Leucines at positions 41 3 and 414 were shown to be imponant for the internalization and lysosomal targeting of CD4 molecules (Shin et a/.. 1991). Recently. this motif has also been shown to be critically required for Nef-induced down-regulation of CD4 (Aiken eta/., 1994). Moreover, the protein kinase C-mediated phosphorylation which occurs mainly on the serine at position 408 is proposed to constitute one of the internalization signals (Shin er at., 1991). In addition. the two cysteine residues located at positions 420 and 422 have been shown to associate with the p56ICkkinase to form a complex which is known to play a critical role in the T-cell antigen receptormediated signaling pathway (Shaw et at., 1990). Our results show that the sequence KRLLSEKKT, located in rhe cytoplasmic domain of CD4. is required for sensitivity to Vpu-induced degradation. This segment also contains the signal for the internalization and lysosomal targeting of CD4 and for Nef-induced down-regulation of CD4 (Aiken er at.. 1994: Shin eta/., 1991). To investigate whether some important amino acids. especially i n the KRLLSEKKT segment. are required for Vpu-induced C04 degradation. we conFIG. 1. Etiec, of atletion of me cytoplasmic dOmatn of CD4 on Vpustructed a series of CD4 substitution mutants. As lneuced aegraaarlon. The svuaure and amino acid sequence of the cfloolasmtc aomam of CD4 are shown at the top of A Plasmids exoressshown in Fig. 2A. serines at positions 408, 415. and mg CD4 w~thCrfferentcytoplasmic truncanons. a s indicated in (A). were 431; leucines at positions 413 and 414; or cysteines coaansfened In COS-7 cells wfth SVCMVVPU' or with SVCMWPUat positions 420 and 422 were. respectively, substiexpressers. 2s ~ n a c x e drn (0).Forry-eight hours pas-nansfecrion. cells tuted for alanines. Also. lysines at positions 417 and were ctrerreated wltn 10 p M BFA prior to and during the labehng wim 418 were substituted for isoleucine and glycine. [i5S]meth~onrnc.The labeled cell Mates were immucopreciDitatecY with :he OKT4 mrrnoclonal antiCD4 antibody and separated by e l € ? c t r ~ g h ~ e - These substitution mutants were tested in COS cells sls on SDS-~lyacrylamide gel followed by auforadiography. in the presence or absence of Vpu. The results of Fig. 28 show that none of these CD4 mutants lost their still sensitive to Vpu-induced degradation. Densitometric sensitivity to Vpu-induced degradation. In addition, scanning of the autoradiograms shows that. as for the the CD4 substitution mutant (KL411.3TP). in which the wild-rype CD4, the amounts of CD4A7 and CD4A14 in positively charged lysine at position 41 1 and the leuThe absence of Vpu were between 2.5 and 4.0 times cine at position 413 were changed to threonine and hisher than that in the presence of Vpu (Fig. 18, compare proline. respectively. still exhibited sensitivity to ?he lanes 1. 3. and 5 to lanes 2,4, and 6). Interestingly. when presence of Vpu (Fig. 38, compare lane 5 to lane 6). the last 23 or 32 aa were deleted from the C-terminus, These results suggest that the resistance of CD4 to both of the resulting truncated CD4s became resistant the Vpu-induced degradation upon deletion of the last to t h e effect of Vpu (Fig. 1%.compare lanes 7 and 9 to 23 amino acids from the C-terminus i s unlikely to relanes 8 and 10).These results suggest that the sequence sult from the loss of a specific amino acid sequence KRLLSEKKT, located between amino acids 41 1 and 419, or charges present in the segment of KRLLSEKKT. contains a signal which is required for Vpu-induced degIndeed. substitution of 6 of 9 amino acids in this segradation. This region overlaps the previously described ment of CD4 still exhibited sensitivity to the Vpu-medireglons LSEKKT and EKKTCQC. which have been shown ated degradation. to be responsible for Vpu-induced degradation in different experimental systems (Vincent et at-, 7993: tenburg Substitution mutants located in the proximal and Landau, 1993). cytoplasmic region of CD4 are resistant to VpuCD4 mutants harboring amino acid substitutions within the KRLLSEKKT segment remain sensitive to Vpu-induced degradation Several amino acids in the cytoplasmic domain of CD4 have been shown to be critical for CD4 biological
induced degradation In order to determine more precisely the CD4 cytoplasmic domain that is responsive to Vpu-induced degradation, we generated. as shown in Fig. 3A. five additional substitution mutants (CD4EM405.7CD;
d
620
YAO
ET
KK417.81G; CD4EM405.7CD: CD4KL411.3TP; CD4M407P; and CD4K411P) either did not exhibit dramatic changes in the predicted alpha-helix conformation (C043S (data not shown); CD4CC420.244 (data not shown); CD4LL413.4AA; CD4KK417.81G; CD4KL41t .3TP; and CD4K411P) or retained pan of this alpha-helix conformation (CD4014: CD4EM405.7CD; and CD4K407P). Interestingly, the C04 EMKLand CD4MK407,ll PP. which were completely resistant to Vpu-induced degradation were unable to retain any alpha-helical conformation in the proximal region of the CD4 cytoplasmic domain. These data suggest that maintenance and/or correct folding of this predicted alpha-helix structure in the proximal cytoplasmic domain of CD4 contributes to C 0 4 sensitiviry to Vpu-induced degradation. DISCUSSION
FIG. 4. Effect of deletion and substitution mutarions in the cyto-
Dtasrnlc domarn of CD4 on rhE pred~ctecalpna-helix conformation. The amrnc acid seuuence of rhe human CD4 cytoplasmic domain and the 3"smon of the alpha-hellx. &sneer and B-turn predicted by computer analyscs are represented at the top of the figure (RG.Robson-Gamier T e i h O C ; CF. Chou-Fasman method]. The effecrs of different deletion and su3st1tuf10r.rnutanons In CDe on the aredined alpha-helix conforas oredicted by the RG method and confirmed by the ANranon) . ( THEPRO program are ~ndicated.
Mutations which confer CD4 resistance to Vpuinduced degradation disrupt a putative alpha-helix structure located between positions 402 and 419 in the membrane proximal region of the CD4 cytoplasmic domain Computer analysis of the CD4 cytoplasmic region predicted the presence of an alpha-helix structure extending from Arg 402 to Thr 419 (Fig. 4). The potential presence of such a conformation has already been reported by Shin el a/. (1991). Indeed, this predicted alpha-helix was reponed to be imponant for endocytosis and lysosomal targeting of CD4 (Shin et at., 1991). To investigate the effect of CD4 truncations and substitution mutants on the conformation of this structure. we performed a computerassisted structural analysis of the mutants described in this study using MacVector software. As shown in Fig. 4, this analysis demonstrates that deletion of 14 amino acids from the C-terminus of CD4. which retained Vpu sensitivity, affects the distal p a n of the alpha-helix structure. Conversely, deletion of 23 amino acids in the Cterminus of the CD4 cytoplasmic domain, which abrogates Vpu-induced degradation, totally abolished the formation of this putative alpha-helix. All CD4 substitution mutants that remained sensitive to Vpu-induced degradation (CD43S; CD4CC420.2A.A: CD4LL413.4AA; CD4-
In addition to the envelope glycoproteins and the Nef protein. HIV-1 encodes another protein, Vpu. which down-regulates the expression of its cellular receptor. the CD4 molecule (Willey er aL, 1992a). Unlike the Nef gene product. Vpu has been shown to mediate a specific degradation of CD4 in the ER as was evident from the significant reduction of the CD4 half-life (Willey et a/., 1992b). So far, Vpu has been reponed to affect at least two independent events during HIV infection, including facilitation of virus release and degradation of trapped CD4 in the ER (Terwilliger et at.. 1989; Willey et at.. 1992a: Yao et at., 1992; Schuben and Strebel. 1994; Friborg et at., 1995). The exact mechanism underlying Vpu-mediated degradation of CD4 is not currently understood. To identify the sequence(s) or structural element(s) within the CD4 cytoplasmic tail necessary for Vpu-induced degradation, we performed a mutational analysis of the cytoplasmic domain of CD4. The results obtained in the present study are in agreement with several recent repons which indicate that the cytoplasmic domain of C04 is involved in Vpu-induced CD4 degradation (Lenburg and Landau. 1993; Vincent et at., 1993). Indeed. analysis of C-terminus-truncated CD4 protein shows that the segment KRLLSEKKT, present in the cytoplasmic domain of CD4. is involved in the sensitivity of CD4 to Vpu. This segment overlaps the segment LSEKKT already identified by Vincent et at. (1993) and parrially overlaps the segment EKKTCQC found by Lenburg and Landau (1993) to be imponant for Vpu-induced CD4 degradation. A previous study has shown that the phosphorylation of serine 408 or substitution mutations of M407 and 1410 or double leucines at positions 413 and 414, respectively. abolished the phorbol 12-myristate 13-acetate-induced internalization of CD4 (Shin er at.. 1991). This doubleleucine motif is also present in the proximal ponion of the CD3y chain and has been shown to be imponant
I
DEGRADATION OF CD4 BY HIV-1
62 1
tion. disrupts totally the conformation of this putative for endocytosis and lysosomal targeting of this protein alpha-helix (Fig. 4). It is of interest to note thar the pre(Letourneur and Klausner. 1992). Recently. Aiken et a/. (1994) have demonstrated that this dileucine motif was viously described CD4 cytoplasmic domain delerion mualso required for Nef-induced CD4 down-regulation. To tants (Env-CD4K1AC and CD4stop418) (Lenburg and determine whether the same CD4 amino acid motifs Landau. 1993; Vincent er al,. 1993). which lost sensitivity were responsible for CD4 internalization/lysosomal tarto Vpu. also severely disrupted this predicted alpha-helix geting and Vpu-induced degradation. a series of substituconformation according to our computer analysis (data tion mutations were generated in the CD4 cytoplasmic not shown). To directly evaluate the imponance of this domain. Our results demonstrate thar substitution mutaputative alpha-helix conformation. we introduced proline tions in the segment KRLLSEKKT. including a dileucine substitution mutation individually or in combination. Promutant (CD411413.4AA) and a CD4 phosphorylarion muline residue has a low helix propensity and has a unique tant (CD43S) (as shown in Figs- 2 and 3) which have distoning effect on alpha-helix conformation (Blaber et a/., 1993). In addition. a substitution mutant of four amino been reponed to abolish the internalization and lysosoma1 targeting of CD4 (Shin eta/., 1991). did not confer acids (EMKL) at positions 405.407.41 1, and 413 for Cys. resistance to Vpu. These results indicate that the mechaAsp. Thr. and Pro (CDTP) was evaluated. Both mutants nism ;nvolved in ihe degradation of CD4 by Vpu in the which had a deleterious effect on the conformation of ER is different from the mechanism involved in the interthe alpha-helix abrogated Vpu-induced degradation of nalization and lysosomal degradation of C04. Moreover, C04. The data obtained with these mutants strongly sugour data suggest that Vpu-induced CDL: degradation and gest that the maintenance or correct folding of the preNef-induced CD4 down-regulation involve two distinct dicted alpha-helix structure located in the membrane mechanisms (Aiken et a/., 1994). proximal regionof the CD4 cytoplasmic domain is necesThe distal ponion in the cytoplasmic domain of CD4 sary for Vpu-induced degradation. However. we cannot contains a 12-amino-acidsegment. extending from lysine rule out the possibility that the C-terminal deletion and 41 7 to threonine 429. which constitutes the association substitution mutation in the CD4 cytoplasmic domain site of CD4 with the lymphocyte tyrosine kinase ~ 5 6 ' ~ . may have affected the structure of the transmembrane Two cysteines at amino acids 420 and 422 play a critical region of CD4 since this region was recently shown to role in this interaction (Shaw et a/.. 1990; Shin et a/.. contribute to CD4 sensitivity to Vpu (Buonocore er at.. 199 1). The domain of CD4 that is responsive to Vpu1994; Raja et a/.. 1994). However, this possibility appears induced degradation is clearly distinct from the binding unlikely since our computer analysis indicated thal none domain of CD4 to p56ICkas a double substitution of of the CD4 substitution or deletion mutants described in Cys420 and Cys422 with Ala, which were reported to this study affected the B-sheet and @-turnconformations lack ~56'~~-binding activity (Shaw era/., 1990), is still senin the transmembrane region (data not shown). sitive to Vpu- Lack of endogenous p56ICkexpression in interestingly. in the membrane proximal portion of the COS cells also suppons this result cytoplasmic domain of CD4. the segment from Arg 400 to Substirution of 6 of 9 amino acids in KRLLSEKKT did Glu 416 is highly homologous to the membrane proximal not abrogate CD4 sensitivity to Vpu. This suggests that ponion found in the cyroplasmic domain of the epidermal neither the sequence specificity nor the amino acid growth factor (EGF) receptor (Shin et aL, 1991). Previous charges in this segment play a critical role in Vpu-instudies have proposed that this region of the EGF recepduced degradation of CD4. Interestingly. one of the subtor was involved in oligomerization of the receptor mole~ t i t ~ t mutations i~fl (named CD4MK407.11 PP), which lies cules with a neu/erbB-2 proto-oncogene product outside the Vpu-responsive region identified by deletion p185neu or with. the 10.4-kDa €3 gene product of adenoanalysis (Lenburg and Landau, '1993; Vincent eta/., 1993). virus (Carlin et a/.. 1989; Stern and Kamps. 1988). The completely abolished Vpu-induced CD4 degradation. heterooligomerization of the EGF receptor with the i0.4This observation suggests that CD4 resistance to VpukDa €3 gene product was also proposed to result in rhe down-regulation of the EGF receptor (Carlin et aL. 19891. mediated degradation resulting from deIetion of the Cterminus may be due to a disturbing effect of these deleThe putative alpha-helix conformation encoded by the tions on structural elements lying in a more proximal corresponding region of the cytoplasmic domain of CD4 cytoplasmic region, may be involved in similar protein-protein interaction A previous study has suggested that the membrane which in turn may be crucial for Vpu-induced degradaproximal region of CD4 is likely to form an alpha-helix tion. Such a protein could be a cellular factor which. in the structure (Shin eta/., 1991). Our structure/function analypresence of Vpu. targets CD4 to a degradation pathway. sis of the cytoplasmic region of CD4 also suggests that Alternatively, this factor could be Vpu itself. although dian alpha-helix can be formed between amino acids 402 rect interaction of Vpu with CD4 has not yet been reand cl9. AS expected. the CD4 deletion mutant poned. Moreover. from the data of this study. we cannor (CD4323), which abrogates Vpu-induced CD4 degradarule out that this putative alpha-helix structure may play
YAO ET
an important role in the overall conformation of the cvtoplasmic domain of CD4. In this case. the alteration of such structure is likely to affect the downstream functional domain in the CD4 cytoplasmic tail. More detailed biochemical studies will be reqirired to investigate how this alpha-helix contributes to Vpu-induced degradation of CD4.
