Proc. Nail. Acad. Sci. USA Vol. 87, pp. 648-652, January 1990 Biochemistry

Oligomeric structure of the human immunodeficiency virus type 1 envelope glycoprotein (retrovirus/AIDS/vaccuiia virus vector)

PATRICIA L. EARL*, ROBERT W. DOMS*t, AND BERNARD MOSS* *Laboratoryiof Viral Diseases, National Institute of Allergy and Infectious Diseases; and tLaboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892

Contributed by Bernard Moss, October 31, 1989

immunodeficiency virus (SIV) env precursors exist transiently as NaDodSO4-resistant dimers, whereas the corresponding HIV-1 env protein remains monomeric. In contrast, cross-linking data consistent with a stable gpl60 tetramer have been obtained by Schawaller et al. (12), and Pinter et al. (13) have reported that gp4l extracted from purified virions with low concentrations of NaDodSO4 migrates as a 160-kDa band on PAGE. In this communication, we describe the use of a set of recombinant vaccinia viruses expressing either wild-type or mutant forms of the HIV-1 env gene to study the oligomeric structure as well as the region of the env protein responsible for oligomer formation and stability. The results obtained with a variety of different but complementary techniques show that gpl60 forms stable homodimers after synthesis and that at least some of these molecules assemble into a higher-order structure, most likely a tetramer composed of two dimers. The oligomeric structure is retained after cleavage although it is less stable to isolation. Oligomer stability was primarily associated with the N-terminal 129 amino acids of gp41.

The envelope (env) glycoprotein of human ABSTRACT immunodeficiency virus type 1 (HIV-1) consists of two noncovalently associated subunits, gpi20 and gp4l, that are formed by cleavage of a precursor molecule, gpl6O. Using velocity gradient sedimentation, polyacrylamide gel electrophoresis, and chemical cross-linking, we show that gpl60 is synthesized as a monomer and subsequently forms stable homodimers. The molecule remains dimeric after cleavage to gpl2O/gp4l but is less stable to detergent solubilization and centrifugation. Analysis of wild-type and mutated env proteins indicated that interactions between the ectodomain regions of adjoining gp4l subunits are important for dimer formation and stability. A higher-order oligomeric form was also recovered, probably a tetramer consisting of two noncovalently associated dimers. The proposed subunit composition of the HIV-1 env protein is identical to that previously observed for the paramyxovirus envelope proteins F and HN. The envelope (env) glycoprotein of human immunodeficiency virus type 1 (HIV-1) plays at least two essential roles in the initiation of infection: it binds virus to the surface of cells that express CD4 and promotes fusion between the viral and plasma membranes; Determining the structure of gpl60 is critical for an understanding of these functions. The env protein is initially synthesized as a single polypeptide precursor termed gpl60 (1, 2). Prior to delivery to the plasma membrane, gpl60 undergoes a single posttranslational cleavage, which creates two noncovalently associated subunits; gpl2O, which is external to the membrane, and gp4l, which possesses both cytoplasmic and transmembrane domains (1-3). The cleavage is essential for infectivity, presumably because it generates the highly conserved and hydrophobic N-terminal domain of gp41, which is likely to play a role in membrane fusion. In this regard, the env protein is similar to many other envelope proteins whose membrane fusion activities are contingent on an analogous cleavage (4). While the functions of the HIV-1 env protein are becoming better understood, little is known about the structure of the molecule. A feature common to many viral and cellular membrane proteins is that they are oligomeric (5). Oligomerization has important structural and functional consequences. Studies on other membrane proteins have shown that assembly is contingent on correct initial folding in the endoplasmic reticulum and is a prerequisite for intracellular transport (5). Molecules that fail to assemble correctly are retained in the endoplasmic reticulum and are eventually degraded (6). Relatively little information is available regarding the oligomeric structure of most retroviral env proteins (7-9). The sedimentation rate of the env protein of Rous sarcoma virus is consistent with a trimeric structure (10). Rey and co-workers (11) reported that the HIV-2 and simian

