demonstrated that PRV gIII and BHV-1 gIll share complementary functions. Virus infection is ..... Robbins, A. K., J. H. Weis, L. W. Enquist, and R. L. Watson. 1984.
Vol. 65, No. 10
JOURNAL OF VIROLOGY, Oct. 1991, p. 5553-5557
0022-538X/91/105553-05$02.00/0 Copyright © 1991, American Society for Microbiology
Pseudorabies Virus glll and Bovine Herpesvirus 1 glll Share Complementary Functionst XIAOPING LIANG,1* LORNE A. BABIUK,12 AND TIM J. ZAMB' The Veterinary Infectious Disease Organization' and Department of Veterinary Microbiology,2
University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N OWO Received 15 January 1991/Accepted 2 July 1991
The glll glycoproteins of bovine herpesvirus 1 (BHV-1) and of pseudorabies virus (PRV) are structurally homologous. Both proteins also play preeminent roles in mediating virus attachment to permissive cells. To directly compare the functional relation between these glycoproteins, we constructed a recombinant BHV-1 in which the BHV-1 gIII coding sequence was replaced by the PRV gene homolog. The resultant recombinant virus efficiently expressed PRV glll and then incorporated it into its envelope. The levels of PRV gIll expression and incorporation were equivalent to those achieved by the wild-type virus for BHV-1 glll. The recombinant virus was fully susceptible to neutralization by anti-PRV glll neutralizing antibody. In addition, the virus attachment and penetration functions, as well as the virus replication efficiency, which were lost by deleting the BHV-1 glll gene, were restored by expressing the PRV glll homolog in its place. These results demonstrated that PRV gIII and BHV-1 gIll share complementary functions. edness between BHV-1 gIIl and PRV gIII, we attempted to construct a recombinant virus in which the BHV-1 gIII coding sequence was precisely replaced with the corresponding sequence from the PRV gIII gene. Because this direct replacement would not perturb the BHV-1 gene regulatory sequences, we anticipated that the amount and timing of PRV gIll expression would be similar to BHV-1 gIII expression demonstrated in the wild-type (wt) virus. Thus, any phenotypic differences ascribed to the recombinant virus must be directly associated with differences in the gIll protein structure. We generated the recombinant BHV-1 by a procedure that began by constructing a transfer vector in which the PRV gIll coding sequence immediately follows the BHV-1 gIII translation initiation codon. Figure 1 shows a schematic representation of the methods used to construct pBPgIII, the transfer vector used in this study. To produce recombinant virus, MDBK cells were cotransfected with naked genomic wt BHV-1 DNA plus the transfer vector described above (13, 23). Virus progeny that resulted from this transfection were screened for the presence of recombinant viruses by a black-plaque assay (9) using antiPRV gIII monoclonal antibodies (MAbs) (5). We found that the PRV gIII-positive plaques constituted about 1% of the total viral population. A recombinant virus, named vBPgIII, was further purified from one of the PRV glll-positive plaques and used for subsequent studies. Restriction endonuclease mapping and Southern blot analyses of vBPgIII confirmed the replacement of the BHV-1 glll coding sequence by the corresponding sequence from PRV (data not shown). Our ability to identify the recombinant virus by PRV MAb screening was the first indication that PRV glll was efficiently expressed by the recombinant BHV-1 and that it was incorporated into the membranes of infected cells. The expression of PRV glll protein was further examined by immunoprecipitation assays (24). wt BHV-1- or vBPgIIIinfected MDBK cells were labelled with [3H]glucosamine and precipitated with MAbs to PRV gIIl (5). The results are shown in Fig. 2A. PRV gIll-specific MAbs precipitated a 95,000-molecular-weight protein from vBPgIII-infected MDBK cells (Fig. 2A, lane 1), but they did not recognize any
