Proc. Nati. Acad. Sci. USA Vol. 88, pp. 2189-2193, March 1991 Medical Sciences

Binding of soluble CD4 proteins to human immunodeficiency virus type 1 and infected cells induces release of envelope glycoprotein gp120 (acquired immunodeficiency

syndrome/transmembrane glycoprotein/gp4l/retrovirus)

TIMOTHY K. HART*t, RICHARD KIRSHt, HARMA ELLENS*, RAYMOND W. SWEET§, DENNIS M. LAMBERT¶, STEPHEN R. PETTEWAY, JR.¶, JEFFRY LEARY$, AND PETER J. BUGELSKI* Departments of *Experimental Pathology, tDrug Delivery, WMolecular Genetics, and lAnti-Infectives, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406

Communicated by John D. Baldeschwieler, December 5, 1990

ABSTRACT Human immunodeficiency virus (HIV) infects cells after binding of the viral envelope glycoprotein gpl20 to the cell surface recognition marker CD4. gpl20 is noncovalently associated with the HIV transmembrane envelope glycoprotein gp4l, and this complex is believed responsible for the initial stages of HIV infection and cytopathic events in infected cells. Soluble constructs of CD4 that contain the gpl20 binding site inhibit HIV infection in vitro. This is believed to occur by competitive inhibition of viral binding to cellular CD4. Here we suggest an alternative mechanism of viral inhibition by soluble CD4 proteins. We demonstrate biochemically and morphologically that following binding, the soluble CD4 proteins sT4, VjV2,DT, and V1[106J (amino acids 1-369, 1-183, and -2 to 106 of mature CD4) induced the release of gpl20 from HIV-1 and HIV-1-infected cells. gpl20 release was concentration-, time-, and temperature-dependent. The reaction was biphasic at 37C and did not take place at 4°C, indicating that binding of soluble CD4 was not sufficient to release gp120. The appearance of free gpl20 in the medium after incubation with sT4 correlated with a decrease in envelope glycoprotein spikes on virions and exposure of a previously cryptic epitope near the amino terminus of gp4l on virions and infected cells. The concentration of soluble CD4 proteins needed to induce the release of gpl20 from virally infected cells also correlated with those required to inhibit HIV-mediated syncytium formation. These results suggest that soluble CD4 constructs may inactivate HIV by inducing the release of gpl20. We propose that HIV envelope-mediated fusion is initiated following rearrangement and/or dissociation of gpl20 from the gpl2O-gp4l complex upon binding to cellular CD4, thus exposing the fusion domain of gp4l.

The envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1) consist of two noncovalently associated subunits, gpl20 and gp41, which are derived from the gpl60 precursor (1-3). Viral attachment to target cells is mediated by gpl20 molecules, which are visible ultrastructurally as electron dense spikes on viral membranes. The gpl20 specifically associates with the human CD4 receptor (4-7), a glycoprotein present on the surface of the T4 subset of lymphocytes and, in this manner, is responsible for the tropism of HIV for this and other CD4' cells. Subsequent to attachment, fusion of viral and cellular membranes occurs; a process thought to involve the transmembrane glycoprotein gp4l. The amino-terminal region of gp41 is implicated in this event, based on sequence homology with the fusion regions of membrane proteins of other viruses (8-10) and amino acid substitution analysis of this region (11). gpl20 and gp4l also

appear to function in a similar manner in HIV-induced syncytium formation, as expression of recombinant versions of these proteins, in the absence of other HIV proteins, was sufficient for cell-cell fusion (12). Soluble CD4 proteins, consisting of all or portions of the external region of human CD4, efficiently inhibit infection and virus-induced syncytium formation by HIV-1 (13-16). This effect has been attributed to association of the soluble CD4 protein with gpl20 on the surface of virus and virusinfected cells and consequent competitive inhibition of viral gpl20 binding to cell surface CD4. However, we recently reported (17) that incubation of HIV-1-infected cells with sT4, a soluble CD4 construct composed of the entire extracellular domain of native CD4 (13), resulted in the reduction of virion envelope glycoprotein spikes and the appearance of gpl20 in the supernatant. The release of gpl20 by sT4 indicated that the inhibitory effect might result, at least in part, in inactivation of virus by removing viral binding proteins. Moreover, this release suggested that a similar rearrangement of envelope glycoproteins may occur upon virus binding to cell surface CD4, which then leads to virus-cell fusion. The importance of events following binding of gpl20 to CD4 to the processes of infectivity and syncytium formation have been demonstrated. Disruption of the CD4 binding site on gpl20 (18, 19) prevents infection of CD4' cells. However, the presence of functional binding sites on CD4 and gp120 is not sufficient for infection. Murine cells expressing human CD4 are not infected by HIV (7). In contrast, syncytium formation is disrupted in cells expressing noncleavable mutations of gp160 which did not inhibit gpl2O-CD4 binding (20). Thus, the events subsequent to gpl2O-CD4 binding are important to the pathogenesis of HIV. The present study examines the events associated with viral gpl20-CD4 interactions. We have now refined our analysis of the soluble CD4-induced release of gpl20 from HIV virions and infected cells by examining the stoichiometry, kinetics, and temperature dependence of this process in relationship to the anti-infectivity aspects of soluble CD4 proteins. This analysis has allowed us to define the regions of CD4 required for gpl20 release and to identify the consequent exposure of a cryptic epitope at the amino terminus of gp41.

