Vol. 66, No. 10

JOURNAL OF VIROLOGY, OCt. 1992, p. 6208-6212

0022-538X/92/106208-05$02.00/0 Copyright © 1992, American Society for Microbiology

Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Molecules Containing Membrane FusionImpairing Mutations in the V3 Region Efficiently Undergo Soluble CD4-Stimulated gpl20 Release EDWARD A. BERGER,* JERRY R. SISLER, AND PATRICIA L. EARL Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892 Received 30 March 1992/Accepted 10 July 1992

The ability of soluble forms of CD4 to induce gpl20 release from the human immunodeficiency virus type 1 envelope glycoprotein complex may reflect molecular events associated with membrane fusion. The third hypervariable (V3) region of gpl20 appears to play a role in fusion independent of CD4 binding. We demonstrate herein that envelope glycoprotein molecules rendered fusion defective by mutations in conserved residues within the V3 region nevertheless undergo efficient soluble CD4-induced gpl20 release.

infectivity and syncytium formation without inhibiting CD4 binding (34). V3 has since been implicated as the site of interaction of fusion-inhibiting sulfated polysaccharides which also do not block CD4 binding (3, 6). Recently, site-directed mutation of V3 has been shown to severely impair infectivity and cell-cell fusion activity without affecting CD4 binding (12, 14, 17, 19, 33, 39). A cluster of highly conserved residues (GPGR) at the tip of the hypervariable loop appears to be particularly critical (12, 14, 17, 19, 33). In view of the apparent involvement of the V3 region of gpl20 in membrane fusion and the proposed relationship between fusion and sCD4-induced gpl20 release, it was of interest to examine the ability of sCD4 to stimulate gpl20 release from HIV-1 envelope glycoprotein molecules containing fusion-impairing mutations in the V3 region. A recombinant vaccina virus expression system was employed to test sCD4 stimulation of gpl20 release from the mutant envelope glycoproteins, as reported previously for the wild type (4). By a variety of criteria, cell fusion mediated by vaccinia virus-encoded HIV envelope glycoprotein and CD4 faithfully recapitulates the specificities observed in HIVinfected lymphocyte cultures (see citations in reference 4). As with HIV virions and HIV-infected cells, sCD4 stimulation of gp120 release in the vaccinia virus-based system is dependent on the specific CD4-gpl2O binding on the basis of the blocking effects of anti-CD4 monoclonal antibodies (4). We tested two specific single-amino-acid substitution mutations in the conserved cluster at the tip of the loop. In the envelope glycoprotein of the HIV-1 strain used in our experiments (HTLV-IIIB, BH8 isolate), these correspond to a glycine-to-glutamic acid substitution at position 314 (G314E) and an arginine-to-glycine substitution at position 315 (R315G) (the third and fourth positions, respectively, of the conserved GPGR motif). These mutations were chosen because they have been shown to abolish syncytium formation in transfection experiments with a CD4+ HeLa cell line, despite normal CD4 binding by the corresponding gpl20 molecules (12). Plasmids were constructed by inserting the 1.3-kb StuI-HindIII envelope DNA fragments from plasmids containing the wild-type sequence or either of the two mutated sequences (12) into the corresponding site of plasmid pPE7H. Plasmid pPE7H contains the HIV-1 envelope

