Human Immunodeficiency Virus Type 1 nef ... - Journal of Virology

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Mar 24, 1992 - and spleen DNA of a second child (patient RH; lanes 3 and. 4) ran as a band ...... Budka, H., C. A. Wiley, P. Kleihues, J. Artigas, A. K. Asbury,.
Vol. 66, No. 9

JOURNAL OF VIROLOGY, Sept. 1992, P. 5256-5264

0022-538X/92/095256-09$02.00/0 Copyright © 1992, American Society for Microbiology

Human Immunodeficiency Virus Type 1 nef Quasispecies in Pathological Tissue BENJAMIN M. BLUMBERG, 13* LEON G. EPSTEIN,"12'3 YOSHIHIRO SAITO,' DOUGLAS CHEN,' LEROY R. SHARER,4 AND RITA ANAND5t

Laboratory of Molecular Neurovirology, Department of Neurology, 1 and Departments of Pediatrics3 and Microbiology & Immunology, 2 University of Rochester Medical Center, 601 Elmwood Avenue, Box 605, Rochester, New York 14642; Department of Pathology and Laboratory Medicine, Division of Neuropathology, UMDNJ-New Jersey Medical School, Newark, New Jersey 071034; and Laboratory of Retrovirology, Center for Biologics Evaluation and Research, Food and

Drug Administration, Bethesda, Maryland 208925 Received 24 March 1992/Accepted 17 June 1992

The role of the nef gene in human immunodeficiency virus type 1 (HIV-1) infection is poorly understood. To provide a basis for studies on the role of nef in AIDS, we used targeted polymerase chain reaction amplification and DNA sequencing to determine the structure of nef genes in pathologic tissue from HIV-l-infected children and adults. We find that the nef reading frame is open in 92% of clones derived from both brain and lymphocytic tissue of children, suggesting that nef is expressed in these tissues. One HIV-1 clone, BRVA, obtained by coculture from the brain of an adult AIDS patient with progressive dementia, was previously shown to contain a duplicated region in nef. We show here that similar duplications are widespread in both adults and children with AIDS. However, coculture strongly selects against the broad spectrum of nef quasispecies found in tissue. These findings suggest functional selection for nefquasispecies in pathologic tissues during HIV-1 infection of the human host.

Among the regulatory genes of human immunodeficiency virus type 1 (HIV-1), only the nef gene remains without a precisely defined function (27). The nef gene product, a myristylated 27-kDa protein (25), was thought to downregulate virus replication and to be important for viral latency (7, 57). However, nef expression is blocked by premature termination in some prototype strains (41), and nef was found to be dispensable for HIV-1 replication in cultured cell lines (26, 34). More recent experiments performed in primary cell cultures show that unblocked BRU nef accelerates HIV-1 replication in primary T lymphocytes (13) and that the natural ELI nef allele promotes efficient HIV-1 infection in primary peripheral blood mononuclear cells (58). Thus, nef appears to be a positive regulatory factor for HIV-1 replication in primary cells (11). The role of nef in HIV-1 infection, replication, and disease is currently being reassessed (see reference 28 and references therein for review). An experimental model for directly studying the role of nef in AIDS pathogenesis is unfortunately lacking, as HIV-1 has high specificity for tissues of its human host. However, the importance of nef expression for biological activity is suggested by experiments in which rhesus monkeys were infected with chimeric simian immunodeficiency virus (SIV) constructs containing open, prematurely terminated, or deleted nef genes (33). The SIV nef mutants containing a premature stop codon rapidly reverted to a full open reading frame and caused disease, while mutants with a deletion in the nef gene (that presumably cannot revert) did not produce clinical disease or pathological changes (4, 33, 50). These experiments demonstrate strong selection for SIV nef *

Corresponding author.

t Present address: Virology Study Section, Division of Research

Grants, National Institutes of Health, Bethesda, MD 20892.

5256

expression and function in vivo and suggest that HIV-1 nef similarly has functional importance in AIDS. Kestler et al. speculate that SIV nef expression allows an increased viral load in the host, leading to pathology (33). SIV and HIV are lentiviruses, whose hallmark is early invasion of the central nervous system, followed by viral persistence; the greatest load of SIV and HIV occurs in brain (50, 54) and is concentrated in productively infected multinucleated giant cells (35, 52). Since nef is expressed early in HIV-1 replication (47), the effect of nef protein activity in the central nervous system may be a critical determinant of disease. In order to provide a basis for studying the role of nef in AIDS, we investigated the structure of the nef gene in HIV-1 isolates cultured from adults and in DNA extracted from pathologic brain and spleen tissues of children, using targeted polymerase chain reaction (PCR) amplification and DNA sequencing. We find that the nef reading frame is normally open in clones derived directly from pathologic tissue, that duplications in nef are prevalent, and that coculture of HIV-1-infected tissue with mononuclear cells strongly selects against the broad spectrum of nef quasispecies found in vivo. We discuss why our findings suggest selection for nef function and the significance of nef quasispecies in pathologic tissue.

MATERUILS AND METHODS Patient materials. (i) Adult patients. Blood and tissue samples from patients NAl to NA5 (2), FC-L, Ni to N4, N9, Nll, and N12 (unpublished data) were collected between 1983 and 1986. These patients presented with primarily neurological symptoms and were selected for detailed molecular studies on this basis. The blood donor from whom isolates D-1, D-2, and D-3 were collected was described in

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BRVA duplication (9053) nef 12

8796

(9261) nef 13

-hb

I

RA-1

RA-2A

(8850)

(8986)

mIFF Is

L_

nef 13A (9289)

3'

LTR

\/7 I

.l

9498R 9532R

I

I_

FIG. 1. Schematic diagram of nef and overlapping 3' LTR region. Arrows denote oligonucleotide primers used for cloning, and sequencing; primer sequences are given in Materials and Methods. Numbering is according to the HXB2 heavy line. RA primers were used for targeted PCR amplification of the BRVA duplication region. Major insertion sites as heavy bars above the genome line; the MN deletion is shown as an asterisk.