MATERIALS AND METHODS Cells and Viruses. The wild-type and several mutant forms ofthe HIV-1 env gene from the BH8 isolate (14) were inserted into vaccinia viruses as described (P.L.E. and B.M., unpublished data). The following viruses were used: vPE16, which encodes the full-length, wild-type molecule; vPE12, which lacks the cleavage site between gpl20 and gp4l; vPE17, which lacks the 104 C-terminal amino acids of the molecule (constituting most of the cytoplasmic domain); vPE18, which lacks the 216 C-terminal amino acids (including the transmembrane domain). PAGE. Samples in 1% NaDodSO4 and 1% 2-mercaptoethanol were heated at 100°C and analyzed by electrophoresis on either 4% or 10% polyacrylamide gels unless otherwise specified. All gel buffers contained 0.1% NaDodSO4. Gradient Centrifugation. BSC1 cells were infected with 30 plaque-forming units of recombinant vaccinia virus per cell. Twenty hours after infection, 3 x 106 cells were harvested and lysed in buffer (100 mM Tris HCI, pH 8.0/100 mM NaCI/1 mM CaC12/250 mM octyl glucoside). After 10 min on ice, the cell debris was removed by centrifugation for 10 min in a microcentrifuge. The supernatants were loaded onto 5-20% sucrose (wt/vol) gradients containing 25 mM octyl glucoside and were centrifuged for 14 hr at 40C at 42,000 rpm in the SW50.1 rotor. Fractions were precipitated with 10%o (wt/vol) trichloroacetic acid, analyzed by 10% PAGE, blotted to nitrocellulose, and incubated with monoclonal antibody 902 (15) and 1251I-labeled protein A (Amersham). SediAbbreviations: HIV-1, human immunodeficiency virus type 1; SIV, simian immunodeficiency virus; DTSSP, 3,3-dithiobis(sulfosuccinimidyl propionate); EGS, ethylene glycol bis(succinimidyl succinate).

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.

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mentation coefficients were calculated by the method of McEwen (16) and Martin and Ames (17), using the influenza hemagglutinin and vesicular stomatitis virus G protein as standards. Cells were pulse labeled 12 hr after infection for 20 min with [35S]methionine (Amersham) and were harvested immediately or after a 4-hr chase. The lysates were loaded onto 5-20% sucrose gradients and centrifuged for 23 hr at 40,000 rpm in an SW40 rotor. Twenty-four fractions were collected per gradient. These were immunoprecipitated with rabbit antisera to gpl20 (PB33; a gift of P. Berman, Genentech) and analyzed by 10% PAGE. Chemical Cross-Linking. Cross-linking reagents were obtained from Pierce. For cross-linking on the cell surface, BSC1 cells were infected and metabolically labeled with [35S]methionine for 16 hr. They were then washed with phosphate-buffered saline (PBS) (pH 8.0), and 3,3-dithiobis(sulfosuccinimidyl propionate) (DTSSP) was added to 10 mM final concentration. After 30 min on ice, glycine was added to 100 mM final concentration. After an additional 15 min on ice, the cells were washed with PBS and lysed as described above. The sample was either immunoprecipitated with HIV1-positive human sera (gift of L. Arthur, Frederick Cancer Research Facility) and analyzed by PAGE or subjected to sucrose gradient centrifugation. For cross-linking of proteins from sucrose gradient fractions, a vPE16-infected cell lysate was centrifuged on a 5-20% gradient for 20 hr at 4°C at 40,000 rpm in an SW40 rotor. Each fraction was cross-linked with 5 mM ethylene glycol bis(succinimidyl succinate) (EGS) for 15 min at room temperature. The reactions were stopped by addition of glycine to a final concentration of 100 mM. After trichloroacetic acid precipitation, the samples were separated by 4% PAGE and were analyzed by Western blotting. Escherichia coli,-galactosidase at a concentration of 10 mg/ml in PBS was cross-linked with 1 mM EGS for 15 min at room temperature and was used as a molecular weight standard.