Human immunodeficiency virus type 1 (HIV-1) infection is initiated by binding of the viral envelope glycoprotein to CD4 molecules on the surface of the target cell followed by direct fusion between the virion and plasma membranes (reviewed in references 8 and 25). In a related process, cells expressing the HIV-1 envelope glycoprotein specifically fuse with CD4+ cells, leading to the formation of multinucleated giant cells (syncytia). The envelope glycoprotein is initially synthesized as a precursor (gpl60) which is proteolytically cleaved to yield a noncovalent gpl2O-gp4l complex; the external gpl20 subunit contains the CD4-binding site, whereas the transmembrane gp4l subunit anchors the complex to the surface. The molecular mechanisms by which the CD4-envelope glycoprotein interaction promotes membrane fusion are poorly understood. Several laboratories have reported that recombinant soluble forms of CD4 (sCD4) stimulate dissociation of the gpl2O-gp4l complex expressed on either the virion or the cell surface, leading to release of the gpl20 (4, 15, 22, 27, 28). These findings support the notion that CD4 might serve not only to mediate binding but also to trigger specific structural changes in the envelope glycoprotein which activate its fusogenic property, possibly by exposing the gp4l hydrophobic amino terminus, which has been directly implicated in the fusion process (see references 8 and 25 for reviews). Genetic engineering, biochemical, and immunological studies have enabled identification of regions within both the gpl20 and CD4 molecules involved in the binding interaction (8, 25). Furthermore, evidence suggesting that there are features of the CD4-envelope glycoprotein interaction critical for membrane fusion that are distinct from those involved in high affinity binding has accumulated (see references 8, 11, 25, and 29 for reviews). Within gpl20, the third hypervariable region (designated V3) appears to play a role in membrane fusion distinct from an involvement in high affinity CD4 binding (reviewed in references 29 and 34). This notion originally emerged from the findings that V3 contains the principal neutralizing determinant; polyclonal or monoclonal antibodies directed against this region block virus *

Corresponding author. 6208

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NOTES WT

G314E

6209

Control

R315G _

200

gp160-0-4_ gp120-0- *

-"*92 _69

_46

FIG. 1. Expression and processing of vaccinia virus-encoded wild-type (WT) and mutant HIV-1 envelope glycoproteins. Western blot analysis was performed on lysates of BSC-1 cells expressing the wild-type HIV-1 envelope glycoprotein or the G314E or R315G mutant (encoded by vaccinia virus recombinants JS-1, JS-2, and JS-3, respectively). The gpl60 and gpl20 bands are indicated on the left, and positions of molecular mass markers (in kilodaltons) are indicated on the right.

glycoprotein gene linked to the vaccinia virus compound early-late P7.5 promoter (42); it also contains the Escherichia coli lacZ gene driven by another vaccinia virus promoter. Vaccinia virus recombinants were selected by homologous recombination into the thymidine kinase locus coupled with 0-galactosidase screening and purified by standard methods (10). All experimental procedures were performed at 37°C in 5% C02-95% air in Eagle's minimal essential medium containing 2.5% (vol/vol) fetal bovine serum plus antibiotics. The expression and processing of the vaccinia virusencoded HIV-1 envelope glycoproteins in BSC-1 cells were examined as previously described (4). Cell monolayers were infected at a multiplicity of infection of 10 with the appropriate virus and then trypsinized 2 to 5 h postinfection. The cell suspensions were incubated overnight (9 to 14 h) to allow envelope glycoprotein accumulation, and cycloheximide (10 ,ug/ml) was added for 4 to 6 h to enable completion of protein processing. The cells were washed and then solubilized in phosphate-buffered saline containing 0.5% (vol/vol) Nonidet P-40 plus protease inhibitors. Aliquots of the cell lysate fractions were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 10% polyacrylamide gels and transferred to nitrocellulose for Western blotting (immunoblotting) and probing with rabbit anti-gp120 antiserum followed by 125I-protein A. As shown in the autoradiogram in Fig. 1, the mutations did not impair total envelope glycoprotein expression; proteolytic precursor processing was also unaffected, as judged by the similar gpl6o/gpl20 ratios. Flow cytometry analysis (EPICS Profile; Coulter) demonstrated that the mutations did not diminish cell surface envelope glycoprotein expression. Major peak mean fluorescence intensities (determined by using antigp160 rabbit antiserum and then fluorescein isothiocyanateconjugated goat anti-rabbit immunoglobulin antiserum [Calbiochem]) were 21.3, 21.1, and 29.2 for cells expressing the wild-type, G314E, and R315G envelope glycoproteins, respectively; the background values (obtained with nonimmune rabbit serum in the first step) were 6.6 to 7.5. These results, showing normal expression and processing of the vaccinia virus-encoded envelope glycoproteins, parallel those reported for transfection experiments with the same mutations (12). To test the effects of the envelope glycoprotein mutations