MATERIALS AND METHODS Proteins and Antibodies. The soluble CD4 proteins sT4 [amino acids (aa) 1-369 of mature human CD4], V1V2,DT (aa Abbreviations: HIV-1, human immunodeficiency virus type 1; HRP, horseradish peroxidase. tTo whom reprint requests should be addressed at: SmithKline Beecham Pharmaceuticals, Mail stop L-62, P.O. Box 1539, King of Prussia, PA 19406.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 2189

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1-183), and V1[106J (aa -2 to 106) and the derivatives were described previously (13, 21). The sT4 and V1V2,DT proteins were prepared in mammalian cells; V1[106] was expressed in Escherichia coli. Concentrations of soluble CD4 proteins were determined by anti-CD4 monoclonal antibodies (21). Recombinant gpl20 from the BH10 isolate of HIV-1 was produced in Drosophila cells (35). Guinea pig anti-gpl20 antibodies were produced against a synthetic peptide analog of amino acids 296-331 from the hypervariable loop of gp120 from HIV-1 strain HXB2. This antibody detected similar amounts of purified recombinant and viral gpl20 in Western blots and ELISAs. Mouse monoclonal anti-gp4l was from Cellular Products, and OKT4a antibody from Ortho Pharmaceuticals. All other antibodies were obtained from Jackson ImmunoResearch. Cell Lines and Virus. H9 and CEM cells chronically infected with HIV-1 strain HTLV-IIIB were used. The MOLT-4 cell line was used as a fusion partner in syncytial assays. Cells were maintained in RPMI 1640 (GIBCO) containing 10% heat-inactivated fetal bovine serum and antibiotics. Western Blot Analysis for gpl2O Release. Chronically infected H9 cells were washed once in culture medium and resuspended at 1-1.5 x 107 cells per ml in medium containing sT4, V1[106], V1V2,DT, or A55F at equimolar concentrations. Control cells were incubated in medium alone. Following the incubations, cell-free supernatants were diluted 1:2 with 2x sample buffer and boiled for 5 min. After SDS/7.5% PAGE, the proteins were transferred to Immobilon-P membranes (Millipore). The blots were probed with a 1:1000 dilution to guinea pig anti-gpl20 antibody followed by alkaline phosphatase-labeled anti-guinea pig IgG or biotinylated anti-guinea pig IgG and streptavidin-horseradish peroxidase (HRP). Visualization of alkaline phosphatase was with nitroblue tetrazolium (Sigma) and HRP was visualized with the ECL detection system (Amersham). The optical density of alkaline phosphatase-stained envelope protein bands was determined using a Magiscan image-analysis system (Joyce-Loebl). Recombinant gp120 was used as a standard, and detection was linear between 0.1 and 5.0 ng of