ACKNOWLEDGMENTS We thank Domini~ueBergeron for helpful discussion and NicoIe Rougeau and lgal Sebbag for technical assistance. Also. we are grateful to Dr. Richard Axel for the pT48 plasmid which was obtained through me AIDS Research Reference Reagent Program. Division of AIDS. NIAID. NIH. X-S.Y.. J.F.. and F.C.are recipienls of studentships from the Nat~onalHealth Research and Development Program (NHRDP). Health and Welfare. Canada. S.G. is a recipient of a studentship from the Medical Research Council of Canada (MRC). EAC. and R.P.S. are. reswctively. recipientsof NHRDP and MAC scientist awards. This work was supponed by a NHADP/MRC grant to EAC.
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between multiple alignments. secondary structure predictions and motif/pattem scanning into proteins. Cornput. ~ p p l B~osci. . 9.87-91. Glaichenhaus. N.. Shastri. N.. Lmman. 0.R.. and Turner. 1. M. (1991). Requirement for association of ~ 5 6 - wifh CD4 in antigen-speclfrc signal transdualon in T cells. Cell 64. 51 1-520. Ho. S. N.. Hunt H. D. Honon. R. M.. Pullen. J. IC. and Pease. L R. (19891. Sitedirected mutagenesis by overlap extension usrng the polymerase chain reaction Gene 77.51 -59. Jabbar. M. A. and Nayak. D. P. (1990). lnuacellular interaction of human immunodeficiency virus rype 1 (ARV-2) envelope glycoprotein gp160 with CD4 blocks me movement and maturation of CD4 w me plasma membrane. I. Wml. 64. 6297-6304. leang. K T. Rawlins. 0.R. Rosenfeld. P. J.. Shero. J. H. Kelly. T. 1.. and Hayward. G. S. (1987). Multiple tandernty repeated binding sites for cellular nuclear faaor 1 mat surround the major immediate-early promoters of Simian and human cytomegalovirus. 1. Viral. 61, 15591570. Lenburg, M. E. and Landau. N. R (1993). Vpu-induced degradation of CD4: Requirement for specific amino acid residues in the cyroplasmic domain of CD4. I. Virol. 67, 7238-7245. Letoumeur. F. and Klausner. R D. (1992). A novel di-leucine motif and a ryrosine-based motif independently mediate lysosomal targeting and endocytosis of CD3 chains. Cell 69. 1143- 1157. Maddon. P. J., Linman. D. R. Godfrey. M.. Maddon. D. E. Chess. L. and Axel. R. (1985). The isolation and nucleotide sequence of a cONA encoding the T cell surface Protein T4: A new member of the immunoglobulin gene family. Cell 42. 93-104. Maldarelli. F.. Chen. M. Y.. Willey. R. L.and Suebel, K (1993). Human immunodeficiency virus type 1 Vpu protein is an oligomeric type 1 integral membrane protein. I. Viral. 67, 5056-5061. Raja. N. U. Vincent M. 1.. Jabbar. M.A (1994). Vpu-mediated proteolys~s of gplW/C04 Chmeric envelope glycoproteins in the endoplasmc reticulum- Requirement of both the anchor and cytoplasmic domains of CD4. Vimlogy 204.357-366. Schubt?~U.. and Suebel. K (1994). Differential activities of the Human immunodeficiency virus type lencoded Vpu protein are regulated by phosphorylation and occur in different cetlufar compartments. J. Virol. 68.2260-2271. Shaw. A. S. Amrein. K E.. Hammond. C.. Stem. D. F.. Seflon. 0. M.. and Rose. J. K (1989). The Ick lyrosine protein kinase interacts wlth the cyroolasmic tail of the CD4 glycoprotein mrough its unlque amino-terminal domain. Cell 59. 627-636. Shaw. A S.. Chalupny. J. Whitney. J. A. Hamrnond. C.. Amrein. K E.. Kavathas. P.. Sefion. B. M.. and Rose. 1. K (1990). Short related sequences In me cytoplasmic domains of CD4 and CD8 med~ate binding to the amino-terminal domain of the ~ 5 ryrosine 6 ~ protein kinase. Mol. Cell. Biol. 10. 1853- 1862 Shin. J. Dunbrack. R. L, Jr.. Lee. S.. and Strominger. I. L (1991). Phosphorylation-dependent down-modulation of CD4 requires a specific structure within the cytoplasmic domain of CD4.J. Biol. Chem. 266, 10658 10665. Stem. 0. F., and Kamps. M. P. (1988). EGFstimulated tyrosine phospnorylation of pl85neu: A potential model for receptor interactlcns. EMBO 1. 7,995-1001. Strebel. K. Klimkait T.. Maldarelli. F. and Manin. M. A (1989). Molecular and biochemical analyses of human immunodeficiency virus type 1 Vpu protein. I. Wml. 63.3784-3791. Terwilliger. E. F.. Cohen. E. A. Lu. Y.-C.. Sodroski. J. G.. and Haseltme. W. A (1989). Funaional ro;e of human immunodeficiency virus rype 1 Vpu. Proc. Natl. Acad. Sci- USA 86. 5 I 63 -51 67. Tremblay. M.. Meloche. S. Granon. S.. Wainberg. M. A. and Sekaly. R. P. (1994). Association of 056" with the woplasmic domaln of CD4 modulates HIV-1 expression. EMBO J. 13. 774-783. Veillene. A. Bookman. M. A. Horak. E. M.. and Bolen. J. B. (1988) The CDs and CD8T cell surface antigens are associated w ~ t hthe ~nternal membrane tyrosine-protein kinase ~ 5 6 ' Cell ~ . 55. 301 -306.
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\
Vincent M. J. Rala. N. U.. and Jabbar. M. A (1993). Human immunodeficiency virus type 1 Vpu protein induces degradation of chimeric envelope glycoproteins bearing the cytoplasmic and anchor domain of CD4: Role of the Cyto~lasmi~ domain in Vpu-induced degradation ir, the endoplasmic reticulum. J. Vim/. 67. 553-5549. Willey. R L. Maldarelli. F.. MaRin. M. A. and Strebel. K (1992a). Human immunodeficiency virus type 1 Vpu protein regulates the formation of intracellular gpl60-CD4 complexes- /. VmL 66,226-234. Willey. R L. Maldarelli. F. Martin. M,A. and Strebel. K (1992b). Human immunodeficiencyvirus type 1 Vpu protein induces rapid degradation of CD4. J. ViroL 66.71 93-7200.
Willey. R L. Buckler-White. A. and Strebel. K (1994). Sequences present in the cytoplasmic domain of C04 are necessary and sufficient to confer s e n s i t i i to me human immunodeficiencyvirus rype 1 vpu pratein I. V7roI. 68.1207- 1212 Yao. X-J.. Gotrlinger. H. Haseltine. W. A. and Cohen. E. A (1992). Envelope glywprotein and C 0 4 independence of Vpu facilitated HIV1 capsid export J. Wml. 66. 5 119-51 26. Yao. X-J-. Ganon. S-. BoisveR F.. Haselline. W. A, and Cohen. E A (1993). The effect of Vpu on HIV-1-induced syncytia formation. I. Acquired Immune D e e Syndr, 6. 135 141.
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Article 4: Structural requirements for the binding of the human immunodeficiency virus type 1 Vpu protein to the cytoplasmic domain o f CD4.
STRUCTURAL REQUIREMENTS FOR THE BINDING OF THE AUMAN IMMUNODEF'ICIENCY VIRUS TYPE 1Vpu PROTEIN TO THE CYTOPLASMIC DOMAIN OF CD4
Jacques Friborg, Xiao-Jian Yao, Angelo Tiganos, Nash G. Daniel and h c A. Cohen*
Laboratoh dc Riaovirologie Humaine, Ixparternent & Mimbiologie et Immunologic, Facult6 de Mcdecine, Universite de Montral, 86128, succursale centre-ville, M o n M , Qudbec, Canada H3C 3J7
*To whom correspondence should be addressed Tel: (5 14) 343-5967 Fax: (5 14) 343-5995 Electronic mail address:
[email protected]~.ca
ABSTRACT The HIV-I encoded Vpu phosphoprotein induces a rapid degradation of CD4 molecules in the endoplasmic reticulum (ER). The molecular mechanisms underlying this biological
activity still remain undefined- Interestingly, a specific interaction between Vpu and CD4 molecules was recently demonstrated in coimmunoprecipitation experiments. Yet, this interaction was shown to be insufficient for uiggering CD4 degradation. In order to delineate sequence(s) orland structural determinant(s) involved in the f d o n of Vpu/CD4 complexes, we performed in this report a mutational analysis of (i) the Vpu protein and (ii) the cytoplasmic domain of CD4. In agreement with previously described experiments, we show that phosphorylation mutants of Vpu unable to induce CD4 degradation retain the capacity to interact with CD4 molecules. However, in contrast to substitution mutations of
both Vpu phosphoacceptor sites Ser 52 and Ser 56, the solely mutation of Ser 56 abrogated herein the binding of Vpu. This result suggests that conformational constraints regulated by the phosphorylation state of Vpu may be relevant in the interaction with CD4 molecules. Moreover, our analysis of a panel of substitution mutants in the cytoplasmic domain of CD4 revealed that the segment LSEKKT (residues 414 to 419) of the protein previously described in deletion studies to be the minimal requirement for Vpu-induced degradation appears herein dispensable for the binding of Vpu. Substitution mutations of 4 of the 6 amino acids in this segment had no effect on the capacity of Vpu to bind CD4 molecules. In contrast, we
demonstrate that a putative a-helix in the proximal cytoplasmic region of CD4 which was previously shown to contribute to Vpu sensitivity is required for efficient binding of Vpu.