FIG. 2. Syncytium formation mediated by vaccinia virus-encoded wild-type and mutant HIV-1 envelope glycoproteins. HeLa cells expressing vaccinia virus-encoded CD4 were mixed with BSC-1 cells expressing the indicated vaccinia virus-encoded envelope glycoproteins. In the control, the BSC-1 cells were infected with the WR strain of vaccinia virus. The photomicrographs were taken 22 h after cell mixing.

on syncytium formation in the vaccinia virus-based system, mixing experiments were performed with cells expressing vaccinia virus-encoded envelope glycoprotein and CD4. Envelope glycoprotein expression in BSC-1 cells was achieved by infection, trypsinization, and overnight incubation as described above. For CD4 expression, HeLa cell monolayers were coinfected at a multiplicity of infection of 10 with each of two viruses: vEB-8, which encodes fulllength CD4 under control of the bacteriophage T7 promoter (1), and vTF7-3, which encodes the T7 RNA polymerase driven by the P7.5 promoter (13). Cells were trypsinized and incubated overnight for protein expression, as noted above for the BSC-1 cells. Equal numbers (105) of envelope glycoprotein-expressing and CD4-expressing cells were mixed in a total volume of 0.2 ml in individual wells of a 96-well flat-bottom microtiter plate (Costar); cultures were examined microscopically at various times. Figure 2 shows the results at 22 h after cell mixing. With cells expressing wild-type envelope glycoprotein, syncytia were observed by 2 h and gradually increased in size and number. Formation of syncytia was strictly dependent on envelope glycoprotein expression, as judged by their absence when a control vaccinia virus (strain WR) was used to infect the BSC-1 cells. In contrast to the results obtained with wild-type envelope glycoprotein, syncytia were not observed with either the G314E or the R315G mutant. The syncytial defects of the mutant envelope glycoproteins were also noted when CD4+ human T-cell lines were directly infected with the vaccinia virus recombinants (data not shown). The vaccinia virus system thus recapitulates the inhibitory effects of these

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NOTES G314E

WT gpl2O-sCD4 (nM):

R315G

0

10

100 1000

0

10 100 1000

0

10

100 1000

=100 E

x E

o 80 U)

60 CN 0

40

CD 0

c 0

cT

20

E

sCD4 Concentration (nM)

FIG. 3. sCD4 stimulation of gp120 release from vaccinia virusencoded wild-type (WT) and mutant HIV-1 envelope glycoproteins. BSC-1 cells expressing the indicated vaccinia virus-encoded envelope glycoproteins were treated for 3 h with various concentrations of sCD4. The sCD4 construct consisted of the four extracellular domains (residues 1 to 369). The medium fractions were analyzed for gp120 by Western blotting. (Top) Autoradiogram of Western blot. Only the gp120 band is shown. (Bottom) Quantitation of Western blot. The relative radioactivity in each gp120 band was determined with a PhosphorImager (Molecular Dynamics). For each envelope glycoprotein, the gp120 released in the absence of sCD4 was subtracted to yield the sCD4-stimulated component; the value obtained at 1,000 nM sCD4 was defined as 100%.

single-amino-acid substitutions on cell fusion previously observed in transfection experiments (12). We then compared the ability of sCD4 to stimulate gpl20 release from the wild type with its ability to stimulate gp120 release from the mutant envelope glycoproteins. BSC-1 cells infected with the recombinant vaccinia viruses and incubated as described for Fig. 1 to allow envelope glycoprotein expression and processing. Cells were washed by centrifugation in medium and then suspended at a density of 106/ml in medium containing cycloheximide (10 ,ug/ml) plus the concentrations of sCD4 indicated in Fig. 3. Reaction volumes were 0.06 ml in individual wells of 96-well microtiter plates. After 3 h, medium and cell lysate fractions were obtained, and gpl20 release was assessed by Western blot analysis (using procedures described for Fig. 1). Control experiments (not shown) indicated that sCD4-stimulated gpl20 release for all three envelope glycoproteins was >90% complete at this time point, as previously reported for the wild type (4). The autoradiogram in Fig. 3 indicates that there was some spontaneous gpl20 release for each envelope glycoprotein. Addition of sCD4 stimulated gpl20 release from the wild-type and the G314E and R315G mutant envelope glycoproteins; the dose dependencies were comparable in all cases (Fig. 3). Analysis of each mutant cell lysate fraction (not shown) indicated that most of the gpl20 was released, whereas the uncleaved gpl60 was not, as previously shown for the wild-type envelope glycoprotein (4). We conclude that for these particular V3 mutants, there is no correlation between loss of membrane fusion activity and sensitivity to sCD4-stimulated gpl20 release.

