Vol. 65, No. 6

JOURNAL OF VIROLOGY, June 1991, p. 3276-3283

0022-538X/91/063276-08$02.00/0 Copyright © 1991, American Society for Microbiology

Human Immunodeficiency Virus Infection and Syncytium Formation in HeLa Cells Expressing Glycophospholipid-Anchored CD4 THOMAS A. KOST,1* JOSEPH A. KESSLER,2 INDRAVADAN R. PATEL,' JOHN G. GRAY,' LAURIE K. OVERTON,' AND STEPHEN G. CARTER2 Departments of Molecular Biology' and Cell Biology,2 Glaxo Research Institute, S Moore Drive, Research Triangle Park, North Carolina 27709 Received 10 January 1991/Accepted 18 March 1991

The CD4 molecule, a glycoprotein expressed primarily on the cell surface of specific T lymphocytes, is thought to function in T-cell antigen recognition and activation. In addition, CD4 serves as a receptor for human immunodeficiency virus type 1 (HIV-1) by a direct interaction with the HIV-1 surface glycoprotein (gpl20). To further characterize the HIV-1-cell interaction, a HeLa cell line was established that expressed a chimeric molecule of CD4 and decay-accelerating factor (DAF). In the chimeric CD4-DAF molecule the transmembrane and cytoplasmic domains of CD4 were deleted and replaced with the carboxy-terminal 37 amino acids of DAF. This resulted in the anchoring of the extracellular domain of CD4 to the cell membrane via a glycophospholipid linkage. The glycophospholipid-anchored CD4 had a molecular size of approximately 56 to 62 kDa and was released following treatment of the cells with phosphatidylinositol-specific phospholipase C. HeLa cells expressing the CD4-DAF hybrid could be infected with HIV-1, as evidenced by reverse transcriptase activity, p24 core antigen content, and infectious virus production. In addition, transfection of the HeLa CD4DAF cells with a plasmid that directs the synthesis of HIV-1 envelope glycoproteins or cocultivation with HeLa cells expressing the virus glycoproteins resulted in syncytium formation. These results indicate that the transmembrane and cytoplasmic domains of the CD4 molecule are dispensable for both HIV infection and syncytium formation.

ization process does not require the intact cytoplasmic domain of CD4. The expression in a CD4- T lymphocyte cell line of a truncated derivative of CD4 that lacks the cytoplasmic region of the molecule resulted in the cells becoming permissive for infection (2). Although these studies have provided information on the role of the extracellular and cytoplasmic domains of CD4 in the infection process, it is not clear whether the intact transmembrane domain of CD4 is required for the internalization of HIV-1. As a means of clarifying the role of the transmembrane domain in the infection process, a HeLa cell line was established that expresses a chimeric protein consisting of the extracellular domain of CD4 fused to the carboxyterminal 37 amino acids of decay-accelerating factor (DAF). DAF is one of a group of cellular proteins that undergo posttranslational modification in which glycophospholipidanchoring structures are substituted for membrane-spanning sequences (for reviews, see references 10, 14, 23, and 24). Previous studies have demonstrated that the carboxy-terminal segment of DAF is sufficient for directing the glycophospholipid anchoring of DAF to the cell membrane (6, 7, 29). This sequence also has been used to anchor the extracellular domain of the CD8 receptor (47) and the normally secreted human growth hormone (7, 19a) to the cell membrane via a glycophospholipid anchor. In this report, we show that HeLa cells expressing a glycophospholipid-anchored form of CD4 can be infected by HIV-1 and form syncytia in response to the interaction with HIV-1 envelope proteins.

