Glycoprotein Gp120 (Acquired Immunodeficiency Syndrome/Transmembrane Glycoprotein/Gp4l/Retrovirus) TIMOTHY K

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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) in- appear to function in a similar manner in HIV-induced fects cells after binding ofthe viral envelope glycoprotein gpl20 syncytium formation, as expression of recombinant versions to the cell surface recognition marker CD4. gpl20 is nonco- of these proteins, in the absence of other HIV proteins, was valently associated with the HIV transmembrane envelope sufficient for cell-cell fusion (12). glycoprotein gp4l, and this complex is believed responsible for Soluble CD4 proteins, consisting of all or portions of the the initial stages of HIV infection and cytopathic events in external region of human CD4, efficiently inhibit infection infected cells. Soluble constructs of CD4 that contain the gpl20 and virus-induced syncytium formation by HIV-1 (13-16). binding site inhibit HIV infection in vitro. This is believed to This effect has been attributed to association of the soluble occur by competitive inhibition ofviral binding to cellular CD4. CD4 protein with gpl20 on the surface of virus and virus- Here we suggest an alternative mechanism ofviral inhibition by infected cells and consequent competitive inhibition of viral soluble CD4 proteins. We demonstrate biochemically and gpl20 binding to cell surface CD4. However, we recently morphologically that following binding, the soluble CD4 pro- reported (17) that incubation of HIV-1-infected cells with teins sT4, VjV2,DT, and V1[106J (amino acids 1-369, 1-183, sT4, a soluble CD4 construct composed of the entire extra- and -2 to 106 of mature CD4) induced the release of gpl20 cellular domain of native CD4 (13), resulted in the reduction from HIV-1 and HIV-1-infected cells. gpl20 release was con- of virion envelope glycoprotein spikes and the appearance of centration-, time-, and temperature-dependent. The reaction gpl20 in the supernatant. The release of gpl20 by sT4 was biphasic at 37C and did not take place at 4°C, indicating indicated that the inhibitory effect might result, at least in that binding of soluble CD4 was not sufficient to release gp120. part, in inactivation of virus by removing viral binding The appearance of free gpl20 in the medium after incubation proteins. Moreover, this release suggested that a similar with sT4 correlated with a decrease in envelope glycoprotein rearrangement of envelope glycoproteins may occur upon spikes on virions and exposure of a previously cryptic epitope virus binding to cell surface CD4, which then leads to near the amino terminus of gp4l on virions and infected cells. virus-cell fusion. The concentration ofsoluble CD4 proteins needed to induce the The importance of events following binding of gpl20 to release of gpl20 from virally infected cells also correlated with CD4 to the processes of infectivity and syncytium formation those required to inhibit HIV-mediated syncytium formation. have been demonstrated. Disruption of the CD4 binding site These results suggest that soluble CD4 constructs may inacti- on gpl20 (18, 19) prevents infection of CD4' cells. However, vate HIV by inducing the release of gpl20. We propose that the presence of functional binding sites on CD4 and gp120 is HIV envelope-mediated fusion is initiated following rearrange- not sufficient for infection. Murine cells expressing human ment and/or dissociation of gpl20 from the gpl2O-gp4l CD4 are not infected by HIV (7). In contrast, syncytium complex upon binding to cellular CD4, thus exposing the fusion formation is disrupted in cells expressing noncleavable mu- domain of gp4l. tations 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 envelope glycoproteins of human immunodeficiency The present study examines the events associated with virus type 1 (HIV-1) consist of two noncovalently associated viral gpl20-CD4 interactions. We have now refined our subunits, gpl20 and gp41, which are derived from the gpl60 analysis of the soluble CD4-induced release of gpl20 from precursor (1-3). Viral attachment to target cells is mediated HIV virions and infected cells by examining the stoichiom- by gpl20 molecules, which are visible ultrastructurally as etry, kinetics, and temperature dependence ofthis process in electron dense spikes on viral membranes. The gpl20 spe- relationship to the anti-infectivity aspects of soluble CD4 cifically associates with the human CD4 receptor (4-7), a proteins. This analysis has allowed us to define the regions of glycoprotein present on the surface of the T4 subset of CD4 required for gpl20 release and to identify the consequent lymphocytes and, in this manner, is responsible for the a of tropism of HIV for this and other CD4' cells. Subsequent to exposure of cryptic epitope at the amino terminus gp41. attachment, fusion of viral and cellular membranes occurs; a process thought to involve the transmembrane glycoprotein MATERIALS AND METHODS gp4l. The amino-terminal region of gp41 is implicated in this and The sT4 event, based on sequence homology with the fusion regions Proteins Antibodies. soluble CD4 proteins of membrane proteins of other viruses (8-10) and amino acid [amino acids (aa) 1-369 of mature human CD4], V1V2,DT (aa substitution analysis of this region (11). gpl20 and gp4l also Abbreviations: HIV-1, human immunodeficiency virus type 1; HRP, horseradish peroxidase. The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed at: SmithKline payment. This article must therefore be hereby marked "advertisement" Beecham Pharmaceuticals, Mail stop L-62, P.O. Box 1539, King of in accordance with 18 U.S.C. §1734 solely to indicate this fact. Prussia, PA 19406.