Thus, taken together, these results strongly suggest that the phosphoacccptor sites of Vpu modulate the binding of CD4 molecules and subsequently their targeting to an undefined degradative pathway. In addition, it is likely that the formation of Vpu/CD4 complexes which interaction appears stabilized through a putative a-helix in the proximal cytoplasmic region of CD4 is of a transient nature due to structural constraints.
INTRODUCTION The human immunodeficiency virus type 1 (HIV-1)vpu gene encodes an 81 amino acid integral membrane protein capable of homo-multimerization (15, 22), that is not
expressed by the closely related HIV-2 or by most simian immunodeficiency viruses (SIVs) (8, 16, 23). Vpu has the topology of a class I protein with a strongly N-terminal
hydrophobic region of 27 amino acids (sta.), which constitutes the membrane-spanning domain, and a charged C-terminal hydraghilic domain facing the cytoplasmic side (15). Furthermore, Vpu is phosphorylated by a casein kinase-IT-related protein (CKII) at serine residues (Ser 52 and Ser 56) located in a highly invariant C-terminus stretch of 12 a-a.
(residues 47-58) of the protein (10, 19, 20). Using a combination of ckular dichroism and
'H nuclear magnetic resonance spectroscopy, the C-terminal domain of Vpu has been predicted to form two amphipathic a-helices joined by a flexible t u n containing the two phosphoacceptor sites of the protein (30). Numerous hctional studies have indicated that Vpu has several biological activities during viral replication. Expression of V p u during HIV-1 infection in CD4+ cells delays
cytopathic effects of the virus by reducing the rate of syncytium formation, and induces W4 degradation in the endoplasmic reticulum (ER) (13,24,28,29). Recent studies revealed that these Vpu-associated activities are dependent on the phosphorylation state of the protein (10,
18). In addittion, Vpu facilitates the release of HN-1 budding virions from infected cells in a manner which is independent of the expression of Env glycoproteins and CD4 receptor molecules (11, 13,22,24,32). These observations have emphasized the possibility that V p u promotes differential biological activities during HlV-1 replication and may in fact have
more than one target in infected cells.
The Vpu-induced CIM degradation has been shown to require the retention of CD4 molecules in the ER either through formation of complexes with Env precursor gp160 or by the treatment of cells with brefeldin A (BFA), a fungal metabolite known to block protein
sorting from the ER to the Golgi apparatus (29). Several mutagenesis analyses have described sequences or motifs both in the transmembrane and cytoplasmic domains of CD4 and in the C-terminal region of Vpu necessary far the Vpu-induced CD4 degradation (6,7, 14, 17, 18,26,27,3 1). However, the mechanism involved in such phenomenon has yet to be defined. Recently, coirnmunoprecipitation experiments have shown that Vpu can
specifically bind 0 4 moleculcs in the ER (5). The weak affinity of the two proteins and/or the high rate of Vpu-induced CD4 degradation may have previously imp&
the detection
of Vpu/CCM complexes in whole cell systems. Interestingly, the use of chimeric proteins
and deletion mutants of CD4 indicated that the cytoplasmic tail of the protein previously shown to be important for Vpu-induced degradation also contains determinants important for the binding of Vpu. Moreover, substitution mutations of Vpu phosphoacceptor sites as well as deletion of five amino acids between residues 47 and 52 which abrogate the capacity
of Vpu to promote CD4 degradation were shown to have no effect on the protein's interaction with CD4 molecules (7, 18). On the basis of these results it was proposed that the binding of Vpu appears necessary but not sufficient to cause CD4 degradation.
In order to gain further insight into the mechanism of Vpu-induced CD4 degradation, we proceeded
in this report to define sequence(s) or structural element(s) of CD4 and Vpu
involved in the formation of VpdCD4 complexes. In an initial experiment (Fig. I), wildtype
(wt)
Vpu or phosphorylation Vpu mutants carrying individual substitution of Ser 52
(SVCMV-~pu52G)or S a 56 (SVCMV-~pu56G)for glycine residues as well as a double substitution mutant ( S V C M V - ~ p u s m )previously described (10) were tested for their ability to form Vp-4
complexes in the ER. Thus, COS-7 cells were cotransfected as
with a mixture of 5 pg of an expression plasmid previously described (31) encoding wt CD4
(SVCMV-CD4) and 12.5 pg of either SVCMV-Vpu', SVCMV-Vpu+ or SVCMV-Vpu mutants expressor using the calcium phosphate coprecipitation technique (9). Subsequently, Vpu-induced CD4 degradation and VpuKD4 complexes were analysed in pulse-chase experiments. Forty eight hours posttransfection, cells were treated with 10 p M BFA 1 h
prior to and during metabolic labeling. Transfected cells were starved in methionine-free
Ddbecco's modified Eagle's medium (DMEM) for 15 min. Cells were then pulse-labeled for 30 min with [3s~]methionine (250 pCi/ml) and chased in complete DMEM supplemented with BFA. At the indicated time p e r i d , labclcd cells were lystd in RIPA buffer containing lOmM Tris-HC1 IpH 7-41, 1mM EDTA, lOOmM NaCI, 1%Triton X-100, 0.1% sodium dodecyl sulfate [SDS], 0.25% dwxycholate and 0.2% phenyl-methyIsulfony1
fluoride. CD4 molecules were immunoprecipitated with an anti--
monoclonal antibody,
separated on a 12.5% SDS-polyacrylamide gel and visualized by a u t d o g r a p h y . The data in Fig. 1 show that the relative levels of C l X were significantly reduced in the presence of
Vpu (SVCMV-Vpu+) when compared to those detected in its absence (SVCMV-Vpu-) (compare lanes 1-4 with lanes 5-8). Since the half-life of CD4 in the presence of Vpu is approximately 10 min, it appears that CD4 molecules have already undergone Vpu-induced degradation during the 30 min pulse-labeling time. Revious reports investigadng the Vpuinduced CD4 degradation have described similar observations in pulse-chase experiments (14, 26). By contrast, phosphorylation mutants of Vpu did not induce the degradation of CD4 molecules as levels detected were comparable to those seen in the absence of Vpu (Fig.
1; compare lanes 1-4 with lanes 9-12, 13-16 and 17-20). Interestingly, coprecipitation of a band migrating at a molecular weight corresponding to Vpu was detected in SVCMVvpus2G and SVCMV-V~USUS~ cell lysates (Fig 1; compare lanes 1 4 with lanes 9-12 and
17-20). Immunoprecipitation with a rabbit anti-Vpu serum confirmed that the coprecipitated band were indeed Vpu products (data not shown). However, most likely due to rapid degradation in these experiments, we were not able to demonstrate direct interaction of CD4 with wt Vpu (lanes 5-8). Surprisingly, substitution of Ser 56 (SVCMV-V~US~G) abolished the ability of protein to bind CD4 since this specific band was not detected (Fig. 1; lanes 13-
16) even though the Vpu product was shown to be stable in cells (data not shown). As
previously reported by Bour ez al. (1995), these data indicate that the binding to CD4 precede the phosphorylation of Vpu and that such posttranslational event will in nxm trigger
a process leading to CD4 degradation- It was recently proposed that the net charge of the C-
terminal portion of Vpu will increase upon phosphorylation of the protein (30). The result obtained with the individual substitution of Ser 56, which phosphorylation by CKII was previously shown to be favoured over Scr 52 (19), suggests that this Vpu mutant is probably unable to interact with CD4 mIecules due to conformational constraints.
CD4 segment involved in the Vpu-induced degradation b not essential in the binding of Vpu. Numerous studies have described sequences in the cytoplasmic region of CD4 involved in the Vpu-induced degradation of the molecule (7, 14, 18,26,27,3 1). An overlapping consensus segment encompassing amino acids 414 to 419 (LSEKKT) of the CD4 cytoplasmic tail can be deduced fiom these reports. However, the exact relevance of
this sequence in Vpu's mode of action rernains unknown. Several residues in this segment have been shown to be critical for CD4 biological activities in T cells. Phosphorylation of CD4 at serine residues 408,415 which is present in the segment LSEKICT, and/or 431 was
proposed to be a prerequisite in the endocytosis and targeting of the protein to a dcgradahve pathway in response to phorbol 12-myristate 13-acetate stimulation (21). In addition, two leucine residues at positions 413 and 4 14 were shown to be important for the internalization and lysosomal targeting of CD4 molecules (21). This motif has recently been shown to be required for the HIV-1 Nef-induced CD4 down modulation (3). Finally, lysine residues at positions 417 and 418 are contained in the sequence KKTC, which can act as ER retention signal (21). Moreover, CD4 molecules have been shown to associate with p56'ck tymsine kinase through cysteine residues located at positions 420 and 423 and to form C~4/p56& complexes. Therefore, a series of CD4 mutants previously described (31) harboring amino acid substitution in these discrete motifs was analyzed for their ability to retain Vpu/CD4
interaction (Fig. 2A). In these experiments, we took advantage of the phosphorylation mutant ~pus*/56to visualize the fannation of VpdCD4 complexes since it was shown to efficiently interact with wt CD4. The CD4 substitution mutants were transfected in COS-7 cells in the presence of S V C M V - V ~ U S W ~plasmid as described earlier. Forty-eight hours
posmansfection. COS-7 cells were labeled with [3%]methionine for 1 h in the presence of 10 pM BFA and Vpu/CD4 complexes immunoprccipitated with the anti-CD4 monoclonal
antibody fkom the cell lysates were visualized by autoradiography. The results of Fig. 2B show that none of these CD4 mutants, which were prtviously shown to be degraded in the presence of wt Vpu (31), lost their ability to interact with VpuSu%. These data suggest that the consensus segment LSEKKT sensitive to Vpu as well as the biological motifs describe above are unlikely to retain the binding sitc for Vpu. A putative a-helix located in the cytoplasmic taiI of CD4 may contribute to the binding of Vpu. A region encompassing residues 402 to 420 in the cytopIasmic tail of CD4
was proposed to provide the binding sitc for Vpu (5). Deletion of 13 amino acids fiom the
C-terminus of CD4 still allowed the formation of VpdCD4 complexes, while removal of the last 22 aa.abrogated Vpu binding. We have previously shown that a putative a-helix in the proximal region of the CD4 cytoplasmic domain is important for Vpu-induced CD4 degradation (31). This predicted a-helix, located between positions 402 and 419, was reported to be essential for endocytosis and lysosomal targeting of CD4 (21). Therefore, we
evaluated the relevance of this secondary structure in the formation of Vpu/CD4 complexes.
