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Coexpression of human immunodeficiency virus envelope proteins and tat from a single simian virus 40 late replacement vector. (recombinant DNA/acquired ...
Proc. Natl. Acad. Sci. USA Vol. 85, pp. 334-338, January 1988 Biochemistry

Coexpression of human immunodeficiency virus envelope proteins and tat from a single simian virus 40 late replacement vector (recombinant DNA/acquired immunodeficiency syndrome/shuttle vector/transfection)

DAVID REKOSH*t, ANDERS NYGREN4, PER FLODBY*, MARIE-LOUISE HAMMARSKI&LD§, AND HANS WIGZELLt Departments of *Biochemistry and WMicrobiology, State University of New York at Buffalo, Buffalo, NY 14214; and tDepartment of Immunology, Karolinska Institute, Box 60 400, S-10401 Stockholm, Sweden

Communicated by Peter Reichard, September 18, 1987 (received for review June 22, 1987)

ABSTRACT A Sal I-Xho I fragment containing the genes encoding tat, art, and the envelope proteins from the BH10 clone of human immunodeficiency virus (HIV) was inserted into a simian virus 40 (SV40)-based eukaryotic expression vector. The vector is a shuttle vector that replicates to high copy numbers in both Escherichia coli and eukaryotic cells permissive for SV40 replication. Transfection of the HIV DNA-containing vector (pSVSX1) into the CV-1 monkey cell line gave high levels of expression of the envelope glycoproteins gp160 and gpl20 in 20-30% of the transfected cells. By several criteria, the proteins were indistinguishable from those produced during infection. The proteins were localized to the cytoplasm and plasma membrane, and some of the gpl20 was shed into the culture medium. Approximately 0.5 pg of envelope protein could be extracted from 106 cells. This is at least 100 times higher than the levels found in HIV-infected H9 cells. In addition, a trans-activation assay performed with pSVSX1 and a plasmid containing the gene for chloramphenicol acetyltransferase under the control of the HIV long terminal repeat demonstrated that a functional tat gene product also was expressed. Thus, this transient vector system provides an abundant source of native envelope protein for purification and characterization and also will be useful for studies dealing with the regulation of HIV gene expression.

interaction (14, 15). The envelope protein formed on the surface of infected cells also mediates cell fusion by interacting with T4 antigen (also called CD4), creating the giant cells often seen during HIV infections (16-18). This eventually leads to cell death. All serotypes of HIV are expected to retain these functions, and it is likely that this functional conservation will be found to physically reside in the conserved regions that exist within the envelope molecule. To rationally develop an effective vaccine against HIV, further studies are needed to define the functional regions of binding and fusion within its envelope protein and to demonstrate the link that hopefully exists between these regions and virus neutralization. These studies require large amounts of the HIV envelope protein, which are difficult to recover from infected cells (2). To try to overcome this problem and to facilitate manipulation of the envelope protein sequence, several vector systems have recently been developed that produce the envelope proteins in eukaryotic cells (3, 17, 19-22). From these studies it has been shown that efficient synthesis of envelope protein from the HIV promoter requires expression of the two regulatory proteins tat and art/trs (19, 22), necessitating cotransfection with at least two plasmids. Production of the envelope protein from heterologous promoters has also been problematical (3), and it has been speculated that sequences near the ATG start codon of the envelope gene, env, are crucial in the regulation of its expression. The situation is further complicated by the fact that the first coding exons of both art/trs and tat are just upstream from the start of the env coding region and by the lack of information about the exact structure of the native envelope mRNA (17, 19). In this report we describe a single recombinant vector that produces a functional tat gene product and large amounts of the HIV envelope proteins. To accomplish this, the Sal I-Xho I DNA fragment from the clone of HIV designated BH10, containing the coding regions of tat, art, and env, was cloned into a simian virus 40 (SV40) late replacement vector.