The CD4 molecule is a glycoprotein found primarily on the surface of a population of mature, thymus-derived T lymphocytes and to a lesser extent on cells of the monocytemacrophage lineage. CD4 is thought to play a role in T-cell antigen recognition through its interaction with both the T-cell receptor and class II major histocompatibility complex molecules (for reviews, see references 4, 22, 37, and 41). Recent studies have also demonstrated the association of the cytoplasmic domain of CD4 with the lymphoidspecific tyrosine kinase p56I1k (38, 46, 48, 49). Thus, the ability of CD4-ligand interactions to generate intracellular signals may be important for its function. The CD4 molecule has a molecular size of approximately 55 kDa. It has four extracellular domains, homologous to the immunoglobulin variable regions, a single membrane-spanning domain, and a short cytoplasmic tail. This structure is consistent with the placement of CD4 in the immunoglobulin supergene family (26). In addition to its role in lymphocyte activation, CD4 can serve as a receptor for human immunodeficiency virus type 1 (HIV-1) (11, 17, 19, 21, 42). Direct evidence for this was provided by the development of a HeLa cell line expressing CD4 (25). In contrast to the parent cells, the HeLa cells synthesizing an intact CD4 molecule were shown to be infectable by HIV-1. The expression of truncated, soluble derivatives of the extracellular domain of CD4 has provided direct evidence that the N-terminal region of CD4 contains important elements for the high-affinity binding of HIV-1 gpl20 (1, 36). Subsequent to the binding of the viral envelope to CD4, the virus enters the cell either by direct fusion of the envelope to the cell membrane (27) or by receptor-mediated endocytosis of a HIV-CD4 complex (33). The virus internal*

MATERIALS AND METHODS Plasmids. Plasmid pSP65-CD4 contains the CD4 receptor cDNA sequence (26). pAGS3 is a mammalian expression vector containing the chicken ,-actin promoter and simian virus 40 (SV40) polyadenylation and splicing signals (30).

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pSV2neo (43), which expresses the aminoglycoside phosphotransferase gene from the SV40 early promoter, was obtained from the American Type Culture Collection. pUC19 (51) was obtained from GIBCO, BRL. pLVWT is a plasmid that expresses the HIV-1 envelope proteins from the HIV-1 long terminal repeat (5). pDOLHIVenv also expresses HIV-1 envelope proteins (15). The plasmid designated pCD4-DAF, designed to express the CD4-DAF fusion protein, was constructed as follows. A 624-bp fragment containing the CD4 receptor signal sequence and the receptor protein-coding sequence through the glutamine at position 150 was isolated from a HaeIII digest of pSP65-CD4. The blunt-ended fragment was ligated into the SmaI site of pUC19, and a clone was selected that contained the HaeIII end of the fragment adjacent to the HindIII site in the pUC19 polylinker region (pCD4-Nterm). The internal portion of the receptor-coding region was obtained by digesting pSP65-CD4 with HaeII and NciI. A 1,129-bp fragment was isolated, and the ends were made blunt with T4 polymerase. The resulting fragment was cloned into the HincII site of M13mpl9 amber (8), and oligonucleotide site-directed mutagenesis (32) was used to create an EcoRV site at the NciI-HincII junction (pCD4Intern). The sequence encoding the 37 carboxy-terminal amino acids of DAF (6) was constructed by synthesizing a set of eight 30- to 49-bp oligonucleotides. The appropriate pairs of oligonucleotides were annealed and treated with kinase by using standard conditions (39). An aliquot of each pair was ligated and digested with SstI and EcoRI, and the 130-bp fragment was purified by gel electrophoresis. The fragment was cloned into SstI-EcoRI-digested M13mpl9 (M13-DAF). For the final construction, the appropriate HindIII-PvuII fragment from pCD4-Nterm, a PvuII-EcoRV fragment from pCD4-Intern, and a Sma-HindIII fragment from M13-DAF were ligated into HindlIl-digested pAGS3. A clone with the correct orientation was selected, and the sequence of critical regions was verified by dideoxynucleotide sequence analysis (40). Transfections. HeLa cells were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics. Cells were trypsinized, diluted, and seeded at 5 x 105 cells per 10-cm culture dish 16 to 24 h prior to transfection. One hour before transfection, the medium was removed and replaced with fresh medium. The cells were cotransfected with a mixture of 10 pug of pCD4-DAF and 1 ,ug of pSV2neo by the calcium phosphate precipitation method (16), using the reagents supplied in kit form by Stratagene. Forty-eight hours after transfection, the cells were trypsinized, diluted in DMEM10% FBS, supplemented with 500 ,ug of G418 (GIBCO) per ml, and replated. After incubation for 2 to 3 weeks with twice-weekly medium changes, colonies of G418-resistant cells were collected by trypsinization and pooled. This cell population was designated HeLa CD4-DAF. Clonal lines were isolated by using cloning cylinders to collect individual G418-resistant colonies. Immunoblot analysis. Flasks (25 cm2) were seeded with 5 x 104 cells in DMEM-10% FBS and incubated for 72 h at 37°C. The medium was changed 1 day before the cells were harvested. The cells were washed twice with Dulbecco's phosphate-buffered saline lacking MgCl2 and CaCl2 (PBS) and scraped into 2 ml of PBS. The cells from two flasks were pooled and centrifuged at 400 x g for 10 min, and the pellets were resuspended in 30 jil of PBS. Ten microliters of each cell suspension was added to 10 ,ul of 2x Laemmli sample buffer (20) and boiled for 5 min. The proteins were separated