Two previously described CD4 mutants which were shown (i) to be unable-to form the C terminal putative a-helical confurmation and (ii) to be resistant to Vpu-induced degradation (3I), were analyzed herein for their ability to interact with Vpu (Fig. 3A). A CD4 mutant
harboring two proline residues at positions 407 and 411 (CD4MK407.11PP)as well as a substitution mutant of amino acids Glu,Met, Lys and Leu at positions 405, 407,411 and
413 for, respectively, Cys, Asp, Thr and Ro ( W E M K L ) were cotransfected in COS-7 cells with SVCMV-~pu5m.Forty-eight hours posttransfcction, the cells were mated far 1 h with 10 pM BFA as described above and subsequently pulse-labeled with [3s~]methionine
for 30 in the presence of BFA. At the indicated time periods, newly synthesized CD4 were immunoprecipitated from cell lysates with an anti-CD4 monoclonal antibody and visualized by autoradiography. As seen in Fig. 3B, the two CD4 mutants retained the capacity to
interact with ~ p u s m .However, the stability of Vpu/CD4 interaction appears affected
since the formation of complexes was barely detectable in these cell lysates (Fig. 3B; compare Ianes 1-4 with lanes 5-8 and 9-12). Dcnsitometric scanning of the bands corresponding to Vpu revealed that the amounts of Vpu/CD4 compltxcs irnmunoprecipitated, according to the level of CD4 present at each indicated time, were
significantly reduced after 30 min. of chase in cells transfccted with CD4MR407.11PP or CD4EMKL when compand to cells aansfected with wt CD4 (Fig. 3C). This result suggests that the putative a-helix in the cytoplasmic tail of CD4 may contribute to the stability of Vpu/CD4 complexes and further reinforces its importance in the Vpu-mediated degradation
of CD4 molecules.
Conclusions. As previodsy reported by Bour et al. (1995), the data presented in this study demonstrate that the cytoplasmic tail of CD4 retains determinants necessary for the binding of the HIV-1Vpu protein. Moreover, this report delineate structural requirements for the
efficient binding of Vpu with CD4 molecules. It is clear from the overall data that the
binding of Vpu is an early step in the degradation of CD4. Neverthless, these events appear separable and may require distinct domains within Vpu and CD4. The exact mechanism by which Vpu interact with CD4 needs to be clarified
Although functional studies have demonstrated that phosphorylation of Vpu is
imponant for the degradation of CD4 (18), the contribution of the two phosphoacceptor sites
(Ser 52 and Scr 56) in the mode of Vpu'action has not been defined. The findings of this study strongly suggest that each phosphoacceptor site of Vpu may contribute to the specific events leading to t& degradation of CD4 molecules. In contrast to v p u S E and V P U ~ ~ , the individual substitution mutation of Ser 56 introduced in Vpu56G has abolished the capacity of Vpu to interact with CD4 moIecules in pulse-chase experiments (Fig. 1). Interestingy, phosphorylation of Ser 56 by CKfI was previously shown to be f a v d over
Ser 52 and may in fact enhances the phosphorylation of the latter (19). The different
behavior between these mutated Vpu proteins could be in part explained by their differing phosphorylation status. This phenomenon is somehow reminiscent of the catalytic regulation of the tyrosine b a s e p56W which also possess the ability to bind CD4. It was shown that phosphorylation/dephosphorylation of a tyrosine residue at position 505 (Tyr 505) of ~ 5 6 f would i affect its structural confixmation, constquently modulating its binding with intracelIular substrates (4). Furthermore,phosphorylation of Tyr 505 was shown to
repress the p56M-associated protein kinase activity (1. 2). Another tyrosine residue at position 394 CTyr 394) was shown to be autophosphorylatrA at a low level in p561ck (2.25).
In contrast to Tyr 505, substitution mutation of Tyr 394 prevents p56U activation, implying that its phosphorylation may upregulate the catalytic activity of the enzyme. It is thmfore tempting to speculate that while the phosphorylation status of Ser 56 m a y modulate the formation of Vpu/CD4 complexes, phosphorylation of Ser 52 will regulate the ability of Vpu to induce CD4 degradation. It is conceivable that the removal of S a 56 in ~ p u 5 6 G
would lead to a conformational state of Vpu inadequate for the proper presentation of sequences or motifs within the protein required for the binding to CD4. In contrast, the substitution mutation of Ser 52 would still permit the formation of Vpu/CD4 complexes as
seen herein (Fig. 1) but in turn affect the bioIogical mechanism unddying the degradation of ax. Alternatively, the segment containing the phosphoacctptor sites of Vpu was recently shown to be flexible and strongly acidic, which acidity will be increase upon phosphorylation of the protein (30). In addition, Vpu/CD4 complexes were proposed to be stabilize through interactions between the cytoplasmic tail of CD4 and two amphipathic ahelical domains (residues 30 to 50 and 57 to 72) joined by the flexible segment in the Cterminus of Vpu (5). Such features can be foreseen with the cellular protein calmodulin (CaM). CaM which has the propensity to bind with high affinity several cellular enzymes is
known to form two a-helices joined by a flexible loop at the C-terminal portion of the protein (12). Phosphorylation of two residues (Thr 79 and Ser 8 1) located in the flexible
segment of the protein is proposed to cause a conformational change that prevent CaM from folding around its target, thus greatly lowering the binding m t y .Since the formation of Vpu/CD4 complexes appear to occur soon aftcr the synthesis of the two proteins, whether or
not phosphorylation of Vpu will regulate the binding affinity of the protein to CD4 remains to be determined Even though Vpu has the capacity to bind CD4 molecules, more detailed biochemical studies will be required to insure the stability of this interaction upon phosphorylation of Vpu and during the per se induced degradation of CD4. The interaction between Vpu and Q)4 may be one of a transient nature regulated by structural constraints. Apart from retaining determinants essential for the Vpu-induced CD4 degradation, the cytoplasmic tail of CD4 was also shown to harbor the binding site for Vpu (5). We show
in this report that several motifs within this domain are not essential for the formation of Vpu/CD4 complexes (Fig. 2). Substitution mutations of 4 of the 6 amino acids present in the consensus segment LSEKKT involved in the sensitivity of CD4 to Vpu (31) had no
effect on the capacity of Vpu to bind CD4 molecules. These data suggest that neither sequence specificity nor d
o acid charges in this central region of CD4 cytoplasmic tail
appear critical for the binding of Vpu. Furthennore and most notable, substitution of the
two cysteine residues (420 and 423), important for the formation of C~4/p56&complexes in the ER and at the surface of the cell, did not affect the ability of Vpu to bind CD4
molecules. Nevertheless, it remains to be define whether the presence of p56" could interfere with the binding of Vpu to CD4 molecules or would allow the formation of heterotrimer structures. Finally, substitution mutations of a di-leucine motif (Leu 4 13-414) or poten tie1 phosphoacceptor sites of CD4 (serine residues 408,4 15 and/or 43 I), which were shown to be important in the internalization and in the targeting of CD4 to a degradative pathway, had no effm on the binding of Vpu. In contrast, alterations in a putative a-helix
in the cytoplasmic tail of CD4, which was shown to affect the Vpu-induced degradation (3I), altered the binding of Vpu (Fig. 3B). Even though Vpu/CD4 complexes can be detected in the early time of pulse-chase experiments (Fig. 3B; compare lane 1 with lanes 5
and 9), the stability of these complexes appear affected indicating that the presence of this a-helical configuration is required far the efficient binding of Vpu. Recently, Wray et d.
(1995) have shown that the net charges of the two amphipathic a-helices located in the Cterminal region of Vpu arc equal and of opposite polarity (4 positive and 4 negative charges, respectively). Thus, similarly to the CaM-binding ability*Vpu may collapse around CD4 to f m a pocket stabilized through salt bridges orland hydrophobic interactions beween these regions. Upon phosphorylation, Vpu may adopt a conformation which will disclose sequences or motifs in the cytoplasmic tail of CD4 sensitive to a undefined degradative pathway.
ACKNOWLEDGEMENTS J. F. ,N. G.D. and X. -J. Y.are d p i e n t s of studentships from the National Health Research
and Development Program (NHRDP),Health and W e M . Canada, E.A. C. is a recipient of a NHRDP AIDS career award. This work was supponed by to EAC.
grants from NHRDP and MRC
LEGENDS F i 1. The formation of VpulCD4 complexes in the ER: phmphaacceptor sites of Vpu modulate the binding to CD4 molecules. SVCMV-CD4 expression plasmid (5 pg) previously described (REF) was comsfected into COS-7 cells with 12.5 pg of either
SVCMV-Vpu- (lanes 1-4), SVCMV-Vpu+ (lanes 5-8). SVCMV-vpu5*G (lanes 9-12), SVCW-vpdffi (lanes 13-16) or SVCMV-vpu-
(lanes 17-20). Forty-eight hours
u dwith 10 pM d BFA for lh. Subsequently, cells were posttransfection, the cells were m
harvested, pulse-labeled for 30 min with [35~]mthionine(250 pCj/ml) and chased for 0,30,
60 or 180 min in complete DMEM in the presence of BFA. Cell lysates were p r e p d at each indicated time period and imrnunoprccipitatcd with an anti-CD4 monoclonal antibody. Immunoprecipitates were separated on a 12.5% SDS-polyacrylamide gel and visualized by autoradiography. The position of CD4 and coimmunoprccipitated Vpu proteins are indicated.
Fig. 2. Effects of mutations introduced in the cytoplasmic tail of CD4 on the capacity to form V p d C D 4 complexes. (A) Schematic representation of wt CD4 and amino acid
sequence of the cytoplasmic tail are shown in the top of the figure. Previously described
substitution mutations introduced in the cytoplasmic tail of wt CD4 (REF)are shown in the bottom pan. (B) COS-7 cells were coaansfected with 5 pg of wt CD4 or CD4 mutants expression plasmid and 12.5 pg of either SVCMV-Vpu- or SVCMV-V~U-
expressor.
Forty-eight hours posttransfection, cells were mated with BFA prior to and during 1 h labeling with [3%]methionine. Subsequently, cell lysates were irnmunoprccipitated with an anti-CD4 monoclonal antibody and Vpu/CD4 cornpiexes analyzed on a 12.5% SDSpolyacrylamide gel. The position of CD4 and coimmunoprccipitated Vpu proteins are indicated.
Fig. 3. A putative whelk in the proximal portion of the cytoplasmic tail of CD4 is
required for the stability of Vpu/CDQ complexes. (A) Schematic representation of wt CD4 and amino acid sequence of the cytoplasmic tail of wt CD4 are shown in the top of the figure. Effects of substitution mutations introduced in the cytoplasmic tail of wt CD4 on a putative a-helical (I secondary ) structure as previously described (REF) are indicated in the
bottom part. (B) COS-7 cells were cotransfa~& with 5 pg of wt CD4 or CD4 mutants expression plasmid and 12.5 pg of S V C M V - V ~ U expressor. ~~/~~
Forty-eight hours
posttransfection, cells were mated with BFA prior to and during pulse-labeling with [3%]methionine. Subsequently, the labeled cells were chase at 0, 30, 60 or 180 min in complete DMEM supplemented with BFA.
Cell lysates were prepared and
immunoprecipitated with an anti-CD4 monoclond antibody and analyzed on a 12.5% SDSpolyacrylamide gel. The position of CD4 and coimmunoprecipitated Vpu proteins are indicated. (C) Quantitation of Vpu/CD4 complexes immunoprecipitated from cell lysates.
The Vpu and CD4 bands detected in panel B were quantified by densitometric scanning of the autoradiography and the ratio of Vpu coprecipitated relative to the amount of CD4
imrnunoprecpitated at each indicated time was expressed as arbitrary unit.
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Schubert, U, P. Heinklein, B. Boldyreff, E-Wingender, K Stnbel, and T. PorJtmPnn. 1994. The human immu11odeficiencyvirus type 1encoded Vpu protein
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Schubert, U., T o Schneider, P.Henklein, K Hoffbn, B. Berthold, H.Hauser, G.