PCR amplification, shown as the are shown (oversize)

genome,

references 1 and 56. A group of Zairean patients from whom isolates were collected was described in reference 55. Molecular studies of these isolates were carried out in several laboratories; see reference 41 and Table 1. (ii) Pediatric patients. Samples were collected from a large number of children seen at University Hospital and Children's Hospital, Newark, N.J. Some case descriptions and molecular studies have been published (51, 52); patient JR showed large amounts of HIV-1 DNA in brain tissue (54). HIV-1 DNA preparation. (i) Adult patients. High-molecular-weight HIV-1 DNA was prepared by coculture of phytohemagglutinin-stimulated HIV-1-infected peripheral blood lymphocytes (PBLs) with minced brain tissue, cerebrospinal fluid, or lymphocytes from adult patients with AIDS and extracted as previously described (1, 3, 55, 56). (ii) Pediatric patients. DNA was extracted directly from minced 0.1- to 0.4-g slices of selected frozen brain or spleen tissue by sodium dodecyl sulfate-proteinase K digestion followed by phenol extraction and was recovered by ethanol precipitation (17). Targeted nef gene amplification by PCR. (i) Adult patients. Targeted PCR amplification of the BRVA duplication region in nef was performed on the above-described DNAs as previously described (42), by using primers RAl (5'-AT GGGTGGCAAGTGGTCA) and RA2A (5'-ACTACTTGTG ATTGCTCC) that flank the duplication region. The expected band size is 138 bp. (ii) Pediatric patients. Nested primers flanking the nef gene were employed under PCR conditions as previously described (17). The outer primers were 5'-TTCGCCACATAC CTAGAAGAATAAGA and 5'-CCGCCCAGGCCACGCC TCCCT; the inner primers were 5'-TTGCTATAAGATGGG TGGCAAGTG and 5'-CGGAAAGTCCCTTGTAGCAAGC TC. Targeted PCR amplification was performed with the RA primer set with the DNA product of the outer primer set as a template, after recovery of this DNA and removal of the outer primers with GeneClean (Bio 101). nef DNA cloning and sequencing. (i) Adult patients. A library of recombinant lambda phage clones containing unintegrated HIV-1 DNAs was established, and full or partial sequences of these clones were determined by chemical sequencing (1, 3, 55, 56). (ii) Pediatric patients. To avoid a loss of sequence information, full-length nef DNA from the second round of PCR amplification with the nested primer set described above was ligated directly into the T/A cloning vector (Invitrogen). This vector contains 3'-overhanging T residues that anneal with 3'-overhanging A residues in the PCR product (8). Dideoxy

many

sequencing was performed with Sequenase (U.S. Biochemicals) on double-stranded plasmid DNA from minilysates. Primers used for sequencing were the SP6 and -40 T/A vector primers (Invitrogen) and 5'-TCCAGTCACACCTC AGG, 5'-ACCT-TGGATGGTGCTA, and 5'-GGTACTAGC TTGTAGCA, corresponding to conserved internal nef sequences. Figure 1 presents a schematic diagram of the nef gene and 3' long terminal repeat (LTR), showing primers used for PCR amplification and sequencing. Nucleotide sequence accession number. The full 3' open reading frame sequences described in this study have been deposited in GenBank. RESULTS Duplications in the nef gene: PCR studies of adult and pediatric patients. We used targeted PCR amplification to determine how many HIV-1 isolates obtained by coculture from adult patients with AIDS contained nef duplications similar to that previously found in BRVA (3). Figure 2 shows that bands migrating with the BRVA duplication were present in PCR products from three other adult patients (panel A, lane 9; panel B, lanes 10 and 13). The samples in Fig. 2B, lanes 8 and 9, also contain duplications (more clearly shown in Fig. 3). Other samples gave bands migrating faster than BRVA but more slowly than the control LAV isolate (Fig. 2A, lanes 6, 10, 11, and 12), suggesting smaller duplications. In all, PCR products amplified from 23 brain, cerebrospinal fluid, and lymphocyte isolates representing 20 adult patients with AIDS showed variations from the reference clone LAV. These results are summarized in Table 1. The same primer set was next used to amplify this region of nef from HIV-1 DNAs extracted directly from brain and spleen tissue of children who died of AIDS with progressive encephalopathy (see Materials and Methods). Figure 2C shows that PCR products were often heterogenous. PCR product from brain and spleen DNA of one child (patient KF; lanes 1 and 2) showed four bands; the top two migrated more slowly than the control (lane NL; arrowhead), suggesting duplications, while the bottom band migrated somewhat faster, suggesting small deletions in the targeted region. A large proportion of the PCR product amplified from brain and spleen DNA of a second child (patient RH; lanes 3 and 4) ran as a band about 20 bp longer than the control. Heterogeneity was clearly visible in PCR product from brain and spleen DNA of a third child (patient KL; lanes 5 and 6). Similar heterogeneity was present in brain DNA of child VP (lane 7), but only a small proportion of the PCR product was

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B

1

23

4

5

6

1OI9

0 11

2 13

BR LAV

c 194

M 1 2 3 4

5

6

I 8 9 10 1f 12 13 NL

FIG. 2. Analysis of nef gene duplications by targeted PCR. (A and B) Adult patients. PCR amplification was performed on unintegrated HIV-1 DNA obtained by coculture as described in Materials and Methods. PCR products were electrophoresed on 12% polyacrylamide gels, and bands were visualized by ethidium bromide staining. (A) PCR product from adult patients NA2 (lane 3), FC-L (lane 5), Ni to N4 (lanes 6 to 9), and Z2 to Z5 (lanes 10 to 13); (B) PCR product from patients NA5 (lanes 1 and 2), NA4 (lanes 3 and 4), N9, Nil, and N12 (lanes 5, 6, and 7), Dl to D3 (lanes 8 to 10), 451 (lane 11), PZ85 (lane 12), and 58 (lane 13). Controls were DNA from uninfected PBLs (lanes U) and PBLs infected with HIV-1 BRVA (lanes BR) and LAV. The control LAV band is indicated by a single arrowhead; the BRVA 30-bp duplication is indicated by a double arrowhead. (C) Pediatric patients. PCR amplification was performed using the same primers on DNA extracted directly from brain and/or spleen tissues, as described in Materials and Methods. PCR products were electrophoresed on an 8% polyacrylamide gel and visualized by ethidium bromide staining. Shown are PCR products from patient KF brain and spleen (lanes 1 and 2), RH brain and spleen (lanes 3 and 4), KL brain and spleen (lanes 5 and 6), VP brain and spleen (lanes 7 and 8), AG brain and spleen (lanes 9 and 10), and JR brain, MW brain, and LF brain (lanes 11, 12, and 13). Markers at left: (X174 HaeIII digest. The control was plasmid pNL4-3 DNA