2189 Downloaded by guest on September 25, 2021 2190 Medical Sciences: Hart et al. Proc. Natl. Acad. Sci. USA 88 (1991) 1-183), and V1[106J (aa -2 to 106) and the derivatives were Syncytial Assays. Chronically infected CEM cells (5 X 103 described previously (13, 21). The sT4 and V1V2,DT proteins per ml) were mixed with uninfected MOLT-4 cells (7 x 105 were prepared in mammalian cells; V1[106] was expressed in per ml) and cultured in the presence or absence of various Escherichia coli. Concentrations of soluble CD4 proteins concentrations of sT4. The number of syncytia in each were determined by anti-CD4 monoclonal antibodies (21). culture was determined at 24-48 hours and percent inhibition Recombinant gpl20 from the BH10 isolate of HIV-1 was at each concentration of sT4 was calculated (24). 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 ofgp120 RESULTS from HIV-1 strain HXB2. This antibody detected similar Cells chronically infected with HIV-1 (strain HTLV-IIIB) were amounts of purified recombinant and viral gpl20 in Western used to study the effects of soluble CD4 proteins on the viral blots and ELISAs. Mouse monoclonal anti-gp4l was from envelope glycoproteins. Infected H9 cells were incubated with Cellular Products, and OKT4a antibody from Ortho Phar- or without sT4, and cell-free supernatants were collected for maceuticals. All other antibodies were obtained from Jack- semiquantitative Western blot analysis for gp120. sT4 caused a son ImmunoResearch. dose-dependent release of gpl20 (Fig. 1). To ensure that sT4 Cell Lines and Virus. H9 and CEM cells chronically in- was not displacing viral particles from the cell surface, the fected with HIV-1 strain HTLV-IIIB were used. The specificity ofthe release ofgp120 was assessed by Western blot MOLT-4 cell line was used as a fusion partner in syncytial analysis ofthe supernatants for the presence ofthe HIV-1 core assays. Cells were maintained in RPMI 1640 (GIBCO) con- protein p24. No increase of this protein was found (data not taining 10% heat-inactivated fetal bovine serum and antibi- otics. shown). Maximal gpl20 release occurred at 1050 nM sT4 (EC50 Western Blot Analysis for gpl2O Release. Chronically in- = 46 nM; level of half maximal release of gp120 by sT4). The fected H9 cells were washed once in culture medium and amount of gpl20 released was quantitated using recombinant resuspended at 1-1.5 x 107 cells per ml in medium containing gpl20. At 250 nM sT4, -50 ng ofgp120 was released from 107 sT4, V1[106], V1V2,DT, or A55F at equimolar concentra- cells in 0.25 ml. This corresponded to a 150-fold molar excess tions. Control cells were incubated in medium alone. Fol- of CD4 over gpl20. lowing the incubations, cell-free supernatants were diluted The structural elements ofsT4 required for release ofgpl20 1:2 with 2x sample buffer and boiled for 5 min. After were mapped using the truncated sT4 proteins V1V2,DT and SDS/7.5% PAGE, the proteins were transferred to Immo- V1[106], which have binding affinities for gp120 equivalent to bilon-P membranes (Millipore). The blots were probed with that of sT4 (21). Both proteins efficiently induced the release a 1:1000 dilution to guinea pig anti-gpl20 antibody followed of gp120, although on a molar basis V1[106] appeared to be by alkaline phosphatase-labeled anti-guinea pig IgG or bio- about half as effective as sT4 and V1V2,DT. tinylated anti-guinea pig IgG and streptavidin-horseradish Release of gp120 required the presence of a functional peroxidase (HRP). Visualization ofalkaline phosphatase was gp120 binding site on sT4. Incubation ofHIV-i-infected cells with nitroblue tetrazolium (Sigma) and HRP was visualized with A55F, identical to sT4 except for an Ala55- Phe point with the ECL detection system (Amersham). The optical mutation that disrupts gp120 binding (21), did not release density of alkaline phosphatase-stained envelope protein gp120. Release could also be inhibited by preincubating bands was determined using a Magiscan image-analysis sys- sT4-containing medium with the