The acquired immunodeficiency syndrome (AIDS) and AIDS-related complex (ARC) are caused by the human immunodeficiency virus (HIV), which has now infected millions of people worldwide (1). A vaccine that would prevent further spread of this virus is desperately needed. At the present time, the envelope glycoprotein of HIV (gpl60/gpl2O) must be regarded as the prime candidate for the creation of a subunit vaccine. In support of its use as a vaccine, studies with purified gp120 have shown that it is highly antigenic in rhesus monkey, goat, and horse and that immune sera against this protein can neutralize the in vitro infectivity of HIV in cultured cells (2, 3). Furthermore, sera from patients with ARC or AIDS frequently contain neutralizing antibodies that are directed against the envelope protein (4-7). While these antibodies cannot clear the infection, it is possible that they would be protective if elicited by vaccination before the first exposure to the virus. Although some regions of the protein are highly variable between different HIV isolates (8), conserved regions also exist that may be able to raise antibodies capable of heterologous neutralization, since there is evidence that reinfection of an individual is a rare event (9). A major function of the envelope protein is to direct the virus to the T4 + lymphocyte in which it replicates (10-13). It has been shown that HIV binds directly to the T4 glycoprotein on the surface of the T4+ lymphocyte via a gp120-T4

MATERIALS AND METHODS Vector Construction. The SV40 late replacement-shuttle vector pSVEpR4 (23) was modified to include a segment from the rabbit 3-globin gene containing an intron and a polyadenylylation signal (24). To do this, pSVEpR4 was digested with Aat II and Xho I, and the fragment containing the SV40 sequences was ligated to the Aat II-Xho I fragment from pA8IG (25) containing the f3-globin and pBR322 sequences. The resulting vector, pBABY, can be used to clone Abbreviations: HIV, human immunodeficiency virus; AIDS, acquired immunodeficiency syndrome; ARC, AIDS-related complex; LTR, long terminal repeat; gp, glycoprotein; SV40, simian virus 40; EBV, Epstein-Barr virus; EBNA, EBV nuclear antigen; CAT, chloramphenicol acetyltransferase. tTo whom reprint requests should be addressed.

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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Biochemistry: Rekosh et al. and express cDNA molecules because it contains a unique Xho I site between the SV40 late promoter and the f3-globin splice and polyadenylylation signals. Transfection of CV-1 Cells. Monkey CV-1 cells were maintained as monolayers in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum. Cells were transfected by using a modification of the DEAEdextran procedure as described (23, 26). A transfection efficiency of 20-30% was routinely obtained. Indirect Immunofluorescence. Cells were removed from plastic dishes by mild trypsinization and washed twice in phosphate-buffered saline. The cell pellets were dispersed in phosphate-buffered saline containing 5% fetal bovine serum at a concentration of 2 x 105 cells per ml, and smears were prepared on glass slides as described (27). The slides were dried in air and fixed in either acetone/methanol, 2:1 (vol/vol), or 100lo methanol at - 20'C for 5 min. The smears were stained with a 1:10 dilution of human sera in phosphatebuffered saline and fluorescein-conjugated anti-human IgG antibodies. All sera were preadsorbed with nontransfected CV-1 cells that had undergone the freeze/thaw cycle twice. To do this, -5 x 106 cells were incubated for 30 min at 40C with 1 ml of diluted serum, which was then cleared by centrifugation. Preadsorption was necessary to reduce nonspecific background fluorescence. [35S]Methionine Labeling of Cells. CV-1 cells were washed twice with serum-free DMEM lacking methionine and labeled for 8 hr with 200 /Ci (1 Ci = 37 GBq) of [35S]methionine per ml in the same medium. At the end of the labeling period, the cells were washed twice with phosphate-buffered saline and harvested by scraping and centrifugation. Immunoprecipitations, Electrophoresis, Immunoblotting, and Chloramphenicol Acetyltransferase (CAT) Assays. These were performed essentially as described (23, 28). Specific conditions are detailed in the figure legends.