Virus infection is initiated through a specific interaction between a viral attachment protein (VAP) and its receptor on a permissive cell surface (12). For alphaherpesviruses, the initial attachment of a VAP to its receptor is followed by virus penetration involving membrane fusion (20). These two steps taken together constitute the virus entry process (20). Accumulating evidence suggests that alphaherpesvirus VAPs are composed of the major viral envelope glycoproteins, including gB, gC, and gD for herpes simplex virus and homologs of these proteins for other members of this virus subfamily (4, 10, 11, 13, 27). gB, gD, and their homologs also appear to participate in the virus penetration process (1, 3, 6-8). With regard to the structures that are recognized by VAPs (i.e., virus receptors), heparinlike molecules present on permissive cell surfaces have been shown to be the attachment sites for herpes simplex virus and pseudorabies virus (PRV) (16, 21, 26). However, the detailed mechanisms by which alphaherpesvirus VAPs interact with permissive cells and lead to productive virus infection have yet to be defined. Previously, we showed that bovine herpesvirus 1 (BHV-1) gIll plays a dominant role in virus attachment (13), a finding that is consistent with the role of the PRV glll homolog (22, 27). To further characterize the glll-mediated virus attachment mechanism, we took an approach that allowed us to examine any functional complementation activity which may exist between the BHV-1 and PRV homologs. We anticipate that defining the functional similarities and dissimilarities between the two glll-mediated virus attachment processes will allow us to determine the precise nature of the virus attachment mechanism mediated by the herpesvirus glll class of molecules. As the first step toward our objective, the present study was carried out to examine the functional complementation between BHV-1 glll and PRV giII by an approach involving gene replacement. In order to unambiguously evaluate the functional relat-
Corresponding author. t Published with permission of the Director as Veterinary Infectious Disease Organization journal series article 110. *
5553
5554
J. VIROL.
NOTES
1113RI.B9g3.0
pNT7/123
BHV-1 gIII coding sequence
PRV gIII coding sequence
S-
NcoI
/I fI
I
PleI I-
Ncol
/d
coRI /I
I
A_ I,-j-SalI
PleI+Klenow NcoI NcoI
SallI
Sail
6+Klenow SalI \ccoRI
Blunt
EcoRI
NcoI
-/A-f r// Sal I ScoRI
Blunt
T4 DNA Ligase
NcoI
pBPgIII
I,
,,
EcoRI I'
/-=
,I
SailI
FIG. 1. Construction of transfer vector pBPgIII. Both the PRV and BHV-1 glll translation initiation codons naturally reside within NcoI restriction endonuclease sites (2, 18). Plasmid pNT7/123 (obtained from Molecular Genetics Inc., Minnetonka, Minn.) is a derivative of ptacNB and p7/123 (18, 19) and contains the entire PRV glll gene. pNT7/123 was digested with PleI, and the protruding ends were repaired with DNA polymerase I Klenow fragment and digested with NcoI. A 1.5-kbp fragment containing the entire PRV glll coding sequence which extends from the translation initiation codon to 58 bp after the translation stop codon was isolated. pll3RI.Bgl3.0 (13, 14), which contains the PHV-1 glll gene, was digested with NcoI and EcoRl in order to isolate a 5-kbp fragment carrying 750 bp of the noncoding sequence immediately flanking the 5' end of the glll gene and the pBR322 backbone. To isolate the flanking sequence 3' to BHV-1 glll, pll3RI.Bgl3.0 was digested with Sall, treated with Klenow fragment to repair the protruding ends, and then digested with EcoRI to produce a 600-bp fragment. The 1.5-kbp PRV fragment and the 600-bp BHV-1 3' glll fragment were simultaneously ligated to the pBR322 backbone carrying the BHV-1 5' gIll sequence to produce the transfer vector, pBPgIII. Solid bars represent the PRV gIll gene fragment, hatched bars represent the BHV-1 gIII gene fragment, and solid lines represent the pBR322 backbone in pll3RI.Bgl3.0. The solid horizontal arrows above the PRV and BHV-1 gene fragments indicate the regions of the corresponding coding sequences.