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CHAPITRE 6 Discussion
Le VIH-1 semble avoir developpC au cours de son Cvolution de multiples stratagkmes h i conf6rant l'un des modes d'infection les plus effrcaces. Grace B son organisation gdnomique plus que complexe, il est capable de mobiliser et de prendre avantage de certaines proprietis de l'hbte afin d'optimiser sa persistance et sa replication. Outre les g h e s gag,pol et env responsables de la formation de la particule virale et cornmun 3 tous ritrovirus, le VIH-1 posside des gines auxiliaires qui agiront t6t ou tardivement au cours de la replication virale. Ainsi, afin de mieux comprendre la progression de la maladie, il devient imp6ratif de definir et de caractCriser l'apport fonctionnel de ces divers gines. Nous nous sornmes donc interesser dans le cadre de ce projet de recherche A l'un de ces gknes, le gkne vpu, qui code pour une prodine unique au cycle de rdplication du VIH de skrotype I. Les premigres etudes fonctionnelles realisees par Terwilliger et al. (1989) ont dCmontr6 que la proteine Vpu facilite le reliichement des particules virales B la surface des cellules infectbes et dirninue les effets cytopathiques du VIH-1 en culture. De nombreux travaux ont par la suite montre que la proteine pouvait Egalement induire une degradation specifique des moEcules CD4 retenues dam le
RE de la cellule infectCe (Willey et al., 1992a; Willey et al., 1992b). L'expression de Vpu semble donc bknifique au VIH-1 puisqu'elle augmente sa propagation au cours de l'infection in vitro. Par quel(s) m&anisme(s) Vpu agit-il? Peut on reconcilier les differentes fonctions de la protiine
un unique mode d'action? Quelle est le rale de
Vpu dans l'infection naturelle et la disst5mination du VIH-I? Afin de rdpondre a ces questions, nous nous sommes domes comme objectif d'effectuer une analyse de structure/fonction de la protdine afin d'identifier le(s) domaine(s) actif(s) de Vpu, et ceci dam le but de mieux ddfinir son r6le au cows de la replication virale. Nous tenterons dam cette section de resumer nos r6sultats et de proposer en tenant compte des travaux publiks dans la littirature un rnodsle reconciliant les diverses fonctions de Vpu.
A prime abord, l'analyse informatique des differents isolats du MI-1 rkv5le
une conservation en acides aminks de prks de 90% entre les protkines Vpu. Les 6tudes biochimiques rialisies par Strebel et al. (1989) ont montri que la protkine est phosphorylie et poss5de la capacid de s'intigrer dam les membranes lorsque traduit in vitro en prisence de CMM. Notre intdrEt initial fut donc d'etudier l'importance de la phosphorylation dam les activites biologiques associkes 3 Vpu. Ainsi, on retrouve quatre risidus serine (acides aminks en positions 23, 52, 56 et 61) et une tyrosine (acide amini en position 29) dam la sequence prkdite d e la protiine qui peuvent s'avkrer des sites potentiels de phosphorylation. Toutefois, des etudes prkliminaires effectuees dam notre laboratoire ont perrni de montrer que Vpu est phosphoryli sur l'un ou plusieurs de ces residus sirine.
En effet, l'analyse tryptique de Vpu
prkalablement marquee l'orthophosphate inorganique [ 3 2 ~ i ]revkle exclusivement la presence de phosphostkine suite au fractionnement par chromatographie sur couche mince (rdsultats non-publiks), Par aiueurs, les travaux de Schubert et al. (1992) ont montrk ultkrieurement que la prottiine est phosphorylke par la CKLI, dont I'activiti enzymatique peut etre specifiquement inhibke dam la cellule suite au traitement & la
5,6-dichloro-1-(P-D-ribofuranosyl)benzimidazole (DRB).
La CKII posstde la
proprikte de phosphoryler des rksidus skrine et/ou thrkonine presents dans le motif, Ser/Thr-XX-Asp/Glu. Or, deux sirines (rdsidus en positions 52 et 56) localisies dans une rkgion hautement conservie de Vpu (acides aminds 47 A 58) se retrouvent dans une sdquence consensus favorable A I'activitk de la CKII.
de difinir les
sites de phosphorylation de Vpu, nous avons donc procedi par mutagtn5se dirigke A des substitutions ponctuelles ou combinkes des risidus sirine en positions 52 et 56 (Ser 52 et Ser 5 6 ) pour des rksidus glycine ou leucine. Les g h e s vpu rnutds 2t ces sites de phosphorylation ont par la suite kt6 inskris d a m un cl8ne molCculaire
infectieux du VM-I, nous pernettant d'effectuer des etudes de rkplication virale I& vitro . Ainsi, dam le premier article (chapitre 2)- nous avons montre que Vpu est effectivement phosphoryle'e sur ces deux rCsidus stkine (figure 2B). Dam un syst&me&expression cellulaire transitoire, il est impossible d'immunoprkipiter la proteine Vpu marquee au [32~i],lorsque c e l l e d est mute sur les Ser 52 et Ser 56. A I'inverse, lorsque ses residus sont substituCs individuellement, la proteine peut &re dktect6e dans les m6mes conditions expdrimentales. Par la suite, nos Ctudes de replication v i d e in vitro ont dimontre que la phosphorylation de Vpu est essentielle
i l'activite biologique de la prodine. Toutes les mutations gCn6rees dans cette etude n'ont eu aucun impact majeur sur la capacite de Vpu de faciliter le rellchement de particules virales tant dans des celldes CD4+ que dam des cellules non-lymphoi'des (figures 2 et 3). Cependant, la double substitution des Ser 52 et Ser 56 ainsi que la substitution ponctuelle de la Ser 56 ont totdement abolit la capacit6 de la protdine j. diminuer les effets cytopathiques du virus. La formation de syncytium n'est plus retardte dans les cultures cellulaires infectees avec des virus exprimant ces prodines mutantes comparativement 2 Itinfection par un virus vpu+ (figure 5). De plus, ces derni$res ne sont plus aptes B diminuer l'expression de la gp120 ii la surface des cellules infectdes de maniere comparabIe 2 la prottine Vpu de type sauvage (tableau
I), expliquant ainsi les effets sur la formation de syncytium. En revanche, la substitution ponctuelle de la Ser 52 pour un residus glycine ou une Ieucine n'a pas eu
un effet aussi drastique tant au niveau de la formation de syncytium que sur I'expression de surface de la gp120. Dans chacun des cas, la mutation ne semble qu'affecter en partie les propridtis de Vpu. Ces rksultats sugg5rent donc que Vpu peut moduler les effets cytopathiques du virus en culture inddpendarnment d u relgchement accru de la progdnie v i d e o b s e d lors de son expression.
Nos observations confiient les resultats rapport& peu de temps avant nous par Schubert et Strebel, (1994). Leurs travaux ont montrt5 que la capaciti de Vpu i augmenter le relilchement de particules virales n'est que partiellernent dkpendante de Mat de phosphorylation de la proteine. Cependant, la double substitution des Ser 52 et Ser 56 inhibe complitement sa capacid B induire la digradation des molecules
CD4 retenues dam le RE. 11 est clair que la r6gion C-terminale de Vpu presente l'un des domaines actifs de la proteine et que M a t de phosphorylation de celle-ci joue un r6le important dam son activit6 biologique. De plus, les auteurs dhontrent que lorsque retenue dam le RE suite au traitement B la BFA, Vpu est incapable d'augmenter le reliichement des particdes virales B la surface des cellules infectees. Ainsi, 1es deux activitds maintenant perpes cornme indipendantes semblent requerir la prksence de Vpu dam des compartiments cellulaires distincts. Toutefois, une Ctude plus approfondie sera necessaire afin de determiner si les propnet& de Vpu d'induire la digradation de CD4 et de diminuer les effets cytopathiques du virus sont fonctionnellement relides. Des etudes fonctionnelles ont montrC que Vpu pouvait moduler le reliichement ou I'assemblage des particules virales en I'absence d'expression des glycoprot6ines d'enveloppe v i d e ou des molicules CD4 (Yao et al., 1992; Geraghty et Panganiban, 1993). Il est donc fort probable que la protkine posscde des domaines distincts lui permettant de rnedier ses diverses fonctions. Vpu est une protCine membranaire intrinskque de type 1 (Maldarelli et al., 1993). Sa region N-terminale d'environ 27 a.a. semble &re responsable de I'intigration de la proteine dam les membranes (Strebel et al., 1989). Ainsi, &in de definir le domaine d'ancrage de Vpu et l'importance de cette region dam l'activite biologique de celle-ci, nous nous sommes consacrer dam nos etudes subs6quentes h une analyse par mutaginkse de 11extrimit6N-terminale de la protiine.
Le deuxikme article (chapitre 3) presente une analyse de structure et de fonction de divers mutants de deletion et de substitution de Vpu. Les mutations introduites dam la region N-terrninale de la prodine furent gentrt5es afin d'affecter une structure secondaire en alpha-hilice pr6dite de se former selon l'dgorithrne de Kyte et doolittle. En resume, nos travaux demontrent clairement que seule cette region est necessaire B l'ancrage de Vpu dam les membranes. La dbldtion de 7 acides aminis (residus 8 B 14) ainsi que la substitution combinke de la valine, de la tryptophane et de la serine, respectivement en positions 20, 22 et 23 ont alter6 la capaciti d'insertion des protCines Vpu mutantes dans les CMM (figure 2B). De plus, ces mutations ont affect6 la stabilitt5 des proteines et aboli leurs propridtCs dam les cellules infectees.
Par ailleurs, des mutations introduites dans une sequence hautement conservee de la region N-terminale de Vpu (residus 6
14) ont affecte l'activite
biologique de la protCine. Deux mutants contenant des triples substitutions. d'une part de deux rCsidus isoleucine respectivement en positions 6 et 8 et de la valine en
position 9, et d'autre part de l'alanine, la leucine et la valine respectivement en positions 10, 11 et 13, ont altCrc5 A des degrks diffkrents la capacitt de Vpu de faciliter le reliichement de particules virales (figure 4). Ces deux mutants ont toutefois conservC les propriCt6s d'induire la degradation des mol6cules CD4 dans le
RE et de diminuer les effets cytopathiques du virus. Nos observations suggerent que la r6gion N-terminale renferme non seulement le domaine d'ancrage de la proteine, mais pourrait egalement presenter le domaine actif ou tout au moins des dkterminants nCcessaires 2 la fonction de la protkine de faciliter le relPchement de particules virales. De plus, ces rc5sultats viennent soutenir la notion de multiples fonctions indbpendantes rnedibes par Vpu au cows de l'infection par le WH- 1. Il est 2 noter toutefois que la region N-terminale ne peut B elle seule moduler la capacite de
la prottine B faciliter le reliichement de particules virales. En effet, bien qu'elle soit
apte 5 s'ancrer d a m les membranes, une protiine d e 45 acides aminks a perdu toutes propriktes lors de la rkplication virale in vitro.
L'association membranaire et
I'intkgrite structurale de Vpu semblent mutuellement requises pour L'activitk biologique de la prodine. Comme nous venons de le voir et tel que prksentk dam la figure 1 de cette discussion, Vpu agit dans des compartiments cellulaires distincts tout au long de son transit intracellulaire, Ainsi, peu apr&s sa synthhe, Vpu induit la degradation des mol&ules CD4 dam le RE. Par la suite, la protiine est tramportee vers l'appareil de Golgi OD elle s'y accumule en abondance (Klimkait et al., 1990). EIle semble alors s1associc5eB des v&icules qui sont rkparties de manitke disparate dms le cytopiasme de la celIule. Bien que la presence de Vpu 2 la membrane plasmique de la cellule reste encore 2 dkrnontrer, on ne peut toutefois en exclure la possibilitk. La proteine peut affecter les concentrations de gp120 exprimkes A la surface de la cellule infectke et faciliter le relgchement de la proghie virale. Tel que nous venons de le decrire, nos resultats suggbrent que ses differentes fonctions sont modulkes par deux domaines distincts. La phosphorylation des risidus serine localisks sur la rkgion Cterminale de Vpu semble jouer un r6le particuliirement dans sa capaciti B induire la dkgradation de CD4 dans le RE et B diminuer les effets cytopathiques du virus. Tandis que la r6gion N-terminale de la protkine semble prgsenter des determinants necessaires ii sa capacit6 de faciliter le relgchernent de particules virales 2 la surface des cellules infectees.
Figure 1 . Reprkntation schhatique du transit intracellulaire de la p r o t h e Vpu du VIH-1. (modifiee de la refirence Geleziunas et aL , 1994)
Au cours de l'infection in vitro par le virus vpu-, on peut denoter l'accumulation de vacuoles renfermant des particules virales matures et immatures
dam le cytoplasme des cellules (Klimkait et al., 1990, Yao et al., 1993). On peut kgalement observer la retention de nombreuses particules bourgeonnants au niveau de la membrane plasmique des cellules infectbes. Ces phknombes peuvent Etre potentiellement toxiques pour la cellule.