RESULTS Velocity Gradient Sedimentation of HIV-1 env Protein. Previous investigations have shown that the env protein synthesized in cells infected with a recombinant vaccinia virus expressing gp160 is glycosylated, cleaved into gpl20 and gp4l, transported to the cell surface, and mediates fusion with CD4-bearing cells (18, 19). In this study, we used recombinant vaccinia viruses that expressed either the wildtype or several mutant forms of the env protein. Thus, vPE16 and vPE12 express the full-length env gene, but in vPE12 the proteolytic cleavage site between gpl20 and gp4l was removed. vPE17 and vPE18 express truncated forms of the env gene lacking the 104 and 216 C-terminal amino acids, respectively. Because of the deletion of the transmembrane domain, most of the env protein made in vPE18-infected cells is secreted. Velocity gradient centrifugation was used to analyze the HIV-1 env protein made by cells infected with the recombinant vaccinia viruses. Cells were infected and lysed in octyl glucoside, and the lysates were centrifuged on 5-20%o sucrose gradients. After fractionation, the distribution of the env glycoprotein across the gradient was determined by PAGE and Western blotting with an antibody to gpl20 (Fig. 1). In cells infected with vPE16 and expressing the wild-type env gene, gpl60 was recovered from two peaks. Much of the gp160 was found in fractions near the bottom of the tube with a sedimentation coefficient of -10.8 S, whereas a smaller amount of gpl6O was recovered from a 7.2S peak. Similar results were obtained when cells were lysed with Triton X-100 or 3-[(3-cholamidopropyl)dimethylammonio]-1-pro-

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Biochemistry: Earl et al.

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FIG. 1. Velocity gradient sedimentation of HIV-1 env glycoproteins. BSC1 cells were infected with recombinant vaccinia viruses encoding the wild type (vPE16), processing mutant (vPE12), or cytoplasmic domain mutant (vPE17). Twenty hours after infection, the cells were lysed and centrifuged. The distribution of env protein across the gradient was determined by PAGE and Western blotting with monoclonal antibody to gpl20 (15). For analysis of secreted gpl20, BSC1 cells were infected with vPE16 and metabolically labeled with [35S]methionine. The medium from infected cells was concentrated and separated on a 5-20o sucrose gradient. Fractions were immunoprecipitated with rabbit antisera to gpl20. For analysis of 8E5 cells, 3 x 106 cells were lysed, centrifuged, and analyzed as described for vaccinia-infected cell extracts.

panesulfonate. We estimate the molecular mass of the 10.8S and 7.2S proteins to be approximately 310 kDa and 167 kDa, respectively, suggesting that gpl60 exists in dimeric and monomeric forms. By contrast, both detergent-solubilized and secreted gpl20 were exclusively recovered from the 7.2S peak. In addition, when cell lysates were boiled in the presence of NaDodSO4 and 2-mercaptoethanol prior to centrifugation, most of the gpl60 also was recovered from the 7.2S peak (data not shown). This indicates that gpl20 as well as NaDodSO4 and heat disrupted gpl60 sediment as monomers.

Sedimentation analyses were also carried out with recombinant viruses that express mutated forms of the HIV-1 env protein that are folded and transported normally (P.L.E. and B.M., unpublished data). Both gpl60 lacking the proteolytic cleavage site (vPE12) and a truncated form of the env protein missing most of the cytoplasmic domain of gp4l (vPE17) also sedimented predominantly at -10.8 S (Fig. 1). In addition, the env protein from extracts of 8E5 cells, which contain a HIV-1 provirus (20), sedimented at 10.8 S (Fig. 1), demonstrating that the pattern of oligomerization is not an artifact of the vaccinia expression system. In many experiments, a small portion of the gpl60 dimer resisted dissociation with NaDodSO4 and 2-mercaptoethanol and migrated with a molecular mass estimated on either 4% or 5-10%o gejs to be =320 kDa. This band is prominent in the vPE12 panel of Fig. 1 and also can be seen in the vPE16 panel. The NaDodSO4-resistant dimer band was only observed upon analysis of the 10.8S material. Its absence from the 7.2S peak indicated that it did not arise from disulfide interchange during sample preparation. This property was more evident when lower concentrations of 2-mercaptoethanol or shorter