protein in the wt BHV-1-infected cell extract (Fig. 2A, lane 2). This finding demonstrated the production of PRV gIII by the recombinant virus and corroborated the results from the black-plaque assay. In addition, it showed that the PRV gIII was glycosylated and had a molecular weight comparable to that of the mature form of authentic PRV gIll (18), suggesting that the PRV gIl was properly processed. PRV is considered to be an exotic pathogen in Canada; thus, government regulations restricted our use of live virus in these studies. We next examined whether the PRV gIll expressed by the recombinant virus was incorporated into mature virions. Integration of the recombinant protein into the virions may indicate that PRV glll shares certain functional properties required for normal intracellular glycoprotein transport and virus assembly with BHV-1 gIII. [3H]glucosamine-labelled, gradient-purified virus was analyzed for its glycoprotein composition by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The recombinant virus was directly compared with wt BHV-1 and a BHV-1 gIll deletion mutant that we described previously (13). Viral samples were run in parallel with immunoprecipitated PRV gIll. The results are shown in Fig. 2B. The wt virus (Fig. 2B, lane 1) produced a broad BHV-1 glll band at a molecular weight of about 93,000 (Fig. 2B, lane 1), which was missing from the glll mutant (Fig. 2B, lane 2). The absence of gIll in the gIll mutant virus revealed the presence of a rather sharp glyco-
protein band which was obscured by glll in the wt virus. On the basis of available data, this band probably represents an uncharacterized BHV-1 glycoprotein (this band is not detected with anti-glll antibody [data not shown]). In contrast to the wt virus and the gIII mutant, vBPgIII (Fig. 2B, lane 3) shows a novel glycoprotein band which has the same electrophoretic mobility as immunoprecipitated PRV gIII, indicating that this band is in fact PRV glll. As measured by densitometer scanning, the band at the PRV glll position and the band at the BHV-1 glll position constituted 34 and 36% of the labelled viral proteins of BHV-1 and vBPgIII, respectively. Since it is unlikely that the expression of the glycoprotein which migrates at the same position as glll differs in wt BHV-1 and vBPgIII, the results from densitometer measurements indicate that the incorporation of PRV gIII in the recombinant virions was as efficient as that of BHV-1 gIII in the wt virus. The existence of PRV gIll in the recombinant virus was also tested by an antibody-mediated neutralization assay. Table 1 shows the results from such an assay using MAbs specific to PRV glll. Neither the wt virus nor the gIII mutant was neutralized by PRV gIII-specific MAbs. However, the recombinant virus was highly sensitive to the MAb neutralization both in the presence and in the absence of complement.
Proper expression and incorporation of PRV glll into the recombinant virus enabled us to perform comparative func-
VOL. 65, 1991
NOTES
5555
TABLE 1. Plaque reduction assay Mean no. of plaques formed (% reduction") Antibodies
Control MAbsb PRV glll MAbs' '
With complement
Without complement wt
gll-
vBPgIII
wt
187 (0) 217 (0)
391 (0) 351 (10)
186 (0) 70 (62)
229 (0) 205 (10)
gIII-
368 (0) 374 (0) Percentages of plaque reduction were calculated by setting the number of plaques formed in the presence of control MAbs as 0% reduction.
vBPgIll 166 (0) 2 (99)
b A mixture of irrelevant MAbs. ' A mixture of PRV glll-specific MAbs (Ml, M6, and M7 [51).
tional studies with the wt and mutant viruses. We previously described a number of functional defects associated with a BHV-1 glll deletion mutant which include reduced attachment to permissive cells, a delay in virus replication, and reduction in the amount of progeny virus shed from infected cells (13). These defects were fully corrected by reconstituting the glll gene via marker rescue of the glll gene-deleted virus (12a), demonstrating various functions of glll in virus replication. Therefore, it was possible to characterize the role played by PRV glll in the process of virus replication by A
B _~
>
=
_
_
F
200
97
697>
~
iwl ~~~
^
46
FIG. 2. Detection of PRV glll expression in vBPglll-infected cells and in vBPglII virions. (A) Immunoprecipitation assay of PRV gIll in vBPgIII-infected cells using either anti-PRV glll MAbs (Ml, M6, and M7 [5]) or anti-BHV-1 glll MAbs (25). MDBK cells were infected with the recombinant virus at a multiplicity of infection of 5 and labelled with [3H]glucosamine. After 18 h of labelling, the cells were lysed and immunoprecipitated for PRV glll with either a mixture of anti-PRV gll MAbs or a mixture of anti-BHV-1 glll MAbs. Samples were separated on an SDS-7.5% polyacrylamide gel under reducing conditions. Lane PRVgIII, sample precipitated with anti-PRV glll MAbs; lane BHVgIII, sample precipitated with antiBHV-1 glll MAbs. (B) SDS-PAGE profiles of [3H]glucosaminelabelled viral proteins. The virus tested was labelled with [3H]glucosamine as described above and purified by a tartrate gradient, and the labelled viral proteins were separated by SDS-PAGE (7.5% polyacrylamide) together with the immunoprecipitated samples. Lane WT, wt BHV-1; lane gIll-, BHV-1 glll deletion mutant; lane vBPgIII, recombinant BHV-1 expressing PRV gIll.