OKT4a antibody, a com- tem (Joyce-Loebl). Recombinant gp120 was used as a stan- petitive inhibitor of CD4 and sT4 binding to gp120 (25). In dard, and detection was linear between 0.1 and 5.0 ng of addition, heat-denatured sT4 was unable to induce the release gpl20. of gp120. These studies indicated that the gp120 binding site Electron Microscopy and Quantitation of Envelope Glyco- protein Spikes. Control and sT4-treated HIV-i-infected cells A Concentration of sT4 mnM) were fixed with 2.5% glutaraldehyde in 0.1 M phosphate H)V 0 11.5 23 115 230 1150 buffer (pH 7.3). Cells were postfixed in 1% OS04 and enve- gpl 20-- M lope glycoproteins were stained with 1% tannic acid followed by aqueous 1% uranyl acetate (22). Samples were processed 5 B c and analyzed as reported (17). The linear surface density of spikes on virions was calculated (spikes per um), and 70-90 to 4 virions were evaluated for each sample. Means, standard 0 deviations, histograms, and statistical analysis (2-tailed t a) ^ tests) were prepared using a Videoplan (Zeiss). Curves were C ... fitted with SigmaPlot (Jandel Scientific, Corte Madera, CA) 1/fo and represent fifth-order regression analyses. >1Con1- and of r 5M 2 Immunocytochemical Staining Quantification gp4l. .vO*." ..,, Cells were washed twice following incubation with or without ~10-1 sT4, then incubated in a 1:50 dilution of antibody in medium, / 'I -P - -- -''-''' washed twice, fixed with glutaraldehyde, and sequentially incubated in biotinylated goat anti-mouse IgG and streptavi- 1 10 100 1000 0 100 din-HRP conjugate. Samples were processed for cytochem- sT4 (nM) ical demonstration of HRP. Quantitative morphometric analysis was performed by the ferritin-bridge technique (23). In FIG. 1. sT4-mediated release ofgpl20from HIV-1 (strain HTLV- brief, cells were incubated in medium with or without 345 nM III) infected H9 cells. Infected cells were incubated for 60 min at sT4 for 60 min at 37°C, washed, incubated with the anti-gp4l 370C in the presence of sT4, V1V2,DT, or V1[106]. (A) Western blot antibody, washed, and fixed. The fixed cells were sequen- showing the concentration-dependent release of gpl20 into the medium following incubation with sT4. (B) Dose-response curves tially incubated with goat anti-mouse IgG antibody, donkey showing the optical density of gpl20 bands vs. sT4 concentration. anti-goat IgG antibody, goat anti-ferritin antibody, and horse Maximal release was found at 1050 nM sT4 (e). Control incubations: spleen ferritin (Sigma). The number of ferritin particles per omission of sT4, A55F (o), preincubation of sT4 with OKT4A (A), virion was evaluated from electron micrographs and com- and heat-denatured sT4 (n). (C) Dose-response curves of sT4 (A), pared with controls by using Student's t test. V1V2,DT (-), and V1[106] (v). Downloaded by guest on September 25, 2021 Medical Sciences: Hart et al. Proc. Natl. Acad. Sci. USA 88 (1991) 2191 in the V1 domain ofsT4 was important for the release ofgpl20 from virions and infected cells. The kinetics of sT4-induced gp120 release were biphasic at 370C (Fig. 2). After addition of sT4, release ofgp120 into the medium was rapid for the first 30 min and then exhibited a slower phase out to 120 min. gp120 release was temperature- dependent and did not occur at 40C. These kinetics suggest an initial binding event followed by a rapid release (tI/2 = 20 min) ofgpl20. The majority ofthe releasable gpl20 appeared in the medium by 60 min. B.'. 