RESULTS Cloning of the HIV Envelope Gene into the SV40 Late Replacement Vector pBABY. pBABY is a shuttle vector designed to replicate to high copy number in eukaryotic cells permissive for SV40 replication as well as in Escherichia coli. The vector is a modification of a previously described expression vector, pSVEpR4 (23), and its construction is detailed in Materials and Methods. pBABY contains sequences from SV40 including the entire early region, the origin of replication, the enhancer, the late promoter, and late RNA start sites up to the Kpn I site at SV40 nucleotide 294. Adjacent to this site are sequences from the rabbit ,B-globin gene containing the splice donor and acceptor surrounding the second intron, as well as the ,B-globin polyadenylylation signal. The Kpn I site at the boundary of SV40 and /8-globin DNA was changed to an Xho I site by linker addition. Any piece of DNA cloned into this site will be transcribed into RNA from the SV40 late promoter. The RNA is expected to be spliced and polyadenylylated because of the rabbit 8-globin signals. The vector also contains sequences from a derivative of pBR322 that lacks the copy control region and the "poison" sequences shown to inhibit replication in eukaryotic cells (29). To create a vector that expresses large amounts of the HIV envelope proteins in eukaryotic cells, we originally used a fragment of HIV DNA from the BH8 isolate (30), which was inserted into pBABY at its unique Xho I site by blunt-ended ligation. The fragment used contains the entire envelope protein coding region (env) and extends from the Sst I site at position 6035 to the Xho I site at position 8920 (Genbank numbering system). It contains about 200 nucleotides of sequence upstream from the start of the open reading frame encoding the envelope protein but does not contain

Proc. Natl. Acad. Sci. USA 85 (1988)

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the complete first coding exons of either tat or art/trs. Attempts to demonstrate synthesis of the envelope proteins with this vector were unsuccessful (data not shown). One possibility that could explain the lack of expression observed from the above vector construction is that tat and/or art are required for efficient env expression even in this heterologous system. For this reason a larger piece of DNA containing the complete first coding exons of both tat and art/trs as well as the env gene was cloned into pBABY. The cloned HIV DNA consisted of a fragment from the Sal I site at position 5820 to the Xho I site at position 8920 (Genbank numbering system) and was derived from the HIV BH10 isolate. The resulting construct was called pSVSX1 (Fig. 1). This construction would be expected to express tat and art/trs as well as env if a splice donor site were provided by the vector. Previous experiments with a similar vector construction containing a cloned gene encoding EpsteinBarr virus (EBV) nuclear antigen 1 (EBNA-1) suggest that this is indeed the case (23). Expression of the HIV Envelope Protein in Transfected Cells. To determine if HIV protein could be obtained in cells transfected with pSVSX1, the vector was transfected into CV-1 monkey cells using DEAE-dextran. The cells were screened at 60 hr posttransfection by indirect immunofluorescence with an AIDS patient's antiserum previously shown to have a high titer against the gpl20 envelope protein. As a control, cells transfected with the parental vector, pBABY, were also screened. Fig. 2 shows examples of the type of fluorescence observed in cells transfected with pSVSX1. In cells fixed with acetone/methanol, two types of fluorescence patterns were commonly seen with approximately equal frequency. Fig. 2 Top illustrates a double nucleated cell containing fluorescent material that is clearly localized to the cytoplasm and the cell membrane. These positively reacting doubly nucleated cells were quite common and probably arose as a consequence of SV40 tumor (T)-antigen expression from the vector. Fig. 2 Middle illustrates the second type of fluorescence and shows a cell where the specific fluorescence is localized to a portion of the surface membrane. When methanol alone was used for (Sal I) ori

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HIV FIG. 1. The structure of pSVSX1. The vector is composed of SV40 sequences containing the entire early region, the origin of replication (ori) and late promoter sequences (nucleotides 2533-294), HIV sequences (BH10 clone) containing the envelope gene (5820-8920), rabbit 13-globin sequences containing the 3' intervening sequence (IVS) and poly(A) signal, and pBR322 (Sal I-BamHI) with a deletion between pBR322 coordinates 1120-2490. AMPr, gene for ampicillin resistance; T antigen, tumor antigen.

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Proc. NatL. Acad. Sci. USA 85 (1988) 1

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FIG. 3. Autoradiograph of immunoprecipitations of ["S]methionine-labeled proteins from transfected CV-1 cells. Immune serum from an AIDS patient was used to immunoprecipitate [35S]cysteinelabeled extracts of HIV-infected cells (lane 1) or [WS]methioninelabeled extracts from CV-1 cells transfected with either pSVSX1 (lane 2) or pBABY (lane 3). In a second experiment, immune serum from another AIDS patient was used to immunoprecipitate the same extract used in lane 1 (lane 4). As a control, serum from a healthy donor was used to precipitate ["S]methionine-labeled extracts from CV-1 cells transfected with pSVSX1 (lane 5) or pBABY (lane 6). Precipitates were analyzed on a NaDodSO4/polyacrylamide gel and visualized by fluorography.