comparing the phenotype of the PRV glll recombinant virus with the phenotypes of the wt and the gIII- viruses. Any restoration of wt function(s) in the recombinant virus expressing PRV gIll (i.e., compared with the glll deletion mutant) would demonstrate complementation between BHV-1 and PRV gIll. Initially, the virus replication efficiency of vBPgIII was examined and compared with the replication efficiencies of the wt and the gIl- viruses. In this experiment, virus was first allowed to adsorb to MDBK cells. After 3 h incubation, unadsorbed virus was removed by washing. Virus replication kinetics were then monitored by determining the cellassociated virus titers at various times postinfection. As shown in Table 2, at all time points assessed, vBPgIII yielded virus titers 0.5 to 2 orders of magnitude higher than the gIII mutant, indicating that expression of PRV glll by the recombinant virus restored replication efficiency lost by deleting the BHV-1 gIll gene. However, at the same time, the wt virus infection yielded higher titers than vBPgIII, indicating that PRV gIII was less efficient than BHV-1 gIII in mediating BHV-1 replication as a whole (Table 2). Since the glll homologs are known to be multifunctional (e.g., both are needed for efficient virus egress from infected cells [22]), it is probable that certain unspecified replication functions determined by BHV-1 glll cannot be efficiently replaced by PRV gIll. Since the glll homologs are required for efficient cellular attachment of either virus, we examined whether PRV glll could replace BHV-1 glll in this important function. MDBK cells were incubated with [35S]methionine-labelled vBPgIII, wt BHV-1, or BHV-1 glll mutant for different lengths of time. After extensive washing to remove unbound virus, cell-associated virus was quantitated by liquid scintillation counting of the infected cells taken at each time point. Figure TABLE 2. Replication of wt, gIll mutant, and vBPgIII in MDBK cells" Virus titer [log (PFU/ml)]
Time (h) wt
0 10 13 24 48
glll mutant
vBPgIII
2.38
2.23
1.92
6.79 7.15 7.03 8.0
5.74 5.74 5.89 4.51
5.81 6.27 6.88 6.95
MDBK cells at confluence in 24-well plates were infected with virus at an approximate multiplicity of infection of 2 in 0.2 ml of inoculum per well. Duplicate experiments were carried out for each virus. After 3 h of adsorption at 37°C, the cells were washed to remove the unadsorbed virus and incubated in 1 ml of culture medium. At the indicated times postinfection, the cells were washed, and the cell-associated virus was harvested and titrated for virus titers. Time zero is the time point immediately after adsorption.