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. C 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 ofgp120 into the FIG. 3. Ultrastructural cytochemistry of HIV envelope glyco- medium by biochemical analysis failed to remove all of the proteins. (A) Envelope spikes (gpl2O; arrows) were visualized after envelope spikes from virions. Results of recent biochemical staining with osmium/tannic acid/lead citrate. Envelope spikes were analyses on isolated viral preparations treated with sT4 found randomly distributed over the surface of virus particles. (B and C) Ultrastructural immunolocalization of gp4l on untreated HIV- a 20% residual fraction of gpl20 following treatment (27). infected cells (B) and cells treated with 1150 nM sT4 for 60 min at The relevance of envelope spike loss and appearance of 37°C prior to immunostaining with anti-gp4l antibody (C). After sT4 free gpl20 in medium following treatment with soluble CD4 treatment, a heavy HRP reaction product was found staining the cell proteins was compared with the biologic activity of sT4 (refs. surface and viral membranes, indicating the exposure of the gp4l 13-16; Fig. 5). Because of differences in experimental con- epitope recognized by this antibody. (Bar = 200 nm.) ditions, 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 Release of gpl20 and loss of envelope glycoprotein spikes density as a function ofsT4-mediated inhibition ofsyncytium from HIV suggested that incubation with sT4 would result in formation showed a strong correlation (r = 0.82). In addition, exposure of the fusion region of gp41. We investigated the an inverse correlation was found between sT4-mediated exposure of this region of gp4l by ultrastructural immuno- inhibition of syncytium formation and the increase in soluble cytochemical staining. The monoclonal antibody used rec- gp120 (r = -0.95). These correlations suggest that release of ognizes an epitope in a highly conserved, immunodominant 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).

A Minutes at 370 C 40 C 5 10 20 30 40 60 120 60 6060 HIV gp1 20 -- (0 B 6-

5 - 0~~~~~ co /Z0 CD> {JZ - 41 0 L- 2 -

1- 0 0

T I __ I- s T I 0I 120 0 20 40 60 s0 100 120 0 10 20 30 40 50 60 Time (minutes) Spikes/nm FIG. 2. Kinetics and temperature dependence of gpl2O release FIG. 4. Ultrastructural morphometric analysis of envelope spike from infected cells incubated in the presence of 1150 nM sT4. (A) loss from cell-associated HIV-1 particles after incubation with var- Western blot ofthe cell-free medium following incubation of0°C and ious concentrations of sT4 for 60 min at 37°C. Incubation with 230 37°C for0-120 min. (B) gp120 band density vs. time. Release ofgpl20 and 2300 nM sT4, which produced 70%6 and 100%6 release of gpl20 (e) was rapid up to 30 min, after which gpl20 continued to accumu- into the medium by biochemical analysis, produced significant (P < late at a slower rate. Incubations without sT4 at 37°C (n) and 4°C (o) 0.05) shifts in the spike density distributions. Incubation with 23 nM and with sT4 at 4°C (o) did not induce gpl20 release over 60 min. sT4 did not. Downloaded by guest on September 25, 2021 2192 Medical Sciences: Hart et al. Proc. Natl. Acad Sci. USA 88 (1991) V strating a time-dependent, 30- to 50-fold increase in the affinity of gp120 following binding to cellular CD4. The third in the dissociation of gp120 from I step cascade, (a the gp120-gp41 complex, has been clearly demonstrated. Ey 0 *- Gelderblom et al. (22) reported that gp120 spikes are lost Xg * C C 00Ew "spontaneously" from the surface of HIV-1 virions as they Q) Eo* mature, and others have reported virus-free soluble gp120 in - the media of infected cultures (26). The results presented 0._ previously by us (17) and Moore et al. (27) as well as in this C-T report indicate that release of gp120 occurs following inter- action with soluble forms of CD4. Although cellular CD4- Co0 induced release ofgpl20 has not been demonstrated, it seems 1 10 100 1000 likely that it would occur. sT4 (nM) Exposure ofthe fusogenic domain ofgp41 following release of gp120 is suggested by two lines of evidence. First, as FIG. 5. Relationships between gp120 appearance in medium, inhibition of syncytium formation following treatment with sT4, and shown in this report, there is a quantitative increase in the envelope spike loss from virions. Envelope spike loss correlates with availability of a gp41 epitope for antibody binding. This sT4-mediated syncytium inhibition (r = 0.82; linear regression anal- epitope lies in the amino-terminal region ofgp41 at or near the ysis). Similarly, release