(arrowhead).

longer than the control band (less than 5% as estimated by the intensity of ethidium bromide-staining on overexposure of the film); heterogeneity was not visible in spleen DNA (lane 8), even on overexposure. The PCR product from brain and spleen DNA of child AG did not appear to be heterogeneous but ran as a single band about 40 bp longer than the control (lanes 9 and 10). No heterogeneity or length variation was apparent in brain samples from children JR and MW (lanes 11 and 12), but PCR product from brain tissue of a third child (patient LF; lane 13) ran as a single band about 15 bp longer than the control. Overall, evidence of heterogeneity was present in PCR product from the BRVA targeted region in 4 of 11 pediatric patients with AIDS, and PCR product from two additional patients showed evidence suggesting homogeneous duplications. These results are summarized in Table 2. Culture selects against HIV-1 nef quasispecies variants. Heterogeneity in the targeted PCR products from pediatric

patients with AIDS (Fig. 2C) reflects the properties of a quasispecies; indeed, the four distinct bands in lanes 1 and 2 and the two distinct bands in lanes 3 and 4 are excellent ex vivo examples of an equilibrated master band and mutant spectrum (16). It is thus striking that heterogeneity is not equally apparent in the adult samples. All of these samples were subjected to PCR using the same primers and reaction conditions; the main difference is that the adult samples represent HIV-1 DNA obtained by coculture of primary PBLs with HIV-1 in infected tissues, whereas the pediatric samples represent HIV-1 proviral DNA in tissue (see Materials and Methods). This suggests that even brief culture selects against the natural spectrum of nef quasispecies that are present in pathological tissue. In vivo selection of a nef duplication. This point is underscored in Fig. 3, where lanes 7 to 12 show selection of a nef duplication in a HIV-1-infected adult. In 1983, the beginning of the study, the patient was seropositive but asymptomatic (56), and the PCR product migrated at the position of LAV (replicate lanes 7 and 8). In 1984, a nef duplication of about 20 bp appeared (replicate lanes 9 and 10). Through 1985 the duplication was maintained (replicate lanes 11 and 12); the HIV-1 antibody titer dropped, and the patient developed frank AIDS with severe neurologic dysfunction. This patient donated blood in 1983; the recipient rapidly developed AIDS. Restriction mapping of cloned 1983 viral isolates from the donor and recipient showed differences in the env and nef region (56). PCR products from other patient isolates in lanes 1, 2, and 5 of Fig. 3 show evidence for in vitro selection of a single nef variant the same as LAV. However, the two weak bands in lanes 3 and 4 may represent maintenance of a quasispecies in culture (note that the lower band in lane 4 is slightly longer than the LAV band). Hints of heterogeneity in lanes 9 and 11 are not present in replicate isolates in lanes 10 and 12. Thus, cultured HIV-1 strains clearly underrepresent the broad spectrum of nef quasispecies that are present in vivo. Definition of nef duplications by DNA sequence studies. The results of targeted PCR studies of adult patients are corroborated by a limited number of published DNA sequences (Table 1). The sequence of BRVA (patient NA1) reveals that the targeted duplication region is made up of a 42-bp insertion at position 76 and a 6-bp deletion at position 22 relative to BRU (see Fig. 4A). Some sequenced clones from the group of adult Zairean patients studied by PCR also show insertions and deletions at similar positions; however, others do not (Table 1). This probably represents selection of particular sequence variants during culture. To confirm the quasispecieslike heterogeneity in pediatric samples suggested by the PCR studies in Fig. 2 and to reveal the presence of mutations too small for detection by PCR or outside the targeted region, we cloned the nef gene-3'-LTR region as shown in Fig. 1, using DNA extracted directly from postmortem brain and spleen tissue of 11 children who died of HIV-1 infection. We established a library of 324 clones and determined the complete nef sequence in 88 clones (Table 2). In 81 (92%) of these sequences the reading frame was fully open, in contrast to sequences of cultured isolates such as MN or HXB2 (BRU) that are prematurely terminated (41). This result suggests that nef is normally expressed in brain and spleen tissue. nef sequences show a mixture of isoforms: one of four brain clones and two of four spleen clones from patient KF showed 6-bp deletions following position 22 of the reference HXB2 sequence. Similarly, two of four spleen clones from patient RH contained small 3-bp insertions inside the tar-

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TABLE 1. Adult patients with AIDS: comparison of PCR and sequence studies with neuropathology Isolatea NA-1 (B) NA-2 (L) NA-3 (L) NA-4 (L) NA-5 (C) FC-L (L) N-1 (C) N-2 (C) N-3 (C) N-4 (C) N-9 (C) N-11 (C) N-12 (C) D-1 (L; 1983) D-2 (L; 1984) D-3 (L; 1985) 451 (L) PZ85 (L) 58 (L) Z-1 (L) Z-2 (L) Z-3 (L) Z-4 (L) Z-5 (L)

pCRb +36 bp +6 bp (het)

+6 bp

+36 bp

+20 bp +20 bp +30 bp +20 bp (het) +30 bp ND +15 bp +15 bp

Sequence

(Dup)f

Neuropathologyd

Reference(s)