HIV INFECTS HeLa CELLS EXPRESSING CD4

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on a 12.5% sodium dodecyl sulfate (SDS)-polyacrylamide gel. Following electrophoresis, the proteins were electroblotted (18) onto a nitrocellulose membrane, and nonspecific binding sites were blocked by incubating the blot for 1 h at room temperature in a solution consisting of 10 mM Tris (pH 8.0), 150 mM NaCl, 0.05% Tween 20 (TBS-T), 3% gelatin, and 3% powdered milk. The blot was rinsed with TBS-T and

incubated for 1 h at room temperature in a 1:25 dilution of anti-Leu 3a + 3b (CD4) (Becton Dickinson) in TBS-T. The antibody solution had been incubated with an acetone precipitate of proteins from HeLa cells to reduce the level of nonspecific binding material (18). The blot was rinsed with TBS-T and then incubated with a 1:7,500 dilution of alkaline phosphatase-conjugated goat anti-mouse antibody (Promega) for 1 h at room temperature. The blot was rinsed with TBS-T and developed for 1.5 h in the dark in a solution of 100 mM Tris (pH 9.5), 100 mM NaCl, and 5 mM MgCl2 containing the substrates nitroblue tetrazolium and 5-bromo4-chloro-3-indolylphosphate (Promega). Fluorescence-activated cell sorting (FACS) analysis and PI-PLC treatment. Cell monolayers were washed once with PBS and removed by Versene (0.53 mM EDTA-PBS) treatment after a 3-min incubation at 37°C and 5% CO2. The cells were washed in 10 ml of cold PBS, centrifuged at 8°C for 10 min at 400 x g, resuspended in 0.8 ml of DMEM-2% FBS, and separated into two equal aliquots. One group received 80 ,ul of phosphatidylinositol-specific phospholipase C (PIPLC; 10-U/ml stock; American Radiolabeled Chemicals, Inc., St. Louis, Mo.) per 400 ,ul of medium, and the second group served as the control, without enzyme. Both groups were incubated at 37°C and 5% CO2 for 1.5 h, washed with 10 ml of cold DMEM-2% FBS, and centrifuged for 10 min at 400 x g. The cell pellets were washed again with cold PBS, resuspended, and washed once in 2 ml of PBS with 0.1% sodium azide and once in cold PBS without sodium azide. The cell pellets (106 cells) were treated with 20 ,ul of either fluorescein isothiocyanate (FITC)-conjugated anti-Leu 3a + 3b (100 ,ug/ml) or FITC-conjugated mouse immunoglobulin Gi (50 ,ug/ml) (Becton Dickinson). After staining for 30 min on ice, cells were washed in cold PBS, resuspended in 1 ml of PBS containing 0.5% paraformaldehyde, stored at 4°C, and analyzed within 24 h. Phenotypic analysis was performed with a FACScan flow cytometer (Becton Dickinson). Virus infection. HeLa and HeLa derivative cell lines were seeded into 25-cm2 flasks and grown to a density of approximately 50% confluency by the day of infection. Briefly, conditioned medium was removed from the cultures and monolayers were washed once with DMEM. HIV-1IIIB (0.01 50% tissue culture infectious dose per cell) was added to each monolayer and allowed to adsorb for 1 h at 37°C and 5% CO2 with occasional rocking. Uninfected control cultures were treated similarly, receiving 0.5 ml of DMEM in lieu of virus for adsorption incubation (mock infection). After adsorption, 4.5 ml of the appropriate medium was added to each flask, and after 2 to 3 days an additional 5 ml was added. Cell-free supernatants were collected weekly from each culture and assayed for reverse transcriptase (RT) activity and p24 core antigen content. H9 cells in log-phase growth were used as target cells for the isolation of virus present in the supernatants of HIV-1infected HeLa cells. Target cells (107) were treated with 25 pug of DEAE-dextran (Pharmacia) per ml in 2 ml of PBS for 20 min at 37°C, centrifuged for 10 min at 400 x g, and then washed in 10 ml of RPMI 1640-10% FBS. After the final wash, the medium was removed and 2 ml of cell-free supernatant from the various HIV-1-infected HeLa cell