ofgp120 into the medium inversely correlates fusion sequence (8, 28). Second, Allan et al. (36) have with the antisyncytial activity of sT4 (r = -0.95). demonstrated that infectivity of a simian immunodeficiency virus, SIVa, is enhanced by preincubation with soluble domain located in the amino-terminal region of gp4l (28). CD4. This enhancement could be a facilitation of virus-cell This region contains the fusogenic domain of gp41 (8-11). fusion due to exposure of the fusogenic domain of the gp4l Immunoperoxidase staining of untreated cells and cell- homolog on SIVagm. Similar enhancements in infectivity have associated virus showed minimal staining oftheir membranes been observed recently with HIV-1 (G. Pantaleo, G. Poli, and (Fig. 3B). However, after incubation with sT4, a substantial A. S. Fauci, personal communication). increase in the intensity of immunoperoxidase reaction prod- The events associated with the fifth step in the cascade uct was observed (Fig. 3C). This increase in staining was remain obscure. The presence of human CD4 on a cell is not quantified by the ferritin-bridge technique (23). Morphomet- sufficient for fusion to occur (7). Furthermore, chimpanzee ric analysis revealed a statistically significant (P < 0.05), CD4, which allows infection by HIV, does not allow syncy- =4-fold, increase in the detection of the gp41 epitope on the tium formation (37). At present, the importance ofaccessory virus after incubation with sT4 [no. of ferritin particles per molecules (proteins, lipids, etc.) is not understood (38, 39). virion (mean + SD): control, 0.51 ± 0.32; with sT4, 2.00 Loss of activity offusion molecules for other viruses, such 1.15; P < 0.05 (paired t test)]. 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 DISCUSSION loss ofgpl20 suggests that numerous gp4l proteins should be These studies show that soluble CD4 proteins induce release exposed. However, only low levels of gp41 immunostaining of gp120 from HIV-1 and HIV-1-infected cells. Further, were observed on control cells/virions, suggesting that the release of gp120 is associated with exposure of a previously epitope is blocked or lost with time following spontaneous cryptic epitope on gp4l and correlates with inhibition of release of gp120. Further, if the fusogenic function of gp41 HIV-1-mediated syncytium formation. These findings, taken were stable, exposure of this region by spontaneous release together with those of others, allow us to propose a stepwise of gp120 would be expected to allow fusion of HIV-infected cascade for infection by HIV-1 which takes place subsequent cells with CD4- cells. This, however, occurs only rarely (30, to of the cell 31). to viral binding and prior penetration target The cascade provides an effec- on virus or cells binds to proposed infection/fusion membrane: (i) gpl20-gp41 complex tive framework for dissecting the molecular events of the CD4 via gpl20; (ii) binding to CD4 induces a conformational initial steps in HIV infection. However, other possible se- change in gpl20; (iii) gpl20 dissociates from the gp4l trans- quences could be considered. For instance, the transient membrane protein; (iv) release of gpl20 exposes the gp4l exposure and detection of the amino-terminal region of gp41 fusion sequence; (v) fusion of viral and/or cell membranes following treatment with sT4 may reflect exposure of the leads to infection or syncytium formation; (vi) if fusion does epitope at the postbinding, conformational-change step, but not occur, the gp4l fusion sequence becomes nonfunctional. prior to release of gpl20 from gpdl. The sequence of these The first step in viral and retroviral infection ofmammalian events, spacing ofmolecules, actions of secondary accessory cells is the binding of envelope or capsid proteins to specific molecules, and correct presentation of fusion proteins to cell surface components (for review see ref. 29). As such, the susceptible membranes are all areas in need of elucidation in attachment ofHIV-1 to target cells is mediated by the specific