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times of incubation at 100'C were used. A similar degree of NaDodSO4 resistance has been observed with the env proteins of HIV-2 and SIV (11) as well as HIV-1 (21). While most noncovalently associated oligomers are sensitive to NaDodS04-induced dissociation, the finding of a discrete NaDodS04-resistant band is likely to reflect a specific, stable interaction unlikely to arise from nonspecific aggregation. HIV-1 env Protein Forms Oligomers Posttranslationally. To determine whether sedimentation of gpl60 in the 10.8S peak was due to posttranslational assembly, we used a pulse-chase protocol. Cells infected with either vPE16 or vPE12 were pulse labeled for 20 min with [355]methionine and were lysed immediately or after a 4-hr chase with excess unlabeled methionine. The lysates were centrifuged in parallel gradients and fractions were immunoprecipitated with rabbit antisera to gp120. As shown in Fig. 2, most of the gpl60 from both vPE16 and vPE12 sedimented in the 7.2S peak immediately following the pulse. After the chase, almost all of the gpl60 from vPE12 and much of the gpl60 from vPE16 sedimented in the 10.8S region. Thus, we concluded that gp160 is synthesized as a monomer and forms dimers posttranslationally. In cells infected with vPE16, gp120 generated during the chase period was found almost exclusively in the 7.2S peak. Since both membrane-associated and secreted gp120 (Fig. 1) were recovered only in monomeric form, dimer formation is either a transient event or the dimer becomes less stable to detergent solubilization and centrifugation after proteolytic cleavage. Chemical Cross-Linking of HIV-1 env on the Surface of Infected Cells. To test whether proteolytic activation to gpl20/gp4l causes the oligomers to dissociate in vivo or simply renders them less stable to detergent solubilization, we performed chemical cross-linking on cells infected with vPE16 and metabolically labeled with [35S]methionine. Cross-linking on the cell surface provides a way to covalently stabilize multimeric proteins prior to detergent solubilization, which might otherwise cause complexes to dissociate. As shown in Fig. 3A, when the cleavable cross-linking reagent DTSSP was used, the gpl60, gp120, and gp4l bands all diminished in intensity and a higher molecular weight band increased. To analyze the higher molecular weight complex, a part of the cross-linked cell lysate was subjected to gradient centrifugation. Samples from each gradient fraction were incubated in the presence or absence of reducing agent and analyzed by PAGE. As shown in Fig. 3B, the cross-linked material was almost quantitatively recovered from the 10.8S peak. When the same samples were reduced prior to electrophoresis, we found that the cross-linked material that sedimented at 10.8 S contained gp120 and gp4l as well as gpl60 (Fig. 3C). Thus, the env protein exists as an oligomer

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FIG. 3. Cross-linking of env protein on the surface of cells. Cells expressing the wild-type env protein (vPE16) were labeled with [35S]methionine for 16 hr. The cells were then cross-linked with 10 mM DTSSP and lysed, and the env proteins were immunoprecipitated with HIV-1-positive human sera (A). An aliquot of the lysate was loaded onto a 5-20%o sucrose gradient and centrifuged as described in Fig. 1. The distribution of env protein across the gradient was determined by immunoprecipitation followed by PAGE under both nonreducing (B) and reducing (C) conditions. 2-me, 2-Mercaptoethanol.