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cultures was added to the H9 cell pellet and adsorbed for 1 h at 37°C and 5% CO2 with occasional mixing. After adsorption, RPMI 1640-10% FBS was added to the virus-treated cells and incubated at 37°C and 5% CO2. Culture supernatant samples were removed weekly for analysis of RT activity and HIV-1 p24 core protein content. RT activity and p24 antigen assays. The microtiter plate assay system for RT measurement has been described elsewhere (9, 35, 44). Briefly, 100 1.l of the cell-free culture supernatant was removed and treated with 11 IlI of lysis buffer (0.5 M Tris hydrochloride [pH 8.0], 1.0 M NaCl, 5% Triton X-100, 10 mM EDTA). Six microliters of the lysed sample was placed in the reaction mixture, and the incorporated radioactivity was reported as counts per minute per 6 ,ul. HIV-1 p24 core antigen production was determined by using an enzyme-linked immunosorbent assay kit (DuPont, NEN) and reported as nanograms of p24 per ml. Cell fusion. HeLa, HeLa CD4, and HeLa CD4-DAF cells in DMEM-10% FBS were seeded into 10-cm culture dishes at a cell density of 2.5 x 105 per dish. The following day the cells were transfected with 10 jig of pLVWT, pDOL HIVenv, or pUC19 as described above. After transfection, the cells were observed on a daily basis for syncytium formation. For the cocultivation assay, HeLa cells were transfected with pLVWT or pUC19. Two days posttransfection, the cells were removed with Versene and added to 25% confluent cultures of HeLa CD4-DAF cells. The mixed cultures were observed daily for syncytium formation. Recombinant baculovirus. Through a series of DNA modification steps, a DNA fragment containing the CD4 receptor through the proline, at amino acid position 370 of the CD4 cDNA, followed by an alanine residue and terminator codon was generated. The resulting DNA, which includes the complete sequence of the CD4 receptor minus the transmembrane and cytoplasmic domains, was cloned into the BamHI site of the baculovirus shuttle plasmid pAc373 (45). A recombinant virus, which directed the expression of a soluble form of the CD4 receptor, was generated and plaque purified as described by Summers and Smith (45). The presence of soluble CD4 in the culture medium of virusinfected cells was demonstrated by immunoblot analysis. RESULTS Isolation and immunoblot analysis of HeLa cells expressing a CD4-DAF fusion protein. The initial step in generating a HeLa cell line expressing a glycophospholipid-anchored form of the extracellular domain of CD4 was the construction of pCD4-DAF (Fig. 1). The important expression elements included in the parent plasmid pAGS3 (30) are the chicken P-actin gene promoter and the splicing and polyadenylation signals of SV40. The chimeric CD4-DAF gene was constructed by fusing the signal sequence and extracellular domain of the CD4 cDNA to a synthetic DNA fragment encoding the carboxy-terminal 37 amino acids of DAF and a termination codon. A five-amino-acid in-frame linker was created at the fusion joint during the gene construction process. The complete CD4-DAF gene fusion was cloned into the unique Hindlll site of pAGS3. HeLa cells were cotransfected with pCD4-DAF and pSV2neo and cultured in the presence of the selection agent G418. A population of cells, designated HeLa CD4-DAF, was selected and maintained in medium containing G418. Immunoblot analysis of the cells using the anti-CD4 antibody mixture Leu 3a + 3b is shown in Fig. 2. The HeLa CD4-DAF cells expressed a protein, recognized specifically by the