BRVA (+) NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA

Rapid progressive dementia Dementia, abnormal CT Headache, degeneration Early dysfunction Early dysfunction GBS-like syndrome Early dysfunction (ARC) Early dysfunction (ARC) Early dysfunction (ARC) Early dysfunction (ARC) Early dysfunction (ARC) Early dysfunction (ARC) Early dysfunction (ARC) Asymptomatic Asymptomatic Progressive dementia, AIDS Unremarkable (Zairean)

2, 3 2 2 2 2 Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished 1, 56 1, 56 1, 56 15

Unremarkable (Zairean) Unremarkable (Zairean) Unremarkable (Zairean) Unremarkable (Zairean) Unremarkable (Zairean) Unremarkable (Zairean) Unremarkable (Zairean)

Unpublished Unpublished 41, 55 41 41 Unpublished Unpublished

451(-) PZ85 (-) 58 (-) Z6 (+) Z2Z6 (-) Z3 (+) NA NA

B, brain; L, PBL or peripheral blood mononuclear cell; C, cerebrospinal fluid. +bp, band longer than LAV control; -, band same length as LAV control; ND, not determined; (het), slight heterogeneity in PCR product. Data obtained from cloned isolate with (+) or without (-) duplication; NA, clone not available. d Contemporary observations on clinical presentation. C-r, computed tomogram; GBS, Guillain-Barre syndrome; ARC, AIDS-related complex. The history and sequences of the Zairean cases are given in reference 41: case Z3 was cloned, and the first 106 nucleotides of nef were published as part of env; infectious clones Z2Z6 and Z321 have been partially sequenced. Case Z-1 was cloned and sequenced as HIV-1 Zr-6. a

b

c

geted region at position 81, as well as outside it at position 196. One clone from patient KL contained a 3-bp insertion at position 193. In six of six clones from patient AG brain and spleen, the expected long 36-bp duplications were found following position 69, while in two of two brain clones from patient JR and three of three clones from patient LF, 12-bp insertions following position 80 were found. No duplications or deletions were found in 33 clones from patients VP, OC, and LS, but two LS brain clones and two RH spleen clones contained stop codons due to point mutations that would not be seen by targeted PCR. An alignment of representative sequences in the duplication region is shown in Fig. 4A. Relative to HXB2, adult clones BRVA and Z2 and pediatric clone KFJ3 show small deletions beginning at position 6, as does the reference strain MN. Since MN is the most common isolate worldwide (32), the deletion following position 6 should perhaps be considered the rule rather than the exception. The major hot spot for duplications clearly follows position 64, and a third minor hot spot is defined by 3-bp insertions in three clones from patients RH and KL after position 193 (Table 2). These hot spots in nef are shown schematically in Fig. 1. Interestingly, as shown in Table 2 and Fig. 4B, clone 113 contains a 15-bp duplication just inside the end of the nef gene. In contrast, in two of two clones from patient MW brain, 25-bp duplications were noted in the LTR, just beyond the normal nef gene stop at position 628, while three clones from patient RH and one from patient KL had identical 22-bp duplications at position 630. These nontriplet insertions do not affect the nef reading frame. Thus, substantial selection pressure is exerted to preserve the nef reading frame, as noted in BRVA by Anand et al. (3).

Mutations in the nef gene suggest selection for function. Although the nef reading frame is normally open in pathologic tissues from children with AIDS, three brain clones and two spleen clones had premature stops due to point mutations, and two spleen clones had truncated reading frames due to a 2-base insertion and a 1-base deletion (Table 2). Frameshift mutations are unlikely to reflect PCR errors and were not found in our previous studies of the env gpi20 V3 domain (17). The nontriplet insertions in the 3' LTR show that HIV-1 reverse transcriptase has no inherent bias favoring mutations in triplets. Therefore, the finding of even a few reading frame interruptions due to insertion or deletion and point mutations suggests that nef is not under strong immune selection like V3; rather, it suggests selection for nef function. Comparison of nef N and C termini, which have been identified as important antigenic epitopes (23, 48), shows that brain and spleen sequences evolve separately within host-determined quasispecies (17), as mutations within these regions are delimited by host rather than tissue (Fig. 5). In sibling clones with deletions, the most highly variable amino acids, 8 to 11 and 14 to 16 at the N terminus, are found in both brain and spleen sequences, as is the deletion itself. These amino acids are also variable in brain and spleen sequences of patient RH sibling clones, as is an additional A residue at position 42 not previously reported (41). In contrast, positions 8 to 14 of patient LS brain and spleen sibling sequences are identical but differ from all other patient N termini, as do sequences that contain duplications. C-terminal amino acids also show considerable variation, mainly at positions 182 and 188. Of interest is that, in sequences with premature stop codons, there is no greater

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TABLE 2. Pediatric patients with AIDS: comparison of PCR and sequencing studies with neuropathology Patienta

No. of clones

PCR (lane)b

Sequenced

Position(s)c

Neuropathologyd

OC-B (D) OC-S (E)

NS NS

7

With mutations (description)e 0

6

0

TM-B (Ff

NS

6

1 (point mutant stop)

311

+++, M

LS-B (G)f LS-S (H)

NS NS

3 3

2 (point mutant stops) 0

64

+++, M

HET (1)

4

1 (6-bp deletion in nef) 1 (15-bp insertion in nef) 2 (6-bp deletion in nef)

22

+++, M

1 (22-bp insertion in LTR) 2 (3-bp insertions in nef) 2 (point mutant stops)

630

2 (22-bp insertion in LTR)

630

1 (22-bp insertion in LTR) 1 (3-bp insertion in nef) 1 (2-bp insertion in net)

630

KF-B

(I)f

KF-S (J)

HET (2)

4

RH-B (K) RH-S (L)

+20 bp (3) +20 bp (4)

4 4

KL-B (M)

KL-S (N)

het (5) het (6)

10

10

1 (1-bp deletion in nef)

+, M

616 22

+++, M

81, 196 250, 256

193 536 541

++, M

VP-B (0) VP-S (P)

HET (7) het (8)