Ainsi, Vpu semble privenir le
bourgeonnernent intracytoplasmique des particules virales etlou pennettre un relachemnet efficace des virions de la surface des cellules infectkes. Par ailleurs, bon nombre de travaux ont montrk que les effets cytopathiques du virus etaient attdnuis en presence de Vpu (Klimkait et al., 1990; Terwilliger et al., 1990; Yao et al., 1993, Fnborg er al., 1995). La protiine est capable de dirninuer le taux de formation de syncytium e n culture. Cette propriCtC semble ftre le resultat d'une diminution de l'expression de la gp 120 B la surface des cellules infect& (Yao et al., 1993). En effet, il est bien c o m u que la glycoproteine de surface gp120 peut moduler la fusion de nombreuses cellules via son interaction avec le rkcepteur CD4 et ainsi former des cellules gkantes multinucl6es dont la durke de vie est lirnitk.
En revanche,
l'expression de Vpu ne semble pas altdrer le taux de mort cellulaire individuelle survenant suite
la rdplication du VIH-1. Nos travaux nous permettent maintenant
de mieux concevoir les phenotypes observQ. Il est clair que Vpu peut influencer de
manikre disparate le taux de replication v i d e dam divers types cellulaires. Grace Zi l'expression de Vpu, le VIH- 1 semble avoir ddveloppb un stratag5me lui permettant
la fois de persister dans la cellule hcte, en retardant la mort cellulaire
et facilitant l'exportation virale. Bien qu'elle ne soit pas indispensable il la replication in vitro, environ 30 B 40% des individus en phase initiale de l'infection p r k n t e n t des anticorps contre Vpu (Scheinder et al., 1990; Reiss et al., 1990). Sa presence dans les diffkrents isolats du VM-1 peut se reveler un atout dans la diss6mination virale. Des etudes recentes ont d'ailleurs montre l'importance de Vpu dans l'ttablissement
d'une infection productive dans les monocytes/macrophages (Balleit et al., 1994). Ces derniers sont perGus comme l'une des cibles majeures du VIH-1 et semblent particuli5rement dfractaires aux effets cytopathiques du virus. Il est B noter que l'on peut observer de nombreuses particules virales associkes B des vacuoles intracytoplasmiques, confdrant ainsi aux macrophages le statut de reservoir du W1. L'expression de Vpu peut donc s'avkrer importante dam la virkrnie si l'on consid5re qu'approximativement 1/1000 des macrophages du sang periphirique sont infect& par le VM-1 chez les individus asymptomatiques (Bagasra et al., 1992). La proprietk que poss&deLa protiine B faciliter le relschement de la progenie virale pourrait s'averer culminante dans la progression du cycle d'infection dam I'organisme.
Par ailleurs, il est frappant de constater que le r6le de Vpu dans la replication virale se rkvgle moins important dans des lymphocytes T du sang pdripMrique, n'influenpnt que tr6s peu le taux de reliichement de particules virales (BaIIeit et al., 1994). La presence de Vpu pourrait avoir une toute autre importance lors de I'infection de cellules CD4+. On sait que le VIH-1 peut attinuer de multiples fagons l'expression de CD4 5 la membrane plasmique (Klatzmann et al., 1984; Stevenson et al., 1987; Clapham st al., 1987). Griice B Vpu, le virus s'est probablernent pourvu
d'un autre moyen pour moduler l'expression de CD4 en induisant sa degradation dam le RE. De nombreux travaux ont rev616 que la protiine Nef du VIH-1 pouvait induire l'internalisation et la dkgradation des mol6cules CD4 exprimees B la surface des cellules (Garcia et Miller, 1991; Aiken et al., 1994; Anderson et al., 1994). Nef possede la capaciti d'interagir autant avec la molkcule CD4 que la p561ck. Suite B ces interactions, cette protkine semble inhiber l'activation et la prolifiration normaie des lymphocytes T infect& (Niedennan et al., 1992; Greenway et al., 1994; Greenway et al., 1995). Bien qu'aucune 6tude n'a encore d6montrt5 une diminution de l'expression
du ricepteur CD4 a la membrane plasrnique en prksence de Vpu, la proteine pourrait
de manikre sirnilaire 2 Nef influencer plusieurs aspects physiologiques de la cellule au cours de I'infection virale.
Ainsi, en altirant les phinomines d'activation
cellulaire, la protCine Vpu pourrait promouvoir la survie des cellules CD4+ infectkes dans l'organisme. L'environnement lymphocytaire normal B l'etat de repos s1av6re non permissif au VIH- 1 et inapte B une surinfection qui est bien souvent associee B la mort cellulaire. De plus, en induisant la degradation des molCcules CD4, Vpu dkstabilise les complexes gp160/CD4 qui se f o m e n t d a m Ie RE (Willey et al., 1992a; Willey et al., 1992b). Ces complexes peuvent bloquer les pores nuclc5aires et
sont potentiellement toxiques pour la cellule (Koga et al., 1991). Plusieurs scenarios peuvent Ctre envisager suite i la degradation des molkcules CD4 induite par Vpu et demanderont une analyse plus approfondie. Les m&xnismes mole'culaires entourant Ies propriktis de Vpu restent encore Enigmatiques. Ainsi, afin de mieux cerner le mecanisme mol6culaire de la protkine, nous avons entarn6 d a m nos travaux subsCquents une 6tude exhaustive du phenomene de degradation de la mol6cule CD4 observd lors de l'expression de Vpu. Plusieurs groupes ont rapport6 l'importance de la queue intracytoplasrnique de CD4 dans les processus conduisants B sa proteolyse (Vincent et al., 1993; Chen er al., 1993; Lenburg et Landau, 1993; Willey et al., 1993). Nous avons donc procidt dans
le troisieme article de cette thhse (chapitre 4), 2 une analyse par mutagdnkse des rc5sidus e t des motifs sur la queue intracytoplasrnique de CD4 pouvant Stre necessaires au processus spkcifique de degradation induit par Vpu. Mon apport ii cette Ctude effectuee par Mr. Xiao-Jian Yao et rdalisde dans Ie cadre de son projet de doctorat fut dam la conception et Ia construction de certains des mutants de CD4 . L'ensernble des etudes rkaliskes par plusieurs groupes ont permi de dklimiter dam la queue intracytoplamisque de GD4 une region minimale entre les acides aminis 414 et 419 confkrant
Zi
la moldcule sa sensibiiite au mode d'action de Vpu
(Vincent et al., 1993; Chen et al., 1993; Lenburg et Landau, 1993; Willey et al.,
1993). Notre analyse effectuee sur une sene de mutants de dklktion de la queue intracytoplasmique de CD4 confirme ses donnies. Une rkgion entre les acides aminks 41 1 et 419 est apparament nkcessaire au processus de degradation induit par Vpu (figure I). D'autre part, I'analyse systkmatique par mutagen5se des r6sidus presents dam cette region a r6vkl6 que la sequence primaire ne peut contribuer 2t elle seule B la degradation des molCcules CD4. En effet, la substitution de 6 des 9 acides amin& Iocalis6s dam cette sequence n'ont pas empSch6 Vpu d'induire la degradation de CD4 (figure 2). De plus, plusieurs motifs dans la queue intracytoplasmique de CD4 jouant des r8les pr6ponddrants dam I'activitC biologique de la mol6cule dam les cellules T se sont egalernent avkrks non essentiels au mode d'action de Vpu. En rCsumk, la substitution des sites de phosphorylation de CD4 (sirines en positions
410, 415 et/ou 4 13) et celle du domaine d'interaction 2 la p561ck (cystdines en positions 420 et 422) n'ont pas aboli la degradation de Ia proteine induite par Vpu. Des r6sultats similaires ont kt6 obtenus lorsque un motif leucine-leucine en positions
413 et 414 de la queue intracytoplasmique, qui est requis dam Itinternalisation et dans la dkgradation des mol~culesCD4 par la proteine Nef, a kt6 substituk. En revanche, nous demontrons qu'une structure secondaire en alpha-h6lice prddite de se former dam cette rkgion de la queue intracytoplasmique, qui fut auparavant decrite par Shin et al. (1991), se r6vkle importante dans la capacite de Vpu h induire la dkgradation de CD4. Des mutations gknirkes afin d'empCcher potentiellement la formation de cette structure qui s'dtend de l'arginine en position
402 ii la thrdonine en position 419 rendent les molkcules CD4 r6sistantes
I'effet de
Vpu (figure 3). La conformation de la queue intracytoplasmique de CD4 semble
donc determinante dans le(s) processus rnkdiee par Vpu menant A la proteolyse de la mol6cule. Les travaux rialis& par Bour et al. (1995) ont grandement contribui A notre cornprkhension de I'effet spbcifique de Vpu sur CD4.
Leurs etudes de co-
immunoprkcipitation ont revel6 la formation de complexes intracellulaires entre les deux protiines. Cette interaction semble se produire A la suite de leur synth5se puisqu'elle s'observe d a m le RE et est un prbrequis B la prot6olyse de CD4. Cependant, I'activation d'un second mecanisme mol6culaire semble nkcessaire afin de promouvoir cette dkgradation. En effet, la del6tion de 5 acides amin6s dans la region 47 B 52 de Vpu ou la substitution des sites de phosphorylation, qui dam les deux cas rendent la protdine inapte A induire la degradation de CD4, nlempEche toutefois pas la formation de complexes VpuKD4.
De plus, la queue
intracytoplasrnique de CD4 renferme des determinants essentiels
la liaison de Vpu
puisqu'en outre leurs analyses moment que la delition des 22 derniers acides aminks de cette region de la mol6cule aboiit leur liaison. Dans le quatrihme et dernier article de cette th5se (chapitre 5 ) , nous prbsentons une analyse de structure ayant pour but de dkfinir les CKments requis dam la liaison entre Vpu et CD4. Nos resultats suggerent fortement que la phosphorylation de Vpu joue un r6le tant dans Itinteraction de la protdine avec CD4 que dam le processus menant A la dkgradation de cette dernikre. La substitution ponctuelle de la Ser 56 de Vpu ne permet plus la formation de complexes VpuKD4 (figure 1). Pourtant, la protCine conserve la capacite de liaison lorsque ces deux sites de phosphorylation sont mutes. On ne peut donc expiiquer cette observation que par la simple substitution de la Ser 56.
La phosphorylation est I'une des modifications post-traductionnelles omniprksentes dans la regulation de divers processus biologiques. Quoique t&s peu
documente, on assume que dam la majorit6 des cas la molkcule adopte une certaine conformation suivant sa phosphorylation pouvant augmenter ou inhiber son activite biologique (Sprang et al., 1988; Hurley et al., 1990). Ainsi, l'enzyme glycoghe phosphorylase, dont la structure tridirnensiomelle a kt6 dkfinie tant sous sa forme phosphorylke que d6phosphorylde, est I'un des exemples concrets de ce tdchanisme de regulation (Sprang et al., 1988). Bri&ement,
suite au processus de
phosphorylation, on observe des changements de conformation dans les r6gions N- et C-terminales de la proteine qui vont permettre la formation de dimgres et du meme coup alterer sa capacid de liaison avec ses substrats. Un p h h o m h e semblable semble se produire egdement avec la p56"
qui posdde tout comme Vpu la capacit6
de lier la moldcule CD4. En effet, M a t de phosphorylation de la tyrosine en position
505 (Tyr 505) de la p561ck peut moduler par un changement de conformation I'affinite de I'enzyme pour ses divers substrats intracellulaire (REF). D'aiUeurs, de nombreux travaux ont montre que I'hyperphosphorylation de la Tyr 505 rdprime I'activite enzymatique de la p561ck (Amreim et Sefton, 1988;Abraham et Veillette,
1990; Abraham et a[., 1991). On peut donc envisager la possibilitk que la phosphorylation de la Ser 56 puisse avoir un effet similaire sur la liaison de Vpu avec CD4.