FIG. 2. Indirect immunofluorescence of pSVSX1-transfected CV-1 cells. Forty-eight hours posttransfection, cells were fixed with acetone/methanol (Top and Middle) or methanol alone (Bottom) and stained with a 1:10 dilution of immune serum from an AIDS patient. Fluorescein isothiocyanate-conjugated goat anti-human IgG was used as the second antibody.

fixation, only cells containing a ring of surface fluorescence were observed (Fig. 2 Bottom). Under optimal transfection conditions, the frequency of positive cells ranged from 20% to 30%. Cells transfected with pBABY were completely negative in this assay (data not shown). To determine the nature of the immunoreactive material made in transfected cells, we performed an immunoprecipitation experiment. CV-1 cells were labeled with [35S]methionine for 8 hr beginning at 48 hr posttransfection, and immunoprecipitated samples were analyzed on a NaDodS04/polyacrylamide gel by autoradiography. Fig. 3 shows that two specific polypeptides migrating at 160 kDa and 120

kDa were precipitated with an AIDS patient's serum from extracts of pSVSX1-transfected cells (lane 2). These polypeptides migrated similarly to polypeptides from HIVinfected cells precipitated by AIDS patients sera (Fig. 3, lanes 1 and 4) and were not precipitated from pBABYtransfected cells (Fig. 3, lane 3) or when serum from a control donor was used (Fig. 3, lanes 5 and 6). The apparent molecular mass of these molecules suggested that they were the gp160 and gpl20 envelope proteins. Under these conditions of analysis, gp4l would not be detected because it has a low methionine content and migrates in the position of the background bands. To further confirm that these bands represent the gp160 and gp120 envelope proteins, a second immunoprecipitation experiment was performed. Cell extracts and culture medium from cells transfected with pSVSX1 were subjected to immunoprecipitation at 72 hr posttransfection by using either a goat serum raised against purified gpl20 (2) or an AIDS patient serum, affinity-purified against gpl20. As controls, normal goat and human sera were used. The precipitates were run on a polyacrylamide gel containing NaDodSO4 and transferred to nitrocellulose by electroblotting. The resulting blot was developed with an AIDS patient's serum and alkaline phosphatase-conjugated second antibody. Fig. 4 shows that precipitates from transfected cell extracts using either the goat anti-gp120 serum (lane B) or the human affinity-purified serum (lane F) contained proteins that migrated at 160 and 120 kDa. In contrast, precipitates from the medium of these cell cultures (Fig. 4, lanes C and G) contained only the 120-kDa band. Both the 160- and 120-kDa bands also were observed when an extract of the transfected cells was run directly on the gel and blotted without prior immunoprecipitation (Fig. 4, lane A). As expected, when control sera were used in the immunoprecipitation step, no specific bands were observed (Fig. 4, lanes D, E, H, and I). Reports from other laboratories have shown that gp120 but not gpl60 can be found in the medium of infected cell cultures (2, 5). Taken together, these results show that the bands observed at 160 and 120 kDa are the gpl60 and gp120 envelope proteins. The amount of envelope protein produced in CV-1 cells transfected with pSVSX1 was estimated on an electrophoretic immunoblot using different amounts of cell extract. The intensity of gpl60 and gpl20 in the diluted cell extracts were

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Proc. Natl. Acad. Sci. USA 85 (1988)

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FIG. 4. Immunoprecipitation and immunoblot analysis of the medium and cells from CV-1 cultures transfected with pSVSX1. Medium and cells were harvested 72 hr after transfection. Immunoprecipitations were then performed on extracts from one-fifth of a dish (106 cells; lanes B, D, F, and H) or from one-fifth of the medium (1 ml; lanes C, E, G, and I). The serum used was one of the following: a goat serum directed against purified gp120 (lanes B and C), a control goat serum (lanes D and E), serum from an AIDS patient (lanes F and G), or control human serum from a seronegative donor (lanes H and I). The precipitates were run on an NaDodSO4/polyacrylamide gel that was electroblotted to nitrocellulose. The blot was developed with a 1:100 dilution of the AIDS patient's serum and alkaline phosphatase-conjugated goat-anti human IgG antibody. Lane A contained extract from 5 x 105 cells, which was run directly on the gel without prior immunoprecipitation.