9 5

0 0

AG-B (S) AG-S (T)

+40 bp (9) +40 bp (10)

3 3

3 (36-bp insertion in nef) 3 (36-bp insertion in nef)

69 69

+

HET (11)

2

2 (12-bp insertion in nef)

84

+

(12)

2

2 (25-bp insertion in LTR)

628

++

JR-B

(U)f

MW-B (V)

+

LF-B (W)

+15 bp (13) 3 3 (12-bp insertion in nef) 80 ++, M a B, brain isolate; S, spleen isolate. In parentheses are given the letter designations of the clones. b Targeted PCR results in Fig. 2C. HET, heterogeneous; het, slight heterogeneity; +bp, band longer than control; -, band same as control; NS, not shown. c Position of difference with respect to the HXB2 sequence. d Descriptions are according to reference 4. +, white-matter pallor and astrocytosis (HIV leukoencephalopathy); + +, white-matter pallor and astrocytosis, with infrequent inflammatory cell infiltrates in gray or white matter; + + +, white-matter pallor and astrocytosis, with numerous inflammatory cell infiltrates in gray or white matter; M, multinucleated (syncytial) giant cells present (HIV encephalitis). e Important mutations compared with the HXB2 sequence. f Case included in reference 51. g Cases included in reference 52; LS = case 3; KF = case 4; JR = case 6.

variation in the untranslated regions than that found in expressed sibling sequences, in part reflecting overlap of the nef C terminus with the 3' LTR. DISCUSSION

nef quasispecies and neuropathology. It is tempting

to

associate the appearance of the nef duplication in serial isolates from the adult blood donor with acceleration of disease and to speculate that the quasispecies variant with the duplication was selected by both the donor and the recipient. A similar process of in vivo selection was seen in a previous study of env variants (40). Also, the identification of nef duplications mainly in brain-derived clones such as BRVA and JRCSF might suggest an association with neuropathology. However, correlations between nef variants and pathology are not clear-cut. Evidence for nef duplications is present in isolates from some, but not all, adult patients with AIDS selected on the basis of prominent neuropathological clinical symptoms and in isolates from a group of Zairean patients who were not selected on this basis (Table 1). Duplications occur in spleen clones from children with

AIDS, all of whom died with clinically severe neurological dysfunction, and those with the most florid neuropathological findings also evidenced deletions and frameshift mutations (Table 2). It appears important that nef is present in pathologic tissues, but it is unclear whether this protein exerts a toxic effect on neural cells or contributes to persistent infection. Structural and functional homologies have been noted among nef, G proteins, and p2lras (25); thus, nef could disrupt cellular activities through interference with transmembrane signalling. Although the biological activities of nef are in dispute (11, 28), in vitro evidence suggests that myristylation is critical for activity (62). Interestingly, the mutational hot spot in the nef N terminus maps to the myristylated alpha-helical membrane anchor domain of a model G protein (38) that is important for biological activity (6). Duplications in this domain would have the effect of extending the membrane anchor, potentially affecting G-pro-

tein activity. If the degree of neuropathology does not correspond with the presence of duplicated regions, what might be the

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3

4 5

6 7 8

9 10 111 2

5261

U BR LAV

FIG. 3. In vivo selection of a nef duplication. Unintegrated HIV-1 DNA prepared from cocultured isolates was PCR amplified and analyzed with primers and conditions as in Fig. 2A and B. Lanes 1 to 3, PCR product from HIV-1 nef in cerebrospinal fluid (lanes 1 and 2) and PBLs (lane 3) of patient NA5 or NA4; lanes 4 and 5, PCR product from PBLs of patients NA3 and NA2; lane 6, PCR product from PBLs cocultured with brain tissue of patient NAl (this isolate was renamed HIV-1 BRVA; cf. BR control). PCR products in paired lanes 7 and 8, 9 and 10, and 11 and 12 represent replicate serial isolates prepared in 1983, 1984, and 1985 from blood donor 1 described in reference 56. Lane U, PCR product from uninfected PBL DNA.

purpose of nef mutability? Evidence that a shift from monocyte/macrophage-tropic strains of HIV-1 to lymphocytetropic strains accompanies progression to AIDS is accumulating (20, 49). However, little is known about nef expression in monocyte/macrophage-tropic HIV-1 strains thought to predominate in brain tissue (39, 43). We have previously proposed that brain is an early site of HIV-1 infection and that V3 sequences in brain represent the original inoculum

strain (17). Determinants for monocyte/macrophage tropism have recently been localized to the V3 domain of gpl20 (31, 53, 60); one marker is a tyrosine at position 283 of the V3 loop (61). Interestingly, nearly all V3 sequences from the brains of patients KF, RH, and JR contain this marker (see Fig. 2 of reference 17; patient JR information is unpublished data), so heterogeneity in the targeted nef PCR products from pediatric patients is representative of macrophage

A

Reference cLones (aduLt cases) 90. 100. 110. 120. 130. 140.. 80 . 60 . 70 . 50 . 30 . 40 . 20 . . ..10 Clone... BRVA CAAAAA ... TGGCTGGATGGTCCACTGTAAGGGAMGM _AGACGAGCTGAGCCAGCAAGGGAGAATGAGACGAGCTGAGCCACGAGCTGAGCCAGCAGCAGATGGGGTGGGAGCAGTATCTCGAGACCTGGAMAAC . TGAGCCAGCAGCAGTAGGGGTGGGAGCAGTATCTCGAGACCTGGAAAAAC CAAAACATAGTGTGMCTGGATGGTCTACTGTAAGGGAMGAATGAGACGAGCTGAGCCAGCAACAGATAGGGTGAGACAAAC .. JRCSF HXB2 CAAAAAGTAGTGTGATTGGATGGCTTACTGTAAGGGAAAGAATGAGACGAGCTGAGCCAG .. . CAGCAGATGGGGTGGGAGCAGCATCTCGAGACCTGGAAAAAC MN CAAAACGT... GTGACTGGATGGCCTACTGTAAGGGAAAGAATGAGACGAGCTGAACCAG ................................. CTGAGCTAGCAGCAGATGGGGTGGGAGCAGCATCCCGAGACCTGGAAAAAC ............................ CAGCAGATGGGGTAGGAGCAGTATCTCGAGACCTGGAAAAAC Z2Z6 CAAAAAGTAGTATAGTTGGATGGCCTGCTATAAGGGAAAGAATAAGAAGAACTGATCCAG .............. .................... GAACTGATCCAGCAGCAGATGGGGTAGGAGCAGCATCTCGAGACCTGGAAAAAC Z6 CAAAAAGTAGTATAGTTGGATGGCCTGCTATAAGGGAAAGAATAAGAAGAACTGTCCAAGAA ........ CAAAACA .... ATGGCCTGCTATAAGGGAGAGAATGAGACGAGCTAGGCCAG .............................. CAGCTGAGCCAGCAGCAGATGGAGTAGGAGCAGCATCTCGAG. Z3