gpl20. Electron Microscopy and Quantitation of Envelope Glycoprotein Spikes. Control and sT4-treated HIV-i-infected cells were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3). Cells were postfixed in 1% OS04 and envelope glycoproteins were stained with 1% tannic acid followed by aqueous 1% uranyl acetate (22). Samples were processed and analyzed as reported (17). The linear surface density of spikes on virions was calculated (spikes per um), and 70-90 virions were evaluated for each sample. Means, standard deviations, histograms, and statistical analysis (2-tailed t tests) were prepared using a Videoplan (Zeiss). Curves were fitted with SigmaPlot (Jandel Scientific, Corte Madera, CA) and represent fifth-order regression analyses. Immunocytochemical Staining and Quantification of gp4l. Cells were washed twice following incubation with or without sT4, then incubated in a 1:50 dilution of antibody in medium, washed twice, fixed with glutaraldehyde, and sequentially incubated in biotinylated goat anti-mouse IgG and streptavidin-HRP conjugate. Samples were processed for cytochemical demonstration of HRP. Quantitative morphometric analysis was performed by the ferritin-bridge technique (23). In brief, cells were incubated in medium with or without 345 nM sT4 for 60 min at 37°C, washed, incubated with the anti-gp4l antibody, washed, and fixed. The fixed cells were sequentially incubated with goat anti-mouse IgG antibody, donkey anti-goat IgG antibody, goat anti-ferritin antibody, and horse spleen ferritin (Sigma). The number of ferritin particles per virion was evaluated from electron micrographs and compared with controls by using Student's t test.

Proc. Natl. Acad. Sci. USA 88 (1991)

Syncytial Assays. Chronically infected CEM cells (5 X 103 per ml) were mixed with uninfected MOLT-4 cells (7 x 105 per ml) and cultured in the presence or absence of various concentrations of sT4. The number of syncytia in each culture was determined at 24-48 hours and percent inhibition at each concentration of sT4 was calculated (24).

RESULTS Cells chronically infected with HIV-1 (strain HTLV-IIIB) were used to study the effects of soluble CD4 proteins on the viral envelope glycoproteins. Infected H9 cells were incubated with or without sT4, and cell-free supernatants were collected for semiquantitative Western blot analysis for gp120. sT4 caused a dose-dependent release of gpl20 (Fig. 1). To ensure that sT4 was not displacing viral particles from the cell surface, the specificity of the release of gp120 was assessed by Western blot analysis of the supernatants for the presence of the HIV-1 core protein p24. No increase of this protein was found (data not shown). Maximal gpl20 release occurred at 1050 nM sT4 (EC50 = 46 nM; level of half maximal release of gp120 by sT4). The amount of gpl20 released was quantitated using recombinant gpl20. At 250 nM sT4, -50 ng of gp120 was released from 107 cells in 0.25 ml. This corresponded to a 150-fold molar excess of CD4 over gpl20. The structural elements of sT4 required for release of gpl20 were mapped using the truncated sT4 proteins V1V2,DT and V1[106], which have binding affinities for gp120 equivalent to that of sT4 (21). Both proteins efficiently induced the release of gp120, although on a molar basis V1[106] appeared to be about half as effective as sT4 and V1V2,DT. Release of gp120 required the presence of a functional gp120 binding site on sT4. Incubation of HIV-i-infected cells with A55F, identical to sT4 except for an Ala55- Phe point mutation that disrupts gp120 binding (21), did not release gp120. Release could also be inhibited by preincubating sT4-containing medium with the OKT4a antibody, a competitive inhibitor of CD4 and sT4 binding to gp120 (25). In addition, heat-denatured sT4 was unable to induce the release of gp120. These studies indicated that the gp120 binding site Concentration of sT4

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showing the concentration-dependent release of gpl20 into the medium following incubation with sT4. (B) Dose-response curves showing the optical density of gpl20 bands vs. sT4 concentration. Maximal release was found at 1050 nM sT4 (e). Control incubations: omission of sT4, A55F (o), preincubation of sT4 with OKT4A (A), and heat-denatured sT4 (n). (C) Dose-response curves of sT4 (A), V1V2,DT (-), and V1[106] (v).

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Proc. Natl. Acad. Sci. USA 88 (1991)