La conformation adoptie par Vpu lorsque la Ser 56 n'est pas
phosphorylde semble emecher la formation de complexes avec CD4 et pourrait de manicre pr6somptueuse rEvEler une interaction de nature transitoire entre Ies deux proteines. Cette cornparaison avec la p561ck va plus loin puisque la phosphorylation d'une autre tyrosine en position 394 (Tyr 394) semble augmenter I'activite enzymatique de la protdine. La Tyr 394 est autophosphorylde B un trks faible niveau et sa substitution inhibe la fonction de la p561ck. Or, nos resultats montrent que la mutation de la Ser 52 permet la formation de complexes mais affecte I1activit6de Vpu. I1 est intkressant de noter que la phosphorylation de la Ser 56 par la CKII est favoriske par rapport B la Ser 52 (Schubert et at., 1994). On peut donc postuler que
la phosphorylation siquentielle des residus serine 52 et 56 sur Vpu permettra tour 2 tour par des changements de conformation sa liaison B CD4 dam le RE et son activation, particulierement dam le phCnornkne de dkgradation de CD4. Enfin, notre analyse de mutagkn2se du domaine intracytoplasmique de CD4 montre entre autres que I1int6grit6de la structure en alpha-helice prCdite de se former dam cette region est importante pour la stabilite des complexes VpuKD4 (chapitre 5 ) . En effet, des mutations altkrant cette structure en alpha-hilice diminue leur
affinite l'une pour l'autre (figure 3). L'ensemble des rksultats prksentCs dans cette article nous laissent envisager la possibilite que cette interaction soit ddpendante en majeur partie de la conformation tertiaire des d e w proteines. Recernrnent, les etudes de NMR rialisies par Wray et al. (1995) ont dtmontrk que la rEgion C-terminale de Vpu forme deux alpha-helices bien definis (rksidus 30 B 50 et 57 3 69, respectivement) liees entre elles par une boucle flexible renfermant les sites de phosphorylation. Ces alpha-helices ont la particularit6 de contenir un nombre dgal d'acides aminis de charge opposk. Selon les auteurs, la phosphorylation de Vpu pourrait augmenter la flexibilite et l'acidite de la boucle dtjB pretminente en raison des rtsidus glutamate et aspartate en positions 50, 5 1, 55, 57 et 59. Ce ph6nombne vient soutenir noue interpretation des rtsultats prdsentis dam le quatribme article de cette these. Nous croyons que Vpu suite ii sa synthkse adopte une conformation lui pennettant de se replier sur la queue intracytoplasmique de
CD4 pour former une poche possiblement stabilisee par des liens hydrophobes et/ou des ponts salin. 11 est plausible de concevoir une telle interaction entre les deux proteines puisque l'on peut noter la presence de regions aptes ii ce phbnom6ne. En effet, la skgrkgation ordonnee de rksidus basiques et hydrophobes sur la premiere alpha-h6lice de Vpu (acides amines 38 2 46) et le partage 6quitable des charges positives et negatives sur chacune des alpha-helices pourrait non seulement avoir un
r6le important dans la multirn6risation de la proteine mais paraissent aussi propices ii la formation de liaisons faibles non covalentes avec CD4. Comme nous I'avons rnentiomd auparavant, M a t de phosphorylation de Vpu sernble activer le mecanisme moliculaire requis dans la d6gradation spicifique de CD4.
De plus, la
phosphorylation de Vpu pourrait causer un changement de conformation de la protkine entrafnant une baisse d'affiniti entre les d e u prot6ines et permettre ainsi de rkviler des 61Cments structuraux et/ou des motifs sur la molkule CD4 essentiels B sa prodolyse. En r&um6, la protkine Vpu peut interagir avec les moltcules CD4 dans le RE et induire spicifiquement l e u degradation. La demi-vie de CD4 passe de 6 heures 5 environ 10 minutes en pr6sence de Vpu (Willey et al., 1992b). La simple retention de la molkcule CD4 par Vpu ne peut 2 elle seule entrdiner sa degradation d a m le RE. Cornme nous venons de le voir, la formation de complexes VpuKD4 n'est pas suffisante pour engendrer la proteolyse de CD4. La phosphorylation de Vpu semble promouvoir les &tapessubsiquentes ndcessaires au processus (Bour et al., 1995). Le
RE est un centre de contr6le dans la biosynthbe et la maturation des protiines naissantes. Ainsi, la structure anormale des protkines provoque leur ritention dans le
RE et les rendent plus sensibles aux attaques protkolytiques de nombreux syst6mes encore ma1 caract6rist5s (Doms et nL, 1993). LI est donc possible que Vpu dirige CD4 vers l'une des quelconques voies de ddgradation suivant s a synth2se. En revanche, Vpu pourrait lui meme activer un systkme protdolytique suivant sa phosphorylation, induisant la dkgradation sdlective de CD4. Dans les deux cas, ces processus laissent envisager une compliciti directe ou indirecte entre Vpu et I'environnernent celldaire.
Par ailleurs, au cours d'etudes fonctio~ellesin vitro, Gottlinger et al. (1993) ont dbmontr6 que Vpu pouvait augmenter le relkhement de particules virales
chim2res fomees ii partir des protkines Gag du VIH-2, mais kgalement de celles de &trovirus aussi divergents que le virus visna de mouton et le virus de la leucdmie murine. Il est interessant de noter que ses r6trovirus presentent non seulement peu d'homologie au niveau de la polyprott5ine Gag, mais different egalement dam leur mode d'assemblage.
I1 est donc fort peu probable que Vpu interagisse
spicifiquernent avec des prodines virales afin d'augmenter le reliichement de virions matures.
Les itudes irnmunohistochimiques r6alisCes par Yao et al., (1993)
montrent clairement que l'expression de Vpu permet un bourgeonnement plus efficace des particules virales autrement retenues B la surface de la cellule infectte et dam des vacuoles intracytoplasrniques. Vpu n'affecte ni Ia synthcse ni l'expression des protiines du VIH-1 mais semble plut6t modifier une voie cellulaire et/ou la physiologie de la cellule permettant de faciliter le bourgeonnement de la progknie virale. Ces observations laissent egalement entrevoir la possibilite que la proteine puisse medier ses differentes fonctions en affectant directement ou indirectement l'enviromement de la cellule h6te. Certains groupes ont fait &at d'une homologie de structure existant entre Vpu et la proteine M2 du virus d'influenza (Klimkait et al., 1990; Schubert et Strebel,
1994). M2 est une protCine membranaire intrinskque de 97 acides aminds que I'on retrouve en abondance 2i la surface des cellules infectkes (Lamb et al., 1985). Sa region N-terminale prksente dewc domaines: une portion extracellulaire de 24 acides axnines et un domaine transrnembranaire de 19 acides amin6s, tandis que sa region C-terminale renfenne son domaine cytoplasrnique. Cette proteine est capable de former minimdement des complexes homo-tdtram&iques et elle pourrait bien &re phosphorylke par la CKII s w des sites encore non identifies (Schubert et al., 1994). Recemment, des 6tudes r6alis6es dam des oocytes de x&noprts laevis ont permis de demontrer que M2 posskdait une activite de canal ionique selective aux ions sodium (Na+) (Pinto et nl., 1992). GrZice 3 cette proprietk, la prot6ine est capable de
promouvoir une baisse de pH au niveau des endosomes et du syst5me trans-Golgi de la cellule infectee nkcessaire au changement de conformation d'une composante de I'enveloppe v i d e , l'hemagglutinine (HA). Cette modification est essentielle aux &apes subsequentes du cycle d'infection de l'influenza revue par: Wharton et al. (1990). La region transmembranaire de M2 semblerait composer le pore du canal
ionique puisque des mutations specifiques dans cette portion de la proteine altsrent son activitE biologique (Wang et al., 1993; Holsinger, et al., 1994). Contrairement B
la proteine M2, Vpu n'est pas exprime ou ne se retrouve qu'en tr2s faible quantite A la surface de la cellule atttnuant la possibilite qu'elle puisse former un canal ionique typique
a la membrane plasmique.
Nianmoins, on peut envisager la formation de
pores membranaires par celle-ci qui, tout cornme M2, est capable d'hornooligom6risation. Ce phdnomkne pourrait &re determinant dans I'activite biologique de Vpu dans Ia cellule, modulant ainsi par un ou plusieurs micanismes rnol&xlaires encore non 6lucidis les diverses fonctions attribukes 2 son expression. Nos travaux de mutagensse sur la region N-terminale de Vpu (chapitre 3) nous ont permis de rnontrer son importance non seulement dam l'ancrage de la protkine mais &dement dans sa capacid de faciliter le relschement de particules virales. Bien qu'aucune etude n'a encore associd jusqu'ii ce jour une activite de canal ionique B Vpu, les avenues d'un tel phinomine devront etre Blabore'es plus en detail au cours de travaux futurs afin de m i e u comprendre les diverses fonctions de Vpu.
Ainsi, des itudes prkliminaires effectukes dam notre Iaboratoire sur des cellules exprimant de mani2re constitutive Vpu ont dimonfri que la protkine pouvait augrnenter la fluidit6 membranaire. De plus, des analyses de cytofluom6trie en flu ont revile que la protkine pouvait affecter la concentration de calcium (Ca++) intracellulaire dam ces cellules (resultats non publiCs). En effet, la stimulation induit une vitro de la cellule par des mitog2nes cornme la phytoh6maggl~tinine~ augmentation du Ca++ libre intracellulaire.
Griice A l'utilisation d'une sonde
fluorescente, le Fluo-3, il nous est possible de mesurer ce phknomkne lors d'analyses de cytofluom6trie en flux. Le Fluo-3 s'avsre un excellent indicateur des fluctuations d'ions calciques libre dam Ie RE (Michelangeli, 1991). Tandis que I'on observe une augmentation de la concentration de Caw libre dans la cellule parentale suivant la rkponse mitogBnique, le niveau de Ca++ libre dam le RE des cellules exprimant Vpu reste B un niveau basal. Bien que la cause et I'amplitude de ces phenomhes nous soient encore inconnue, ils pourraient influencer l'activid biologique de la proteine. Plusieurs Ctudes ont montr6 que la dkgradation de divers proteines dans le RE est Ctroitement like ii la diminution de Ca++ dam le compartiment cellulaire (Lodish et Kong, 1990; Wileman et al., 1991; Lodish et a[., 1992). La degradation de
rkcepteurs cellulaires tels le CD3 et le TCR est selective et sernble etre causie par la conformation qu'adopte les protiines en rdponse la baisse du niveau de Ca++ libre dans le RE (Wileman et al., 1991). Par ailleurs, les travaux de Bonifacino et al. ( 1990) demonwent que la region transmembranaire de la molc5cule TCR joue un r6le
important dans sa prot6olyse intracellulaire.
Bien qu'aucune Ctude n'a encore
rapportCe un tel processus sur la mol6cule CD4, on peut n4anmoins envisager la possibilite que Vpu puisse induire indirectement sa ddgradation par la baisse de Ca* libre dans le RE. Les travaux realis& tour ii tour par Raja et al. (1994) et Buoconore et ai. (1994) ont montrk ii h i d e de mol~culeschimh-es de CD4 que la r6gion
transmembranaire poss&dedes determinants importants dam la degradation induite par Vpu.
Nos Btudes de mutagenhe sur la queue intracytoplasmique de CD4
(chapitres 4 et 5) ont riv&5 l'importance d'une structure secondaire en alpha-helice
dans les processus conduisants la molecule B sa dkgradation. L'ensernble de ces observations font que tout c o m e les molCcules CD3 et TCR,la degradation de CD4 pourrait &re le resultat d'une baisse de Ca* libre dans le R E induite par I'expression de Vpu.
Par ailleurs, le r6le d~ Ca++ dans les processus d'exocytose dans la cellule est 6galement t r h bien document6 (revue par Almers, 1990). Bri&vement, lors de l'augmentation du Ca*+ dam le cytoplasme, on observe le transport massif de v6sicules de sicrktion de I'appareil de Golgi vers la surface de la cellule oh elles fusionneront avec la membrane plasmique pour y lib6rer leur contenu.