on the cell surface even after activation to gp120/gp41. However, as shown in Fig. 1, this form of the molecule is not stable to centrifugation. HIV-1 env Forms a Higher-Order Oligomer. Close examination of the results in Fig. 2 indicates that while the gpl60 remaining after the chase period sedimented mostly in the 10.8S region, some material sedimented more quickly, with s2ow values ranging from 14.0 to 16.3 S. The latter was more evident in cells expressing the mutant env protein, which was not activated to gpl20 and gp4l (vPE12). To analyze the structure of the gpl60 sedimenting at >10.8 S, we used chemical cross-linking with the noncleavable reagent EGS. A lysate of vPE16-infected cells was centrifuged so that the 10.8S region would sediment approximately one-half of the way to the bottom. Each fraction was then cross-linked and

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FIG. 2. Posttranslational assembly of env protein dimers. Cells expressing the wild-type (vPE16) or processing mutant (vPE12) env proteins were pulse labeled for 20 min and then lysed immediately or after a 4-hr chase. The lysates were centrifuged and each fraction was immunoprecipitated with rabbit antisera to gpl20 and analyzed by PAGE.

Biochemistry: Earl et al.

Proc. Natl. Acad. Sci. USA 87 (1990)

analyzed by Western blotting. To estimate the size of large oligomeric complexes, the samples were treated with NaDodSO4 and separated by 4% PAGE with cross-linked E. coli /-galactosidase used as a molecular weight marker. As shown in Fig. 4, three forms of the protein were recovered. The gp160 in the 7.2S peak could not be cross-linked into higher-order structures consistent with it being monomeric. Material sedimenting in the 10.8S region was quantitatively cross-linked into a single, broad band of 300-400 kDa, consistent with it being dimeric. Finally, material sedimenting >10.8 S was cross-linked into a broad band of 550-600 kDa. This higher-order oligomer had a sedimentation coefficient from 14.0 to 16.3 S. Both the s20,w value and the approximate molecular mass estimated for the higher-order form by PAGE are between what would be expected for trimeric and tetrameric gp160. Thus, we were unable to determine the exact composition ofthis highest-order form of the HIV-1 env protein. However, since dimers were very stable both by PAGE in NaDodSO4 and by density-gradient centrifugation, it seems most likely that the higher-order form is a tetramer composed of two dimers, rather than a trimer composed of a dimer and a monomer. gp4l Is Responsible for Dimer Stability. Since stable dimers of gp160 were observed under different conditions including NaDodSO4 PAGE and density-gradient centrifugation in the presence of a variety of detergents, several approaches were taken to determine which subunit was most important for dimer stability. Cells infected with vPE16 and metabolically labeled with [35S]methionine were subjected to chemical cross-linking under conditions designed to reveal less extensively cross-linked intermediates. As shown in Fig. 5, a prominent band of -80 kDa was observed after incomplete cross-linking. When this band was excised and reduced, it migrated as gp4l. By contrast, cross-linked gpl20 dimers were not observed upon analysis of higher molecular weight bands. The absence of cross-linked gpl20 dimers suggested that gp4l is primarily responsible for dimer stability. This conclusion was supported by the finding that secreted gpl20 sedimented only in monomeric form (Fig. 1). Also, as shown in Fig. 1, gpl60 displayed partial resistance to NaDodSO4induced dissociation. Taking advantage of this property, we determined whether other forms of the env protein displayed resistance to NaDodSO4. In Fig. 6 we show that vPE17,

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which lacks the cytoplasmic domain of gp4l, likewise gave NaDodSO4-resistant dimers, as did vPE18, which lacks the transmembrane as well as the cytoplasmic domain. In addition, both ofthese molecules formed oligomers as determined by velocity-gradient sedimentation (Fig. 1; unpublished observations). Since neither the transmembrane nor the cytoplasmic domain of gp41 is absolutely required for dimer formation and stability, we conclude that the N-terminal domain of gp4l is both sufficient and necessary for assembly.