KFJ3 RHL7 AGS2 AGT7 JRU5 LFW3

Representative cLones (pediatric cases) CAAAAA .... TGGTTGGATGGTCTACTGTAAGGGAAAGAATGAGACGAGCTGAGCCAGCA .GCAGATGGGGTGGGAGCAGTATCTCGAGACCTGGMAAAC CAAAACGTGGTGTGGATAAATGGCCTGCAGTAAGGGAAAGAATGAGACGAGCTGAGCCAGCAGCA ....................................... GCAGATGGAGTGGGAGCAGTATCTCGAGACCTGGAAAAAC CAMAAGTAGTATAGTTGGATGGCCTGCTATAAGGGAAAGAATGAGAAGAGCTAGACCTGAGCCAGCAGCAGTGAGAAGAGCTAAA ...... GCTGAGCCAGTAGCAGATGGGGTGGGAGCAGTATCTCGAGACCTGGGAAAAC CAAAMGTAGTATAGTTGGATGGCCTGCTATAAGGGAAAGAATGAGAAGAGCTAGACCTGAGCCAGCAGCAGTGAGMGAGCTAAA ...... GCTGAGCCAGTAGCAGATGGGGTGGGAGCAGTATCTCGAGACCTGGGAAAAC ....................... GCAGAGCCAGCAGCAGAGGGGGTGGGAGCAGTATCTCGAGACCTGGAAAAAC CAAAACGTAGTGTGGGTGGATGGGCTACTGTAAGGGMAGAATGAGACGAGCTGAGCCAGCA ....... CAAAACGTGTTGGGGATGGATGGTCTACTGTAAGGGAAAGAATGAGACGAGCTGAGCCAGCA .............................. GCTGAGCCAGCAGCAGATGGGGTGGGAGCAGCATCTCGAGACCTGGGAAAAC

Reference cLones (aduLt cases) B 50 . 60 . 70. 80.... 40 . 30 . 20 . 10 . CLone .. TTGCTACAAGGGACTTTCC BRVA CCGGAGTACTACMGAACTGC ....... TGACATCGAGC .................. TTGCTACAAGGGACTTTCC HXB2 CCGGAGTACTTCAAGAACTGC ....... TGACATCGAGC .................. TTGCTACAAGGGACTTTCC CCGGAGTACTACAAGAACTGC ....... TGACATCGAGC .................. MN JRCSF CCGGAGTACTACMGGACTGAC ...... TGACACCGAGCTTTCTACM ................ TTTCTACAAGGGACTTTCC ....... TTTCTACAAGGGACTTTCC CCGGAGTTCTACAAAGACTGC ....... TGACACCAAGT ........... Z6 Z321 CCGGAGTTCTACAAAGACTAAAGATGC. TGACACAGAAGTTGCTGACAGGGAC .......... TTTCCGCTGGGGACTTTCC

Representative cLones (pediatric cases) ......... TGCTGACATCGAGCTTGCTACAAGGGACTTTCC KFI 13 CCGGAGTACTACMGAACTAAGMCTGCTGACAT ....... RHK3 CCGGAGTACTACAAGAACTGC ....... TGACATCGAGCTCCAGA.. .ACTGCTGACATCGAGCTTGCTACAAGGGACTTTCC KLM4 CCGGAGTACTACAAGAACTGC ....... TGATATCGAGCTCCAGA.. .ACTGCTGACATCGAGCTTGCTACAAGGGACTTTCC MN2 CCGGAGTATTACAAGGACTGC ....... TGACATCGAGATCTGCAGGGACTGCTGACATCGAGCTTGCTACAAGGGACTTTCC

FIG. 4. nef duplications defined by DNA sequence studies. (A) Comparison of nef sequences in the targeted region. Sequences of reference clones are taken from reference 41, which describes their provenance; that of Z2Z6 (our patient Z2) represents unpublished data. The Z3 isolate appears to represent a defective virus; a partial nef sequence covering the targeted region begins at position 2604, overlapping the normal env termination site. Underlined sequences are AP-1 homologous sites (21) (motif of TgAgtcAg, where capital letters are invariant and lowercase letters are preferred variants). Dots represent gaps inserted to align sequences. (B) Comparison of insertions in late nef and 3' LTR. Reference sequences are taken from reference 41. Sequences were aligned visually on the TGA termination codon (underlined) and end with the NF-KB site (19) (motif of GGGAC(-lJ7CC). Dots represent gaps inserted to align sequences.

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BLUMBERG ET AL. Reference clones 20 ...10.20 30 . 40 . 50 CLone . ....... MGGKWSKR .VTGWPTVRERMRRAEPA ........... ELAA.DGVGAASR MN BRVA MGGKWSK ..MAGWSTVRERMRRAEPARERMRRAEPRAEPAA.DGVGAVSR JRCSF MGGKWSKHSVPGWSTVRERMRRAEPATDRVRQT .... EPAA.VGVGAVSR BRU MGGKWSKSSVVGWPTVRERMRRA ....... ....... EPAA.DGVGAASR RF MGGKWSKSKMGGUPAVRERMQKA ....... ....... EPAA.DGVGAASR

KF-B KF-B KF-S KF-S

Representative sibLing cLones with deLeted 116 MGGKWSK ..MVGWPTVRERMRRA ........... 114 MGGKWSKSSVIGWPAVRERMRRA .......... J2 MGGKWSK ..MVGWPTVRERMRRA ........... J7 MGGKWSKSSVIGWPAVRERMRRA ..........