in the V1 domain of sT4 was important for the release of gpl20 from virions and infected cells. The kinetics of sT4-induced gp120 release were biphasic at 370C (Fig. 2). After addition of sT4, release of gp120 into the medium was rapid for the first 30 min and then exhibited a slower phase out to 120 min. gp120 release was temperaturedependent and did not occur at 40C. These kinetics suggest an initial binding event followed by a rapid release (tI/2 = 20 min) of gpl20. The majority of the releasable gpl20 appeared in the medium by 60 min. To assess the specific effect of soluble CD4 proteins on HIV-1 virions, we analyzed the density of envelope spikes. Envelope spikes are the morphological equivalent of gpl20 (22) and were analyzed on cell-associated mature virus particles following visualization by cytochemical staining of the carbohydrates with osmium/tannic acid/lead citrate (Fig. 3A). On mature HIV-1 virions, envelope spikes appear randomly distributed over the viral membrane (17, 22, 26). Morphometric analysis of the envelope spike distribution on mature HIV-1 virions following sT4 treatment demonstrated a concentration-dependent decrease in spike density (Fig. 4). This decrease corresponded to the increase in free gpl20 observed in the medium. However, treatment with concentrations of sT4 that caused maximal release of gp120 into the medium by biochemical analysis failed to remove all of the envelope spikes from virions. Results of recent biochemical analyses on isolated viral preparations treated with sT4 found a 20% residual fraction of gpl20 following treatment (27). The relevance of envelope spike loss and appearance of free gpl20 in medium following treatment with soluble CD4 proteins was compared with the biologic activity of sT4 (refs. 13-16; Fig. 5). Because of differences in experimental conditions, it is not possible to compare these parameters directly. However, it is possible to assess the tendencies shown by the data. Linear regression analysis of the spike density as a function of sT4-mediated inhibition of syncytium formation showed a strong correlation (r = 0.82). In addition, an inverse correlation was found between sT4-mediated inhibition of syncytium formation and the increase in soluble gp120 (r = -0.95). These correlations suggest that release of gpl20 is a mechanism by which sT4 inhibits viral infectivity in vitro. Loss of viral infectivity following sT4 treatment of purified virions has been observed by ourselves (unpublished data) and by Moore et al. (27). Minutes at 370 C 10 20 30 40 60 120 60

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FIG. 3. Ultrastructural cytochemistry of HIV envelope glycoproteins. (A) Envelope spikes (gpl2O; arrows) were visualized after staining with osmium/tannic acid/lead citrate. Envelope spikes were randomly distributed over the surface of virus particles. (B and C) Ultrastructural immunolocalization of gp4l on untreated HIVinfected cells (B) and cells treated with 1150 nM sT4 for 60 min at 37°C prior to immunostaining with anti-gp4l antibody (C). After sT4 treatment, a heavy HRP reaction product was found staining the cell surface and viral membranes, indicating the exposure of the gp4l epitope recognized by this antibody. (Bar = 200 nm.)

Release of gpl20 and loss of envelope glycoprotein spikes from HIV suggested that incubation with sT4 would result in exposure of the fusion region of gp41. We investigated the exposure of this region of gp4l by ultrastructural immunocytochemical staining. The monoclonal antibody used recognizes an epitope in a highly conserved, immunodominant

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Spikes/nm FIG. 4. Ultrastructural morphometric analysis of envelope spike loss from cell-associated HIV-1 particles after incubation with various concentrations of sT4 for 60 min at 37°C. Incubation with 230 and 2300 nM sT4, which produced 70%6 and 100%6 release of gpl20 into the medium by biochemical analysis, produced significant (P < 0.05) shifts in the spike density distributions. Incubation with 23 nM sT4 did not.

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Proc. Natl. Acad Sci. USA 88 (1991) V

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FIG. 5. Relationships between gp120 appearance in medium, inhibition of syncytium formation following treatment with sT4, and envelope spike loss from virions. Envelope spike loss correlates with sT4-mediated syncytium inhibition (r = 0.82; linear regression analysis). Similarly, release of gp120 into the medium inversely correlates with the antisyncytial activity of sT4 (r = -0.95).

domain located in the amino-terminal region of gp4l (28). This region contains the fusogenic domain of gp41 (8-11). Immunoperoxidase staining of untreated cells and cellassociated virus showed minimal staining of their membranes (Fig. 3B). However, after incubation with sT4, a substantial increase in the intensity of immunoperoxidase reaction product was observed (Fig. 3C). This increase in staining was quantified by the ferritin-bridge technique (23). Morphometric analysis revealed a statistically significant (P < 0.05), =4-fold, increase in the detection of the gp41 epitope on the virus after incubation with sT4 [no. of ferritin particles per virion (mean + SD): control, 0.51 0.32; with sT4, 2.00 1.15; P < 0.05 (paired t test)]. ±