the HIV infection process, and possibly for viruses in gen- association between gpl20 and CD4 (4-7). With few excep- eral. tions, HIV-1 does not infect CD4- cells (30, 31). The first step Our results with the soluble CD4 proteins sT4, V1V2,DT, of our proposed cascade is well established. and V1[106] demonstrate that the structural elements of CD4 Separation ofbinding from entry as well as conformational required for release ofgpl20 lie within the V1 domain ofCD4, changes in viral coat proteins following binding to target cells which contains the genetically determined binding site for have been documented for a number of viruses including gpl20 (41-44). The ability of OKT4a antibody to block poliovirus (32, 33) and influenza virus (34). We have shown soluble CD4-induced release ofgpl20 and the inability of the that gpl20 release is a relatively slow process that can be A55F protein to release gpl20 strongly suggest that specific, separated from binding by the temperature and time of high-affinity binding to gp120 is necessary for release to incubation, thus suggesting a rearrangement (conformational occur. A single amino acid substitution at position 87, which change) subsequent to binding. The occurrence of a post- does not appear to affect the binding of gp120 to CD4 or the binding conformational change in gpl20 is also supported by kinetics of viral infection, markedly reduced the fusion recent studies (H. Repke, personal communication) demon- between HIV-infected cells and uninfected CD4' cells (37). Downloaded by guest on September 25, 2021 Medical Sciences: Hart et al. Proc. Natl. Acad. Sci. USA 88 (1991) 2193 Preliminary results comparing CD4 proteins with and without Hirsch, M. S., Schoofey, R. T. & Flavell, R. A. (1988) Nature this mutation (kindly provided by B. Seed, Massachusetts (London) 331, 76-78. General Hospital) indicate involvement of this region in 16. Hussey, R. E., Richardson, N. E., Kowalski, M., Brown, CD4-induced release of gp120 (T.K.H., unpublished obser- N. R., Chang, H.-C., Siliciano, R. F., Dorfman, T., Walker, vations). B., Sodroski, J. & Reinherz, E. L. (1988) Nature (London) 331, 78-81. A further implication ofrelease ofgpl20 is that soluble CD4 17. Kirsh, R., Hart, T. K., Ellens, H., Miller, J., Petteway, S. A., can act in a viricidal manner, and not as a simple competitive, Lambert, D. M., Leary, J. & Bugelski, P. J. (1990) AIDS Res. reversible inhibitor. This suggestion is supported by work Hum. Retroviruses 6, 1215-1218. (26, 27) demonstrating the irreversible inhibition of HIV-1 18. Lasky, L. A., Nakamura, G., Smith, D. H., Fennie, C., Shi- and HIV-2 by soluble CD4 proteins. We propose that soluble masaki, C., Patzer, E., Berman, P., Gregory, T. & Capon, D. J. CD4 proteins act by enhancing, in the absence of a target cell (1987) Cell 50, 975-985. membrane, the premature removal of gpl20 from the viral 19. Kowalski, M., Potz, J., Basiripour, L., Dorfman, T., Goh, envelope. In the absence of an appropriate target membrane, W. C., Terwilliger, E., Dayton, A., Rosen, C., Haseltine, W. loss of gpl20 leads to inactivation of gp4l, thereby perma- & Sadroski, J. (1987) Science 237, 1351-1355. nently inhibiting the virus particle from subsequent fusion 20. Kieny, M. P., Lathe, R., Riviere, Y., Dott, K., Schmitt, D., Girard, M., Montagnier, L. & Lecocq, J.-P. (1988) Protein Eng. events and infection. This property of soluble CD4 proteins 2, 219-225. has important implications for the feasibility of these mole- 21. Arthos, J., Deen, K. C., Chaiken, M. A., Fornwald, J. A., cules as therapies for AIDS and AIDS-related complex. Sathe, G., Sattentau, Q. J., Clapham, P. R., Weiss, R. A., McDougal, J. S., Pietropaolo, C., Axel, R., Truneh, A., Mad- We thank Drs. G. Morgan and J. Dent for helpful discussion and don, P. J. & Sweet, R. W. (1989) Cell 57, 469-481. review of the manuscript; A. Klinkner, C. Bratby, J. Miller, D. 22. Gelderblom, H. R., Hausmann, E. H. S., Ozel, M., Pauli, G. Gennaro, and T. 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