LS-B LS-B LS-S LS-S

Representative sibling cLones with point mutation stop co G2 MGGKWSKSSVIGWPAVRERMR*a .......... epaa.dgvgavsr G29 MGGKWSKSSVIGWPAVRERMRRA ...........E.EPAA.DGVGAVSR H4 MGGKWSKSSVIGWPAVRERMRRA .............. EPAA.DGVGAVSR H6 MGGKWSKSSVIGWPAVRERMRRA .............. EPAA.DGVGTVSR

RH-B RH-B RH-S RH-S RH-S

AG-B AG-S JR-B LF-B

Kl K3 L6 L7 L9

180 . 190 . 200 . 210 HPMSQHG(frameshift) HGMDDPEREVLQWRFDSRLAFHHMARELHPEYYKNC HGMDDPEKEVLVWKFDSKLALHHVARELHPEYYKDC HGMDDPEREVLEWRFDSRLAFHHVARELHPEYFKNC HGMDDPEKEVLVWKFDSRLAFHHVAREKHPEYYKDC .......

region EPAA.DEVGAVSR EPAA.DGVGAVSR EPAA.DGVGAVSR EPAA.DGVGAVSR

HGMEDPEREVLEWRFDSRLAFHHVARELHPEYFKNC HGMEDLEKEVLQWKFDSRLAFHHVAQELHPEYYKNC HGMEDPEKEVLVWRFDSRLAFHHVARELHPEYYKNC HGMDDPEREVLEWRFDSRLAFHHVARELHPEYFKNC

hgmddperevLewrfdsrlafhhvarelhpeyfknc HGMDDPEREVLEWRFDSSLAFHHVarELHPEYFKNC HGMDDPEREVLEWRFDSRLAFHHVARELHPEYFKNC HGMDDPEREVLEWRFDSRLTFHHVARELHPEYFKNC

MGGKWSKSSVIGWPAVRERMRRA .............. EPAA.DGVGAVSR - - - - - - - - - - - HGMDDPEREVLEWKFDSRLAFHHVARELHPEYFKNC MGGKWSKRSMDKWPAVRERMRRA ..............EPAA.DGVGAISQ - - - - - - - - - - - HGMDDPEREVLVWKFDSLLAFHHMARELHPEYYKNC MGGKWSKRSEDKWPAVRERMRRA .............. EPAA.DGVGAVSR - - - - - * - - - - - hgmedperevLvwkfdshLaLrhmareLhpeyyknc MGGKWSKRGVDKWPAVRERMRRA ..............EPAADGVGAVSR - - - - - * - - - - - hgmddperevLvwkfdsLLafrhmareehpeyyknc - - - - - - MGGKWSKRSEDKWPAVRERMRRA .............. EPTAADGVGAVSR -HGMDDPEKEVLAWKFDSRLAFHHMARELHPEYYKDC

Representative cLones with dupLicated regions S2 MGGKWSSKSSIVGWPAI RERMRRARPEPAAVRRAKA. .EPVA.DGVGAVSR T7 MGGKWSKSSIVGWPAI RERMRRARPEPAAVRRAKA. .EPVA.DGVGAVSR U5 MGGKWSKRSVGGWATVRERMRRAEPAA .......... EPAR . EGVGAVSR W3 MGGKWSKRVGDGWSTVRERMRRAEPAA ..........EPAA.DGVGMSR

-

-

- - - - - - - - - - - - - - - - - - - - -

- - - - - - -

- - -

- - - - - - - - - - -

HGMDDPEKEVLAWKFDSRLAFHHMARELHPEYYKDC HGMDDPEKEVLAWKFDSRLAFHHMARELHPEYYKDC HGMEDPEKEVLMWKFDSRLAFHHMARELHPEYYKNC HGMDDPEKEVLMWRFDSRLAFHHMARELHPEYYKNC

FIG. 5. Comparison of nef amino- and carboxy-terminal sequences. Partial amino acid sequences derived from adult and pediatric patient nef clones are shown. C-terminal numbering is according to HXB2. Dots represent gaps inserted for alignment; dashes represent sequences not shown. Asterisks indicate sites of premature termination; the continued sequence in lowercase letters is hypothetical.

quasispecies. The spectrum of nef quasispecies thus reflects macrophage-tropic HIV-1 strains that infect various tissues of the human host (10, 17). Significance of the BRVA duplication. Insertions likely reflect reverse transcriptase strand switching, a mechanism intrinsic to retrovirus replication (9, 24, 30). Recombination between HIV-1 genomes is a likely mechanism for the independent evolution of nef and LTR (12), and there is direct evidence for recombination between HIV-1 env variants involving a duplicated region (29). One feature of the mechanism is that the "promiscuous jumping polymerase" retains the nascent strand during the jump (45); as the point of reattachment is uncertain, defective genomes often result (36). The 3' LTR contains several functional binding sites for

transcription factors (19). The nontriplet insertions in the 3' LTR may be triggered by polymerase contact with one or more of these proteins (Fig. 4B), and the regulatory elements and promoter sequences are obvious candidates to guide reattachment (44). Interestingly, the BRVA duplication region contains multiple sequences (underlined in Fig. 4A) homologous with the functional AP-1 site in the 3' LTR (18). Another feature of interest is the presence of two homopolymeric stretches of A residues flanking this region. Recent studies have shown that poly(A) is a good acceptor for HIV-1 reverse transcriptase during strand switching (5). We propose that the BRVA duplication occurs because the homopolymeric A stretches guide reassociation of the nascent strand-bearing polymerase, frequently with displace-

ment along the template (consistent with this, the 5-base A stretches are reduced to 4 A's in some clones).