La
diminution du niveau de Ca++ dans Ie RE aurait pour consiquence d'activer un signal provoquant un influx compensatoire d'ions calciques ii travers la membrane plasrnique (Putney, 1990). Ainsi, il est B noter qu'en absence de Vpu on remarque une accumulation importante de vacuoles contenants un grand nornbre de particules virales matures et immatures dans Ie cytoplasme et B la membrane plasrnique de la cellule infectie (Klimbait et al., 1990; Yao et al., 1993). De plus, la prksence de Vpu dans des vksicules lors de son transit cellulaire (figure 1 de la discussion) Iaisse entrevoir un r61e de la protkine dans les processus d'exocytose. ~ t a n donne t la capacitC de Vpu B augmenter le reliichement de diffkrents ritrovirus B la surface des cellules (Gottlinger et al., 1993) et comrne la region N-terminale de la p r o t h e semble jouer un role dans la fonction de reliichernent (chapitre 3), une fluctuation directe ou indirect de la concentration de Ca*
intracellulaire pourrait concilier ses
observations. Finalement, outre le r6le prepondkrant du Ca++ dans la regulation de divers processus biologiques, plusieurs 6tudes ont r6vili son importance dam le cycle replicatif de nombreux virus dont le VIH-1- En effet, les travaux de Dimitrov et al. (1993) ont montrt5 que l'absence d'ions calciques lors de l'infection par le VM-1 inhibe la formation de syncytium en culture. Toutefois, le r61e du Ca++ dans les phdnornhes de fusion mernbranaire reste encore higmatique. Par ailleurs, une itude effectu6e sur le virus Sendai, un virus envelopp6 de la famille des paramyxoviridae, a d6montr6 que la diminution du niveau de Ca++ d a m le R E emptche le transport des glycoprotdines d'enveloppe de I'appareil de Golgi B la
surface de la cellule sans toutefois altCre'e la synthbe des protiines virales (Kawakita et Ono, 1994). Peut on envisager un phinomhe similaire lors d e la replication du
VIH-1 en prisence de Vpu? Nous avons montre lors de nos e'tudes que la proteine pouvait moduler la concentration d e la gp120 exprimee B la surface de la cellule. Nous ne pouvons exclure la possibilitt5 que cette observation soit like au relgchement accru de particules virales en prisence de Vpu.
Cependant, nos re'sultats de
mutag$n&se(chapitre 2) sugg2rent que les deux fonctions sont independantes l'une de l'autre. 11 devient donc attrayant d'associer les observations rapporte'es par Dimitrov et al. (1993) aux effets d e Vpu sur la formation de syncytium.
En affectant
indirectement le Ca++ intracellulaire d a m le RE, l'expression de Vpu pourrait moduler le transit cellulaire des glycoproteines d'enveloppe et ainsi diminuer le taux de formation de syncytiurn lors de l'infection v i d e .
Une etude plus dhille'e sera ne'cessaire a f h de rnieux caractkriser les effets de l'expression de Vpu sur la physiologie de la cellule. Meme si Vpu ne possgde pas
les caract6ristiques d'un canal ionique, elle pourrait tout au moins par la formation de structures complexes dans les membranes (ER, visiculaire ou plasmique), affecter la fluctuations d'ions calciques d a m la cellule directement ou indirectement par un mkcansime mol6culaire encore inditemine.
CHAPITRE 7 Conclusions et perspectives
Le virus du SIDA a su divelopper au cows de son ivolution de multiples statag6mes afin de persister et de rendre sa reproduction chez I'h6te des plus efficaces. L'une des grandes complCxit& du cycle replicatif du VM-1 rkside dam son organisation ghomique particulih-e. Ainsi, des proteines accessoires cornrne Vif, Vpr Vpu et Nef, ne sont pas essentielles pour la rkplication virale in vitm mais pourraient jouer un r61e important dans la propagation v i d e in vivo. Les travaux rkalises au cours de ce projet de recherche nous ont pennis de mieux comprendre l'apport fonctionnel d a m la replication du VIH-1 de I'une de ces protiines accessoires, Vpu. Nous avons ddrnontre que la proteine Vpu pouvait mCdier diffirentes fonctions lors de l'infection par le VM-1 et possiblement au dipend de la cellule. Notre dtude de mutag6nSse a r6vkl6 i'existence de deux domaines distincts sur la proteine qui semblerait moduler son activitC biologique (figure 1 de la conclusion). Dans un premier temps, nous avons monut5 que la phosphorylation de rksidus serines IocalisCs dans la rkgion C-tenninale de Vpu joue un r6le important dans sa capacitd de diminuer les effets cytopathiques encourues lors de l'infection v i d e in vitro. La reduction du taux de formation de syncytium que l'on observe en presence de Vpu pourrait Ctre le resultat d'une modulation de I'expression de la gp120 ii la surface des cellules infectees induite par la protdine. D'autres part, l'etat de phosphorylation de Vpu semble Stre rggulateur de la liaison et de la dkgradation specifique des moldcules CD4 d a m le RE. Le second domaine actif, sit& au niveau de la region Nterminale, contient des ddtemiinants importants dans la capacitii de Vpu de faciliter le relgchement de la progdnie virale. Ce domaine permet dgalement I'ancrage de la proteine, indispensable
son activitk biologique. Ainsi, tout au long de son transit
dam la cellule infectke, Vpu semble agir distincternent par I'intermddiaire de ses deux domaines.
Nos travaux et quelques ividences rapportees jusqu'g prisent, laissent toutefois presumer que l'intigrite stnrcturale de Vpu est dCterminante dam son activitC. Bien que la region N-terminale sernble jouer un r61e dam la capacite de faciliter le relschement de particules virales, une prot6ine Vpu tronquee n'exprimant que les 45 premiers acides amines ne peut assumer cette fonction. Par ailleurs, il est fort probable qu'un seul micanisme moIkculaire encore non identifie soit Zi la base de I'activit6 biologique de la proteine.
Les rksultats de Gottlinger et al. (1993)
dkmontrent clairement que la fonction de Vpu sur le reliichement de particules virales n'est pas specifique au VIH-1, se pretant aussi B divers rdtrovirus. Nous envisageons donc la possibilite que Vpu puisse agir sur des composantes intrinsgques de la cellule afin de midier ces fonctions.
Plusieurs points restent encore
inigmatiques et dernandent des analyses structurales et fonctionnelles plus approfondies. Ainsi, dam les etudes subsCquentes de mutagkn*se, nous esperons identifier B prime abord les acides aminks ou motifs sur Vpu qui pourraient Ctre requis dam la rnultimCrisation de la protiine.
Comme nous I'avons vu dans les chapitres
preckdents, deux alpha-hdlices prkdites de se former dam la rkgion C-terminale (risidus 30 B 50 et 57
Zi
69) apparaissent c o m e des sites propices A cette analyse
(figure 1 de la conclusion). L'identification de telles siquences nous permettra donc de mieux ddfinir la nature encore ndbuleuse de I'oligom6risation et son importance
dans l'activite biologique de Vpu. De plus, nous avons proposC que les deux alpha-hklices dam la region Cterminale de Vpu pouvaient igzlement Etre impliqudes dam la stabilitk des liaisons avec la mol6cule CD4 dam le RE. Une meilleure caractkrisation des complexes VpuKD4 sera importante dans notre comprkhension des phenom&nesde destruction de CD4 et dans leurs ripercussions biologiques au niveau de la physiologie de la
cellule infectCe. Quel est la contribution de Vpu dam la diminution d'expression de surface du recepteur CD4 au cours de l'infection virale? D'autre part, L'activation cellulaire n'a lieu que si la moKcule CD4 est associB 2 la p561ck. Nous avons montr6 que la formation de complexes VpuKD4 n'itait pas dependante des deux risidus cystiines situis en positions 420 et 422 de la queue intracytoplasmique de CD4. Cependant, qu'advient-il de la liaison avec la p56fck et du processus d'activation de la cellule.
Doit-on s'attendre 2 la formation de complexes trimiriques entre les
protdines ou encore 2 un encombrement empechant la liaison de la p56lck. Des itudes de transcomplimentation d a m les cellules COS qui sont depourvues d'expression de CD4 et de la p56Lck permettraient de repondre en parti 2 de telles questions. 11 serait aussi intiressant d'analyser la stimulation in vitro des cellules CD4+ en presence et en absence de Vpu. L'ensemble de ces donnkes pourraient etre importantes d a m la comprehension des phenomhes de persistence et de transmission v i d e productive chez l1h6te. Par ailleurs, nos resultats dimontrent que la phosphorylation de Vpu est
importante pour induire une diminution de l'expression de la gp120 B la membrane plasmique. Tandis que Ies observations rapportkes par Schubert et Strebel (1994) indiquent que cette modification post-traductiomelle est importante dam l'induction des processus conduisant A la protkolyse spdcifique de CD4 par Vpu. Puisque la moldcule CD4 peut former des complexes dam le RE avec la glycoprot6ine gpl60, peut-on faire une correlation entre les deux fonctions de Vpu? Nous croyons que malgrt5 la formation de tels complexes, les deux phknomenes sont independants et relkvent du mode d'action gbnirale de Vpu. Toutefois, afin mieux comprendre l'activite biologique de la protbine, il serait important d'analyser le transit de la gp I60 lors de la dkgradation de CD4.
Finalement, comme nous l'avons vu prdcddemrnent, I'analogie de structure avec la protiine M2 d'Muenza ainsi que nos resultats preliminaires dam des Ligndes cellulaires exprimant Vpu, laissent entrevoir un effet de la proteine sur la fluctuation d'ions calciques dans le RE. La nature de cet effet nous est encore inconnu et demande une analyse plus detaillde des courants ioniques intracellulaire. Ainsi, l'utilisation d'ionophores, c o m e le bromo-ionophore A23187 qui poss5de la proprikt6 d'augmenter la perxn6abilitC de la membrane plasmique aux cations Ca++ et
Mg*, ou encore d'inhibiteurs des pompes calciques ATPase comme la thapsigargine qui agit au niveau de la membrane du RE, powait nous permettre de comprendre du
point de vue physiologique ce phknomsne dam les lignees cellulaires exprimant Vpu- D'autre part, tout comme dam les etudes rCalis6es sur M2, l'utilisation des oocytes peut se rev&lerune approche intdressante du point de vue moI6culaire afin de caracteriser les flux ioniques intracellulaires alt6r6s par Vpu. Ce systkme cellulaire est attrayant en raison de sa vaste utilisation dans I'etude des courants ioniques et des potentiels de membrane. D'ailleurs, des travaux prt5liminaires nous ont permis de montrer par irnmunopr6cipitation que I'on pouvait exprimer Vpu dans des oocytes x6nopus laevis (rksultats non publids). En rdpondant 2i ces divers questions, nous
espirons definir le micanisme d'action de Vpu a f h de concilier ses fonctions au cows de l'infection par le VIH-1. Puisque Vpu n'est pas essentiel B la replication v i d e in vitro, il est difficile de congevoir une implication thirapeutique ii nos Ctudes. Cependant, tous les divers du virus du SIDA doivent Ctre entrevues. Peut-on expliquer la dissemination mondiale plus rapide du VM-1 par l'absence de Vpu chez le VIH de serotype 2? Le VM-1 semble avoir dt5veloppt5 par l'expression de Vpu un moyen d'attbnuer les effets cytopathiques encourues Iors de sa r6plication lui permettant ainsi d'optimiser sa production v i d e pendant une pdriode prolongk. Que ce soit en facilitant le relschement de la progenie virale ou en riduisant la formation de syncytium in vitro,
de tels phtnomhes doivent Ctre envisager dam la propagation du virus. Une meilleur perception du r61e de Vpu et son mode d'action permettra d'evaluer la contribution de ce gbne dam la propagation virale et dans la pathogenbse associt i
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