NOTES
5556
J. VIROL.
40
130
0 20
E o
0
100 I00-
80
0
0-
60
."a to 0
40 20 0
0
10
20
30
40
50
60
Time (min) FIG. 3. (A) Binding of radiolabelled wt, gIll mutant, or vBPgIIl recombinant virus to MDBK cells. Virus was labelled with [35S]methionine, purified through a tartrate gradient, and diluted to 105 cpm/ml in 1% bovine serum albumin in phosphate-buffered saline (BSA-PBS) before use. Calculated from the DNA contents of the virus (17), the specific activities of the labelled viruses were 2.08 x 10-5 cpm per particle for wt virus, 4.49 x 10-5 cpm per particle for the gIll mutant, and 1.66 x 10-5 cpm per particle for vBPgIII. The viral infectivity levels were 4.29 x 10-4 PFU per particle for the wt virus, 2.69 x 10-' PFU per particle for the glll mutant, and 9.73 x i0-5 PFU per particle for vBPgIII. MDBK cells at confluence in 24-well culture plates were cooled to 4°C and blocked with BSAPBS for 30 min. Virus (0.1 ml) was then added to each well, and the cells were incubated at 4°C for the times indicated. After incubation, unbound virus was removed, the cells were washed three times with PBS and harvested by detergent lysis, and the total cell-associated radioactivities were determined (13). The results represent means of triplicate samples. (B) Penetration kinetics of the wt, the gIll mutant, and vBPgIII on MDBK cells. MDBK cells in six-well culture plates were cooled to 4°C, inoculated with approximately 200 PFU of virus in 0.4 ml of minimal essential medium, and adsorbed at 4°C for 1 h. After adsorption, the virus inocula were removed, and the cells were returned to 37°C. At specified times, the cells were washed with glycine-HCl, pH 3.0. The cells were then overlaid with agarose. The viral plaques were counted 4 days later. The number of viral plaques formed in the wells without the acid wash was used as 100% penetration for calculating the percentages of penetrated virus at the specified time points. The results represent the means standard deviations of quadruplicate samples. Sym-
bols:
0,
wt;
A,
gIll mutant; *, vBPgIII.
3A shows the results from this experiment. The wt virus and the recombinant virus expressing PRV gIll showed significantly higher binding efficiency than the BHV-1 gIll mutant. For example, at 120 min, wt BHV-1 and vBPgIII showed 4.3- and 6.3-fold-higher binding efficiencies than the gIll mutant. Differences between wt BHV-1 and vBPgIII were also noticed in this particular experiment. However, since the differences, at all time points assessed, were rather small (less than 0.5-fold) and were not observed in another similar experiment, the significance of the differences between wt
BHV-1 and vBPgIII remains to be determined. In any event, these data demonstrate that PRV glll is at least as efficient as BHV-1 glll in mediating virus attachment. Previously, a PRV glll deletion mutant has been shown to exhibit a lower penetration rate than wt PRV (15). We have observed a similar phenomenon with our BHV-1 gIll mutant (unpublished data). Therefore, we were interested in testing whether the substitution of the PRV glll gene for the BHV-1 homolog, which was fully complementary for virus binding, was also compiementary for virus penetration. In this experiment, virus was first allowed to preadsorb to MDBK cells at 4°C. After unattached virus was removed, the cells were incubated at 37°C. At different times after transfer to 37°C, the infected cells were washed with glycine-HCl, pH 3.0, to inactivate unpenetrated virus. Virus that had completed the penetration process was then quantitated by plaque assays. Samples which were not washed with the acid solution were used as controls, and penetration percentages were calculated for each time point, with the penetration of the untreated controls set at 100% (Fig. 3B). Both the wt virus and the recombinant virus expressing PRV glll showed significantly higher penetration rates than the glll mutant, indicating that PRV glll is also capable of complementing BHV-1 glll for the virus penetration process. The results from this study demonstrate that (i) PRV glll expressed by a recombinant BHV-1 was efficiently incorporated into BHV-1 virions, (ii) the recombinant virus was susceptible to anti-PRV glll antibody-mediated virus neutralization, (iii) the recombinant virus showed significantly higher replication efficiency than a BHV-1 glll gene deletion mutant, and (iv) PRV glll expressed by the recombinant virus was as efficient in mediating virus attachment and penetration as was BHV-1 gIll in the wt virus. On the basis of these results, we concluded that PRV glll and BHV-1 gIll share complementary functions. REFERENCES 1. Cai, W., B. Gu, and S. Person. 