Absent direct evidence that AP-1 proteins bind HIV-1 nef, this proposal must be regarded as speculative. However, AP-1 proteins exist free in the cytoplasm where they can potentially bind HIV-1 RNA, as can a number of other cytoplasmic RNA-binding proteins (46), and there are sites in the duplication region for another transcription factor that binds the HIV-1 LTR, TCF-alpha (motif, CTNAG [59]). Also, strand switching, which would be expected to occur mainly during reverse transcription of HIV-1 genomic RNA (22), occurs at a high level with either RNA or DNA as a template (37). Reversion of point mutations in blocked SIV nef (33) complements our results showing that the natural HIV-1 nef reading frame is open and is conserved even in the few cases that are prematurely terminated by point mutation; taken together, these findings emphasize the importance of selection for nef function. The apparent inability of SIV nef deletion mutants to revert or cause pathology led to speculation that such mutants might be useful for laboratory work or development of attenuated HIV-1 vaccines (14). However, SIV strains do not contain insertions analogous to the BRVA duplication (41). Although at this time we cannot assign a definite function to nef, our data caution that the functionality of even a deleted nef gene could be restored in a vaccine recipient through duplication or recombination, given the widespread prevalence of HIV-1 nef quasispecies.

VOL. 66, 1992

HIV-1

ACKNOWLEDGMENTS We gratefully acknowledge the technical support of Ginger Carney (CBER, FDA), Kirk Dzenko, Robert White, and Nurcan Ergin and thank Stephen Dewhurst, Robert Bambara, Jeff DeStefano, and Howard Gendelman for helpful discussions. B.M.B. and L.G.E. are also indebted to Anthony deRonde and Jaap Goudsmit for inviting us into the forum on nef and for sharing their preliminary results. This work was supported by grants 500044-8-PG from AMFAR and NS28754 from NIH. REFERENCES 1. Anand, R., J. Moore, H. Jaffe, P. Feorino, R. Weinstein, J. Curran, and A. Srinivasan. 1987. DNA and protein heterogeneity in serial isolates of human immunodeficiency virus (HIV-1): indication of change in vivo. Microbios 52:191-201. 2. Anand, R., F. Siegal, C. Reed, T. Cheung, S. Forlenza, and J. Moore. 1987. Non-cytocidal natural variants of human immunodeficiency virus isolated from AIDS patients with neurological disorders. Lancet ii:234-238. 3. Anand, R., R. Thayer, A. Srinivasan, S. Nayyar, M. Gardner, P. Luciw, and S. Dandekar. 1989. Biological and molecular characterization of human immunodeficiency virus (HIV-lBR) from the brain of a patient with progressive dementia. Virology 168:79-89. 4. Budka, H., C. A. Wiley, P. Kleihues, J. Artigas, A. K. Asbury, E.-S. Cho, D. R. Cornblath, M. C. Dal Canto, U. DeGirolami, D. Dickson, L. G. Epstein, M. M. Esiri, F. Giangaspero, G. Gosztonyi, F. Gray, J. W. Griffin, D. Henin, Y. Iwasaki, R. S. Janssen, R. T. Johnson, P. L. Lantos, W. D. Lyman, J. C. McArthur, K. Nagashima, N. Peress, C. K. Petito, R. W. Price, R. H. Rhodes, M. Rosenblum, G. Said, F. Scaravilli, L. R. Sharer, and H. V. Vinters. 1991. HIV-associated disease of the nervous system: review of nomenclature and proposal for neuropathology-based terminology. Brain Pathol. 1:143-152. 5. Busier, R. G., J. J. DeStefano, L. M. Mallaber, P. J. Fay, and R. A. Bambara. 1991. Requirements for the catalysis of strand transfer synthesis by retroviral DNA polymerases. J. Biol. Chem. 266:13103-13109. 6. Buss, J. E., S. M. Mumby, P. J. Casey, A. G. Gilman, and B. M. Sefton. 1987. Myristoylated alpha subunits of guanine nucleotide-binding regulatory proteins. Proc. Natl. Acad. Sci. USA 84:7493-7497. 7. Cheng-Mayer, C., P. Iannello, K. Shaw, P. A. Luciw, and J. A. Levy. 1989. Differential effects of nef on HIV replication: implication for viral pathogenesis in the host. Science 246:16291632. 8. Clark, J. M. 1988. Novel non-templated nucleotide addition reactions catalyzed by procaryotic and eucaryotic DNA polymerases. Nucleic Acids Res. 20:9677-9686. 9. Coffin, J. M. 1978. Structure, replication, and recombination of retrovirus genomes: some unifying hypotheses. J. Gen. Virol. 42:1-26. 10. Coffin, J. M. 1986. Genetic variation in AIDS viruses. Cell 46:1-4. 11. Cullen, B. R. 1991. The positive effect of the negative factor. Nature (London) 351:698-699. 12. Delassus, S., R. Cheynier, and S. Wain-Hobson. 1991. Evolution of human immunodeficiency virus type 1 nef and long terminal repeat sequences over 4 years in vivo and in vitro. J. Virol. 65:225-231. 13. deRonde, A., B. Klaver, W. Keulen, L. Smit, and J. Goudsmit. Natural HIV-1 NEF accelerates virus replication in primary human lymphocytes. 1992. Virology 188:391-396. 14. Desrosiers, R. C., and E. Hunter. 1991. AIDS biosafety. Science 252:1231. (Letter.) 15. Devare, S. G., A. Srinivasan, C. A. Bohan, T. J. Spira, J. W. Curran, and V. S. Kalyanaraman. 1986. Genomic diversity of the acquired immunodeficiency syndrome retroviruses is reflected in alteration of its translational products. Proc. Natl. Acad. Sci. USA 83:5718-5722. 16. Eigen, M., and C. K. Biebricher. 1988. Sequence space and quasispecies distribution, p. 211-245. In E. Domingo, J. J. Holland, and P. Ahlquist (ed.), RNA genetics, vol. III. CRC

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