CD4

DAF

uwNeC

FIG. 1. Schematic representation of pCD4-DAF. The plasmid utilizes the chicken P-actin promoter (AG promoter) and SV40 splicing and polyadenylation signals. The CD4-DAF fusion gene was constructed as described in Materials and Methods and cloned into the HindlIl site of the expression plasmid pAGS3. The lettering in the CD4-DAF block represents the single-letter amino acid code. The CD4-DAF fusion gene initiates at the methionine codon and continues through the proline residue at position +370 of CD4, effectively removing the transmembrane and cytoplasmic coding regions. This is followed by a five-amino-acid in-frame sequence generated during the construction of the fusion gene and the carboxy-terminal 37 amino acids of the DAF gene.

antibody, which migrated as a diffuse doublet with a molecular size of 56 to 62 kDa. As expected, HeLa cells did not contain detectable cell-associated CD4, whereas HeLa CD4 cells synthesized a CD4 molecule having a molecular size of approximately 55 kDa (26). A sample of a soluble form of CD4 (46 kDa) produced by recombinant baculovirus-infected insect (SF9) cells was included as a positive control. FACS analysis of CD4 content. The HeLa CD4-DAF cells shown to express the CD4 protein by immunoblot analysis (Fig. 2) were subjected to FACS analysis. The HeLa, HeLa CD4, and HeLa CD4-DAF cells were stained with the FITC-conjugated Leu 3a + 3b monoclonal antibody to

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o

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.

9769-

30-0 46 -

of mdiufrm SF cels nfce wit a rcmbntbauois membrane.~~~~U CD4 was deetdb Uecig h ebaewt 30-

FIG. 2. Immunoblot analysis of HeLa, HeLa CD4, and HeLa CD4-DAF cells. Suspensions of the various cell lines and an aliquot of medium from SF9 cells infected with a recombinant baculovirus expressing a soluble form of CD4 were electrophoresed through a 12.5% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. CD4 was detected by reacting the membrane with anti-Leu 3a + 3b antibody. Positions of size standards are shown in kilodaltons on the left.

HIV INFECTS HeLa CELLS EXPRESSING CD4

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-

m

20

z

0

-i

3279

Pi PLC -

I

0 w

0

80

a

60 +

Pi PLC -

FLUORESCENCE INTENSITY FIG. 3. FACS analysis and PI-PLC treatment. HeLa (A and D), HeLa CD4 (B and E), and HeLa CD4-DAF (C and F) cells were analyzed ) or FITC-conjugated antifor cell surface CD4 expression. Cells were labeled with either FITC-conjugated anti-CD4 antibody ( immunoglobulin Gl antibody ( ..... ). (D to F) Effect of P1-PLC treatment on the amount of CD4 present on the cell surface.