compared to a serial dilution of purified gpl20 run on the same gel (Fig. 5). From the titration, the amount of envelope protein (gp160 and gpl20) produced in the transfected cells was estimated to be -2.5 pug per 90-mm culture dish containing 5 x 106 cells. Expression of Functional tat in Transfected Cells. In order to determine if pSVSX1 expresses the tat gene product in addition to producing the envelope proteins, a transactivation assay was performed to detect tat function (17). To do this, pSVSX1 was cotransfected into CV-1 cells together with a vector containing the gene for CAT under the control of the HIV long terminal repeat (LTR). CAT activity was measured 48 hr after transfection as described (28). The results of such an experiment, together with appropriate controls, is shown in Fig. 6. No detectable CAT activity was observed when the LTR-CAT construction was transfected 2

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into cells either alone (Fig. 6, lane 5) or together with pBABY (Fig. 6, lane 4). In contrast, cotransfection with pSVSX1 and LTR-CAT resulted in high CAT activity (Fig. 6, lane 2) equivalent to that observed in cotransfections of LTR-CAT and pSVTAT, a vector known to produce tat (Fig. 6, lane 3). In both of these cases, the CAT activity obtained was higher than that from pSV2CAT, which expresses CAT from the SV40 early promoter (Fig. 6, lane 1).

DISCUSSION By several criteria the envelope proteins produced by the vector system described in this paper behave identically to those produced in infected cells. From their sizes, it appears that the proteins are properly glycosylated and, as in the natural system, gpl60 is transported to the plasma membrane where it is cleaved. Some of the gpl20 product thus obtained is shed into the medium of the transfected CV-1 cells. Moreover, our estimates of the quantity of envelope protein made show that transfected cells produce -200 times more protein than HIV-infected cells (2). Since the proteins are transiently expressed from a vector that can be modified easily by site-directed mutagenesis, it will be possible to rapidly produce altered forms of the proteins. Thus, this system can provide an abundant and convenient source of native and mutated envelope proteins for purification and further characterization. pBABY is an SV40 late replacement vector. Since vectors of this type contain the entire early region of SV40 and the origin of replication, they replicate to high copy numbers in cells permissive for SV40. This leads to template amplification and high levels of expression of the cloned gene (23). Such vectors were first used to produce the influenza hemagglutinin, another viral membrane protein, in CV-1 cells (31, 32). The yields from this system were comparable to ours, and the protein was expressed on the cell surface. However, cleavage of the hemagglutinin did not occur unless trypsin was added to the culture medium of transfected cells. The parent vector of pBABY, pSVEpR4, which lacks the p-globin splice and polyadenylylation signals, has been used to express large amounts of EBNA-1 (23) and the human myc protooncogene product (33). In the case of EBNA-1, -500-1000 times more protein was produced in transfected CV-1 cells compared to lymphoid cells latently infected with EBV. Both EBNA-1 and myc were properly localized to the

4 6

FIG. 5. Quantitation of envelope protein in pSVSX1-transfected CV-1 cells. Cells were harvested 72 hr after transfection. Extracts were made, and different dilutions were run on a NaDodSO4/ polyacrylamide gel (A) together with different amounts of purified gpl20 (B). The gel was immunoblotted with an AIDS patient's serum using the same conditions as described in the legend to Fig. 4. The individual lanes in A contained extracts from the following amounts of transfected cells: 1, 7 x 105; 2, 2 x 105; 3, 7 x 104; 4, 2 x 104. The amounts of gpl20 in the lanes in B were: 1, 1.8 jig; 2, 0.45 ,ug; 3, 0.11 ,ug; 4, 0.028 ,ug; 5, 0.007 Ig. By comparing the intensities in lanes 1 and 2 of A to the standards, we estimate that the transfected cells contain at least 0.1 ,tg of envelope protein per 2 x 105 cellsi.e., 2.5 ,ug per 90-mm dish (5 x 106 cells).