DISCUSSION These studies show that soluble CD4 proteins induce release of gp120 from HIV-1 and HIV-1-infected cells. Further, release of gp120 is associated with exposure of a previously cryptic epitope on gp4l and correlates with inhibition of HIV-1-mediated syncytium formation. These findings, taken together with those of others, allow us to propose a stepwise cascade for infection by HIV-1 which takes place subsequent to viral binding and prior to penetration of the target cell membrane: (i) gpl20-gp41 complex on virus or cells binds to CD4 via gpl20; (ii) binding to CD4 induces a conformational change in gpl20; (iii) gpl20 dissociates from the gp4l transmembrane protein; (iv) release of gpl20 exposes the gp4l fusion sequence; (v) fusion of viral and/or cell membranes leads to infection or syncytium formation; (vi) if fusion does not occur, the gp4l fusion sequence becomes nonfunctional. The first step in viral and retroviral infection of mammalian cells is the binding of envelope or capsid proteins to specific cell surface components (for review see ref. 29). As such, the attachment of HIV-1 to target cells is mediated by the specific association between gpl20 and CD4 (4-7). With few exceptions, HIV-1 does not infect CD4- cells (30, 31). The first step of our proposed cascade is well established. Separation of binding from entry as well as conformational changes in viral coat proteins following binding to target cells have been documented for a number of viruses including poliovirus (32, 33) and influenza virus (34). We have shown that gpl20 release is a relatively slow process that can be separated from binding by the temperature and time of incubation, thus suggesting a rearrangement (conformational change) subsequent to binding. The occurrence of a postbinding conformational change in gpl20 is also supported by recent studies (H. Repke, personal communication) demon-

strating a time-dependent, 30- to 50-fold increase in the affinity of gp120 following binding to cellular CD4. The third step in the cascade, dissociation of gp120 from the gp120-gp41 complex, has been clearly demonstrated. Gelderblom et al. (22) reported that gp120 spikes are lost "spontaneously" from the surface of HIV-1 virions as they mature, and others have reported virus-free soluble gp120 in the media of infected cultures (26). The results presented previously by us (17) and Moore et al. (27) as well as in this report indicate that release of gp120 occurs following interaction with soluble forms of CD4. Although cellular CD4induced release of gpl20 has not been demonstrated, it seems likely that it would occur. Exposure of the fusogenic domain of gp41 following release of gp120 is suggested by two lines of evidence. First, as shown in this report, there is a quantitative increase in the availability of a gp41 epitope for antibody binding. This epitope lies in the amino-terminal region of gp41 at or near the fusion sequence (8, 28). Second, Allan et al. (36) have demonstrated that infectivity of a simian immunodeficiency virus, SIVa, is enhanced by preincubation with soluble CD4. This enhancement could be a facilitation of virus-cell fusion due to exposure of the fusogenic domain of the gp4l homolog on SIVagm. Similar enhancements in infectivity have been observed recently with HIV-1 (G. Pantaleo, G. Poli, and A. S. Fauci, personal communication). The events associated with the fifth step in the cascade remain obscure. The presence of human CD4 on a cell is not sufficient for fusion to occur (7). Furthermore, chimpanzee CD4, which allows infection by HIV, does not allow syncytium formation (37). At present, the importance of accessory molecules (proteins, lipids, etc.) is not understood (38, 39). Loss of activity of fusion molecules for other viruses, such as influenza (40), has been documented. Experimental evidence supporting loss of functional activity of the fusogenic domain of HIV-1 is largely circumstantial. The spontaneous loss of gpl20 suggests that numerous gp4l proteins should be exposed. However, only low levels of gp41 immunostaining were observed on control cells/virions, suggesting that the epitope is blocked or lost with time following spontaneous release of gp120. Further, if the fusogenic function of gp41 were stable, exposure of this region by spontaneous release of gp120 would be expected to allow fusion of HIV-infected cells with CD4- cells. This, however, occurs only rarely (30, 31). The proposed infection/fusion cascade provides an effective framework for dissecting the molecular events of the initial steps in HIV infection. However, other possible sequences could be considered. For instance, the transient exposure and detection of the amino-terminal region of gp41 following treatment with sT4 may reflect exposure of the epitope at the postbinding, conformational-change step, but prior to release of gpl20 from gpdl. The sequence of these events, spacing of molecules, actions of secondary accessory molecules, and correct presentation of fusion proteins to susceptible membranes are all areas in need of elucidation in the HIV infection process, and possibly for viruses in general. Our results with the soluble CD4 proteins sT4, V1V2,DT, and V1[106] demonstrate that the structural elements of CD4 required for release of gpl20 lie within the V1 domain of CD4, which contains the genetically determined binding site for gpl20 (41-44). The ability of OKT4a antibody to block soluble CD4-induced release of gpl20 and the inability of the A55F protein to release gpl20 strongly suggest that specific, high-affinity binding to gp120 is necessary for release to occur. A single amino acid substitution at position 87, which does not appear to affect the binding of gp120 to CD4 or the kinetics of viral infection, markedly reduced the fusion between HIV-infected cells and uninfected CD4' cells (37).