were

The present findings, coupled with recent data in the literature, raise questions about the relationship of gp120 release to the membrane fusion process. Most primary HIV-1 isolates are now known to be highly refractory to sCD4-stimulated gp120 release (26); nevertheless, they infect cells by a CD4-dependent route. The present study using fusion-defective envelope glycoprotein mutants, coupled with an earlier study using a fusion-inhibiting anti-gp120 monoclonal antibody (28), indicates that sCD4 stimulation of gpl20 release can occur under conditions which are nonpermissive for fusion. Thus, the gross structural change measured in the gp120 release assay is neither necessary nor sufficient for the fusion process. While gpl20 release per se may not be critical, this in no way negates the working proposal that CD4 activates the envelope glycoprotein for fusion, possibly by triggering more subtle conformational changes. Release of gp120 may simply be a readily measurable, though not essential, consequence of such changes. In this regard, it has been reported that sCD4 binding is associated with enhanced exposure of specific regions on both gp120 (9, 35) and gp41 (15, 35), as judged by monoclonal antibody binding and protease digestion experiments. The relationship of such changes to the fusion process is an intriguing question. The role of the V3 region in membrane fusion mediated by the CD4-HIV-1 envelope glycoprotein interaction remains unclear. One notion is that V3 interacts directly with specific regions of the CD4 molecule. The present findings, coupled with the failure of an anti-V3 monoclonal antibody to inhibit sCD4-induced gpl20 release (28), suggest that V3 does not participate directly in this particular structural change. However, it is still possible that V3 associates directly with specific regions of CD4 in a manner not revealed by the gp120 release assay. Indeed, synthetic peptides containing V3 sequences have been reported to bind to cell surface CD4 (2); conversely, evidence that a synthetic peptide derivative based on a sequence in domain 1 of CD4 binds to V3 has been presented (3). The recent discovery that the determinants responsible for the reduced sCD4 neutralizability of primary HIV-1 isolates are associated with a region of gp120 containing V3 (31) raises additional intriguing questions about the role of V3 in the CD4-gp120 interaction. As an alternative (though not mutually exclusive) notion, V3 may interact functionally with other molecular components on the surface of the target cell. The possibilities that V3 is recognized by a specific target cell protease(s) and that proteolysis plays an essential role in the fusion process have been considered (9, 16, 20, 21, 23, 30). As a final point, V3 has been implicated not only in fusion per se but also as a possible factor in the selective tropisms of different HIV-1 isolates for different CD4+ cell types (5, 7, 18, 24, 31, 32, 36-38, 40, 41). Whether such fusion selectivities are associated with additional specific molecular components of the CD4+ target cell and whether the V3 region interacts with such components remain critical questions for further study. This work was supported in part by the NIH Intramural AIDS Targeted Antiviral Program. The excellent technical assistance of P. Kennedy and N. Cooper is gratefully acknowledged. We thank E. 0. Freed, (University of Wisconsin, Madison) for plasmids pHenv, pHenv319GE, and pHenv320RG (12); S. J. Johnson (Upjohn, Kalamazoo, Mich.) for sCD4; and R. L. Willey (NIAID, Bethesda, Md.) for rabbit antisera to gpl20 and gpl60. The assistance and helpful comments of C. C. Broder and B. Moss (NIAID, Bethesda, Md.) are gratefully acknowledged.

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