DISCUSSION We have used a variety of techniques alone and in combination to determine the oligomeric structure of the HIV-1 env protein. Our data indicate that the env glycoprotein is synthesized as a monomer and then assembles posttranslation-

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FIG. 4. Cross-linking of env protein following centrifugation. Twenty hours after infection, cells expressing the wild-type env protein (vPE16) were lysed and subjected to centrifugation at 40,000 rpm at 4°C for 20 hr in an SW40 rotor. Each fraction was cross-linked by addition of 5 mM EGS for 15 min at room temperature. The reactions were quenched by addition of glycine and the proteins were analyzed as described in Fig. 1, but on a 4% acrylamide gel. E. coli ,8-galactosidase, cross-linked with EGS, was used as a molecular weight standard.

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Proc. Natl. Acad. Sci. USA 87 (1990)

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FIG. 6. NaDodSO4 resistance of env proteins. Lysates of BSC1 cells infected with either vPE16 (lane 1), vPE17 (lane 2), or vPE18 (lane 3) were separated by PAGE and analyzed by Western blotting with monoclonal antibody 902 (15). ally into dimers and higher-order oligomers. The failure of Rey et al. (11) to detect HIV-1 gpl60 dimers, whereas they did detect HIV-2 and SIV dimers, is probably due to the greater stability of the latter (S. Chakrabarti, R.W.D., and P.L.E., unpublished observations). In our studies, the dimeric form was detected by three independent assays: gradient centrifugation, resistance to NaDodSO4-induced dissociation, and chemical cross-linking. Given the agreement between these different approaches, there seems to be little doubt that the dimer represents a particularly stable form of the HIV-1 env protein. However, a higher-order structure that sedimented in the leading edge of the dimeric peak on sucrose gradients also exists. Due to the large size of this complex, it is difficult to ascertain its structure with absolute certainty. Well-characterized molecular weight markers of sufficiently large size are not available for NaDodSO4/ PAGE. While the cross-linked f-galactosidase used in our studies gave good results, we note that cross-linked proteins may migrate more quickly in NaDodSO4 gels than non-crosslinked forms (22). This, coupled with the additional ambiguity imposed on the estimation of the molecular mass of the env protein due to its high carbohydrate content, makes determination of the absolute size even more difficult. However, several considerations lead us to conclude that the higherorder form is tetrameric. First, consistent recovery of stable dimers suggests that these form the "building blocks" of any higher-order structure. Thus, a tetramer would consist of two noncovalently associated dimers. Second, the estimations of molecular mass and sedimentation coefficient are closer to what would be expected for a tetrameric rather than a trimeric molecule. Third, the F env protein of paramyxoviruses (23), with which the HIV-1 env protein shares some structural and functional homology, is also a tetramer composed of two dimers. A subunit structure of A2A2 would explain our inability to consistently observe a partially crosslinked trimeric intermediate, although such a structure was found under the cross-linking conditions used by Schawaller et al. (12). Thus, given all of the considerations, we believe that the env protein forms dimers that in turn assemble into tetramers composed of two dimers each. Important questions that remain to be addressed include where and with what efficiency tetramers are formed and whether both dimers and tetramers are fully functional entities that can be transported to the cell surface and incorporated into virus. Several lines of evidence indicate that gp4l, more specifically the N-terminal 129 amino acids, is responsible for