1

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__ FIG. 6. Trans-activation of expression from the HIV-LTR in transfected CV-1 cells. Cells (5 x 106) were transfected with the various plasmids and harvested 48 hr after transfection. For each assay, extracts corresponding to 1 x 106 cells were incubated with 0.2 uCi of [14C]chloramphenicol for 60 min, and half of each sample was then analyzed by thin-layer chromatography. A fluorograph of the thin-layer plate is shown after exposure for 72 hr with an intensifying screen. The extracts were from cells transfected with: 2 ug of pSV2CAT (lane 1); 2 ,ug of LTR-CAT and 5 ,ug of pSVSX1 (lane 2); 2 jig of LTR-CAT and 5 jig of pSVTAT (lane 3); 2 ,ug of LTR-CAT and 5 ,ug of pBABY (lane 4); and 2 ,ug of LTR-CAT (lane 5).

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nucleus of transfected cells. Large amounts of these proteins are also expressed after transfection of several human lymphoid cell lines because of efficient replication of the vectors in these cells. Thus, it should be possible to use pSVSX1 to obtain expression of the HIV envelope protein in cell lines other than CV-1, including several human lymphoid cell lines. Several groups have reported the expression of fragments of the envelope protein in bacteria by using a variety of expression vectors (18, 34, 35). While these products have been useful for antibody production and mapping, they cannot be used for studies of native protein structure and function because proper posttranslational modifications occur only in selected eukaryotic cell lines. Production of the envelope protein in eukaryotic cells has previously been described. One system involves the use of recombinant vaccinia virus (20, 21). In a second case, a Chinese hamster ovary cell line secreting an altered form of gpl20 was created (3). The protein produced was a chimera composed of the hydrophobic amino-terminal signal sequence from the herpes simplex virus glycoprotein D (50 amino acids) and amino acids 61-531 of the envelope protein. The protein was secreted into the medium. Attempts to make a cell line producing a native envelope protein resulted in poor expression for unknown reasons. A third type of vector system utilizes the HIV LTR joined to sequences encoding the envelope protein (17, 19, 22). These transient vectors have been designed mainly to map viral functions and to study gene regulation. From these studies it has been concluded that expression of the envelope protein can only be obtained in the presence of the transactivating factors tat and art/trs. The vector system that we have described places the tat, art/trs, and env regions of HIV under the control of heterologous promoter. We have demonstrated that this construction gives efficient production of the products of at least two of these genes, gpl60 and tat. We do not yet know whether art/trs is made or if its expression plays a role in the synthesis of gpl60 in this system. However, the absence of gp160 expression from the BH8 construction, which lacked both tat and art/trs, suggests that one or both of the products of these genes may be essential. The expression of gpl60 from pSVSX1 presents an interesting enigma because the protein is encoded in the third coding region downstream from the SV40 late promoter. Either gpl60 is efficiently translated from a polycistronic mRNA, a situation not often found in eukaryotic cells, or the regions including the first exons of tat and art/trs are spliced out of the mRNA used to make gpl60. However, no splice acceptor site has yet been identified in the region between tat, art/trs, and env, that would enable such a splice to occur. These same problems of env gene expression also exist in the virus when transcription is under the control of the HIV LTR. Thus, the pSVSX1 system provides not only a source of large quantities of envelope protein but also a convenient model to elucidate HIV gene regulation. We are grateful to G. Robey (National Cancer Institute, Frederick, MD) and T. Matthews (Duke University, Durham, NC) for gifts of purified gpl20, antisera, and labeled cell extracts; to R. Gallo (NationalCancer Institute, Bethesda, MD) for HIV BH8 and BH10 DNA; and to L. Bachelor (DuPont) for LTR-CAT and pSVTAT vectors. We thank J. Heimer for expert technical assistance. D.R. is a recipient of Research Career Development Award CA-00905 from the National Cancer Institute. D.R. and M.-L.H. are members of the Center for Applied Molecular Biology and Immunology of the State

University of New York at Buffalo.

1. Institute of Medicine (1986) Confronting AIDS (National Academy Press, Washington, DC).

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