Medical Sciences: Hart et al. Preliminary results comparing CD4 proteins with and without this mutation (kindly provided by B. Seed, Massachusetts General Hospital) indicate involvement of this region in CD4-induced release of gp120 (T.K.H., unpublished observations). A further implication of release of gpl20 is that soluble CD4 can act in a viricidal manner, and not as a simple competitive, reversible inhibitor. This suggestion is supported by work (26, 27) demonstrating the irreversible inhibition of HIV-1 and HIV-2 by soluble CD4 proteins. We propose that soluble CD4 proteins act by enhancing, in the absence of a target cell membrane, the premature removal of gpl20 from the viral envelope. In the absence of an appropriate target membrane, loss of gpl20 leads to inactivation of gp4l, thereby permanently inhibiting the virus particle from subsequent fusion events and infection. This property of soluble CD4 proteins has important implications for the feasibility of these molecules as therapies for AIDS and AIDS-related complex. We thank Drs. G. Morgan and J. Dent for helpful discussion and review of the manuscript; A. Klinkner, C. Bratby, J. Miller, D. Gennaro, and T. Covatta for expert technical assistance; and members of SmithKline Beecham Pharmaceuticals Research and Development for experimental materials. We also thank Drs. J. P. Moore, J. A. McKeating, R. A. Weiss, and Q. J. Sattentau for allowing us to review their manuscript prior to publication. This work was supported by SmithKline Beecham Pharmaceuticals. 1. Allan, J. S., Coligan, J. E., Barin, F., McLane, M. F., Sodroski, J. G., Rosen, C. A., Haseltine, W. A., Lee, T. H. & Essex, M. (1985) Science 228, 1091-1094. 2. Veronese, F. D., DeVico, A. L., Copeland, T. D., Oroszlan,

S., Gallo, R. C. & Sarngadharan, M. G. (1985) Science 229, 1402-1405. 3. Wiley, R. L., Bonifacino, J. S., Potts, B. J., Martin, M. A. & Klausner, R. D. (1988) Proc. Nati. Acad. Sci. USA 85, 95809584. 4. Dalgleish, A. G., Beverley, P. C. L., Clapham, P. R., Crawford, D. H., Greaves, M. F. & Weiss, R. A. (1984) Nature (London) 312, 763-767. 5. Klatzmann, D., Champagne, E., Chamiret, S., Gruest, J., Guetard, D., Hercend, T., Gluckman, J.-C. & Montagnier, L. (1984) Nature (London) 312, ?67-768. 6. McDougal, J. S., Mawle, A., Cort, S. P., Nicholson, J. K. A., Cross, G. D., Schepper-Campbell, J. A., Hocks, D. & Sligh, J.

(1985) J. Immunol. 135, 3151-3162. 7. Maddon, P. J., Dalgleish, A. G., McDougal, J. S., Clapham, P. R., Weiss, R. A. & Axel, R. (1986) Cell 47, 333-348. 8. Gallaher, W. R. (1987) Cell 50, 327-382. 9. Brasseur, R., Comet, B., Burny, A., Vandenbranden, M. & Raysschaert, J. M. (1989) AIDS Res. Hum. Retroviruses 4, 83-90. 10. Berman, P. W., Nunes, W. M. & Haffar, 0. K. (1988) J. Virol. 62, 3135-3142. 11. Freed, E. O., Myers, D. J. & Risser, R. (1990) Proc. Natl. Acad. Sci. USA 87, 4650-4654. 12. Lifson, J., Feinberg, M. B., Reyes, G. R., Rabin, L., Banapour, B., Chakrabarti, S., Moss, B., Wong-Stall, F., Steimer, K. S. & Engleman, E. G. (1986) Nature (London) 323, 725728. 13. Denn, K. C., McDougal, J. S., Inacker, R., Folena-Wasserman, G., Arthos, J., Rosenberg, J., Maddon, P. J., Axel, R. & Sweet, R. W. (1988) Nature (London) 331, 82-84. 14. Traunecker, A., Luke, W. & Karjalainen, K. (1988) Nature (London) 331, 84-86. 15. Fisher, R. A., Bertonis, J. M., Meier, W., Johnson, V. A., Costopoulos, D. S., Liu, T., Tizand, R., Walker, B. D.,

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