oligomerization. This includes our inability to detect gpl20 dimers, whereas uncleaved molecules lacking the C-terminal 216 amino acids readily oligomerize. In addition, we have confirmed the oligomerization of free gp4l (ref. 13; unpublished results). The HIV-1 env protein is thus similar in this regard to the Rous sarcoma virus env protein, which is stabilized by the ectodomain region ofits membrane spanning unit (10). The assembly of gpl60 into dimers and tetramers is likely to be important for intracellular transport and stability. One obvious functional consequence of oligomerization is that the molecule has the potential for binding several CD4 molecules simultaneously, which might increase the avidity of virus binding. This, in turn, could affect its membrane fusion activity. Finally, the oligomeric structure of the HIV-1 env protein may have important implications for the design of antiviral agents and vaccines as well as for attempts to create biologically relevant, water-soluble molecules for more detailed structural studies. We thank N. Cooper for cells and viruses, P. Berman (Genentech) for rabbit antisera to gp120, L. Arthur (Frederick Cancer Research Facility) for HIV-1 positive human sera, and J. White (University of California at San Francisco) for discussing results prior to publication. 1. Allan, J. S., Coligan, J. E., Burin, 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. Willey, R. L., Bonifacino, J. S., Potts, B. J., Martin, M. A. & Klausner, R. D. (1988) Proc. Natl. Acad. Sci. USA 85, 9580-9584. 4. Stegmann, T., Doms, R. W. & Helenius, A. (1989) Annu. Rev. Biophys. Chem. 18,187-215. 5. Rose, J. K. & Doms, R. W. (1988) Annu. Rev. Cell Biol. 4, 257-288. 6. Lippincott-Schwartz, J., Bonifacino, J. S., Yuan, L. C. & Klausner, R. D. (1988) Cell 54, 209-220. 7. Takemoto, L. J., Fox, C. F., Jensen, F. C., Elder, J. H. & Lerner, R. A. (1978) Proc. Nat!. Acad. Sci. USA 75, 3644-3648. 8. Pinter, A. & Fleissner, E. (1979) J. Virol. 30, 157-165. 9. Racevskis, J. & Sarkar, N. H. (1980) J. Virol. 35, 937-948. 10. Einfeld, D. & Hunter, E. (1988) Proc. Nat!. Acad. Sci. USA 85, 8688-8692. 11. Rey, M.-A., Krust, B., Laurent, A. G., Montagnier, L. & Hovanessian, A. G. (1989) J. Virol. 63, 647-658. 12. Schawaller, M., Smith, G. E., Skehel, J. J. & Wiley, D. C. (1989) Virology 172, 367-369. 13. Pinter, A., Honnen, W. J., Tilley, S. A., Bona, C., Zaghouani, H., Gorny, M. K. & Zolla-Pazner, S. (1989) J. Virol. 63, 2674-2679. 14. Ratner, L., Haseltine, W., Patarca, R., Livak, K. J., Starcich, B., Josephs, S. F., Doran, E. R., Rafalski, J. A., Whitehorn, E. A., Baumeister, K., Ivanoff, L., Petteway, S. R., Jr., Pearson, M. L., Lautenberger, J. A., Papas, T. S., Ghrayeb, J., Chang, N. T., Gallo, R. C. & Wong-Staal, F. (1985) Nature (London) 313, 277284. 15. Chesebro, B. & Wehrly, K. (1988) J. Virol. 62, 3779-3788. 16. McEwen, C. R. (1967) Anal. Biochem. 20, 114-149. 17. Martin, R. G. & Ames, B. N. (1%1) J. Biol. Chem. 236, 1372-1379. 18. Chakrabarti, S., Robert-Guroff, M., Wong-Staal, F., Gallo, R. C. & Moss, B. (1986) Nature (London) 320, 535-537. 19. Lifson, J. D., Feinberg, M. B., Reyes, G. R., Rabin, L., Banapour, B., Chakrabarti, S., Moss, B., Wong-Staal, F., Steimer, K. S. & Engelman, E. G. (1986) Nature (London) 323, 725-728. 20. Folks, T. M., Powell, D., Lightfoote, M., Koenig, S., Fauci, A. S., Benn, S., Rabson, A., Daugherty, D., Gendelman, H. E., Hoggan, M. D., Venkatesen, S. & Martin, M. A. (1986) J. Exp. Med. 164, 280-290. 21. Berman, P. W., Riddle, L., Nakamura, G., Haffar, 0. K., Nunes, W. M., Skehel, P., Byrn, R., Groopman, J., Matthews, T. & Gregory, T. (1989) J. Virol. 63, 3489-3498. 22. Doms, R. W. (1990) Methods Enzymol. 127, in press. 23. Sechoy, O., Philippot, J. R. & Bienvenue, A. (1987) J. Biol. Chem. 262, 11519-11523.