1988. Role of glycoprotein B of herpes simplex virus type 1 in viral entry and cell fusion. J. Virol. 62:2596-2604. 2. Fitzpatrick, D. R., L. A. Babiuk, and T. J. Zamb. 1989. Nucleotide sequence of bovine herpesvirus type 1 glycoprotein gIll, a structural model for gIll as a new member of the immunoglobulin superfamily, and implications for the homologous glycoproteins of other herpesviruses. Virology 173:46-57. 3. Fitzpatrick, D. R., T. J. Zamb, and L. A. Babiuk. 1990. Expression of bovine herpesvirus type 1 glycoprotein gI in transfected bovine cells induces spontaneous cell fusion. J. Gen. Virol. 71:1215-1219. 4. Fuller, A. O., and P. G. Spear. 1985. Specificities of monoclonal and polyclonal antibodies that inhibit adsorption of herpes simplex virus to cells and lack of inhibition by potent neutralizing antibodies. J. Virol. 55:475-482. 5. Hampl, H., T. Ben-Porat, L. Ehrlicher, K.-O. Habermehl, and A. S. Kaplan. 1984. Characterization of the envelope proteins of pseudorabies virus. J. Virol. 52:583-590. 6. Hughes, G., L. A. Babiuk, and S. van Drunen Littel-van den Hurk. 1988. Functional and topographical analysis of epitopes on bovine herpesvirus type 1 glycoprotein gIV. Arch. Virol. 103:47-60. 7. Johnson, D. C., R. L. Burke, and T. Gregory. 1990. Soluble forms of herpes simplex virus glycoprotein D bind to a limited number of cell surface receptors and inhibit virus entry into cells. J. Virol. 64:2569-2576. 8. Johnson, D. C., and M. W. Ligas. 1988. Herpes simplex viruses lacking glycoprotein D are unable to inhibit virus penetration: quantitative evidence for virus-specific cell surface receptors. J. Virol. 62:4605-4612. 9. Johnson, D. C., M. R. McDermott, C. Chrisp, and J. C. Glorioso.
VOL. 65, 1991 1986. Pathogenicity in mice of herpes simplex virus type 2 mutants unable to express glycoprotein C. J. Virol. 58:36-42. 10. Johnson, D. C., M. Wittels, and P. G. Spear. 1984. Binding to cells of virosomes containing herpes simplex virus type 1 glycoproteins and evidence for fusion. J. Virol. 52:238-247. 11. Kuhn, J. E., M. D. Kramer, W. Willenbacher, U. Wieland, E. U. Lorentzen, and R. W. Braun. 1990. Identification of herpes simplex virus type 1 glycoproteins interacting with the cell surface. J. Virol. 64:2491-2497. 12. Lentz, T. L. 1990. The recognition event between virus and host cell receptor: a target for antiviral agents. J. Gen. Virol. 71:751-766. 12a.Liang, X. Unpublished data. 13. Liang, X., L. A. Babiuk, S. van Drunen Littel-van den Hurk, D. R. Fitzpatrick, and T. J. Zamb. 1991. Bovine herpesvirus 1 attachment to permissive cells is mediated by its major glycoproteins gI, glll, and gIV. J. Virol. 65:1124-1132. 14. Mayfield, J. E., P. J. Good, J. J. VanOort, A. R. Campbell, and D. E. Reed. 1983. Cloning and cleavage site mapping of DNA from bovine herpesvirus 1 (Cooper strain). J. Virol. 47:259-264. 15. Mettenleiter, T. C. 1989. Glycoprotein glll deletion mutants of pseudorabies virus are impaired in virus entry. Virology 171: 623-625. 16. Mettenleiter, T. C., L. Zsak, F. Zuckermann, N. Sugg, H. Kern, and T. Ben-Porat. 1990. Interaction of glycoprotein gIll with a cellular heparinlike substance mediates adsorption of pseudorabies virus. J. Virol. 64:278-286. 17. Nemerow, G. R., F. C. Jensen, and N. R. Cooper. 1982. Neutralization of Epstein-Barr virus by nonimmune human sera. J. Clin. Invest. 70:1081-1091. 18. Robbins, A. K., R. J. Watson, M. E. Whealy, W. W. Hays, and L. W. Enquist. 1986. Characterization of a pseudorabies virus glycoprotein gene with homology to herpes simplex virus type 1
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