determine the amount of CD4 present on the cell surface. The data (Fig. 3A to C) indicate that the normal HeLa cells did not express detectable CD4, while HeLa CD4 and HeLa CD4-DAF cell lines produced high levels of CD4. A second facet of the FACS analysis was to confirm that the chimeric CD4 molecule was actually anchored to the cell membrane via a PI-PLC-sensitive glycophospholipid linkage. The same three cell lines were treated with PI-PLC and then analyzed as described above for cell surface CD4 content (Fig. 3D to F). Expression of the CD4 molecule on the HeLa CD4-DAF cells was decreased by approximately 90% following the treatment with PI-PLC, indicating that the linkage of the CD4-DAF protein to the HeLa membrane was through a glycophospholipid anchor. The incomplete removal of the CD4 by PI-PLC treatment is not unexpected since resistance of DAF to PI-PLC treatment appears to be cell type dependent (23). Between 60 and 90% of DAF expressed by leukocytes and epithelial cells has been shown to be cleavable by PI-PLC treatment (12, 50). In contrast to the HeLa CD4-DAF cells, the HeLa CD4 cells, which express a normal full-length CD4 molecule, were not sensitive to the action of PI-PLC. Infection with HIV-1. The HeLa, HeLa CD4, and HeLa CD4-DAF cells and a clone isolated from the HeLa CD4DAF population (clone 1) were infected with HIV-1 to determine whether expression of the hybrid CD4-DAF protein was sufficient to support the infection of the HeLa cells. RT activity and HIV p24 core protein data (Table 1) indicate that productive infections were established with HeLa CD4 as well as with HeLa CD4-DAF and HeLa CD4-DAF clone 1 by week 3 postinfection. The culture supernatants from these cells, as well as the virus-treated HeLa cells, were incubated with the H9 human T-lymphocytic cell line, and

virus production was monitored. The virus produced from the HeLa cells expressing the chimeric CD4 molecule was capable of recognizing and infecting a natural CD4 receptorexpressing cell (Table 1). Cell fusion. The infection of cells expressing cell surfaceassociated CD4 results in cell fusion leading to the formation of multinucleated syncytial cells (17, 21, 25, 28). Syncytium formation has also been observed following the transfection of CD4-positive cells with plasmids expressing HIV-1 envelope glycoproteins (15). The infection of HeLa CD4-DAF cells with HIV-1 appeared to initiate syncytium formation. This observation was investigated further by transfecting HeLa, HeLa CD4, and HeLa CD4-DAF cells with either pLVWT, which directs the synthesis of HIV-1 envelope proteins (5), or pUC19. Following transfection, the cells were observed microscopically for syncytium formation. The HeLa CD4 and HeLa CD4-DAF cells transfected with pLVWT formed large syncytia by 2 days posttransfection, whereas no syncytia were observed in the cells transfected with pUC19 (Fig. 4). The same cells transfected with pDOL HIVenv also formed syncytia, whereas transfected HeLa cells did not (data not shown). In a separate experiment, HeLa CD4-DAF cells were cocultivated with HeLa cells transiently transfected with pLVWT. Within 24 h syncytial cells were seen in the mixed cell cultures (data not shown). DISCUSSION The infection of CD4+ cells by HIV-1 is a complex multistep process initiated by the interaction of the HIV envelope glycoprotein gpl20 with the cell surface CD4 molecule (11, 17, 19, 21). The extracellular domain of CD4 is composed of four tandem immunoglobulinlike domains (26).

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TABLE 1. Infection of HeLa and HeLa cell derivatives with Sample

Treatment

HIV-1111B and rescue of the HeLa-produced virus in H9 cell culturesa Wk postinfection

Activity 1

HeLa

HIV-1 Mock

HeLa CD4

HIV-1

Mock

HeLa CD4-DAF

HIV-1 Mock

HeLa CD4-DAF clone 1

HIV-1 Mock

RT p24 RT p24 RT p24 RT p24 RT p24 RT p24 RT p24 RT p24

3 ~~~2 ~

647 18.0 334 ND 15,017 175.0 121 ND

217 ND 121 ND 4,302 110.0 117 ND

82 ND 160 ND 2,854 50.0 49 ND

1,904 33.0

9,116 162.0

5,782

135 ND 856 13.0 298 ND

272 ND 764 ND 102 ND

99.0 95 ND

1,990 26.0 117 ND

H aPE HeLa CPE

H9 virus isolation

4islto 114 ND 57

ND 3,298 62.0 99 ND 8,100 135.0 115 ND

10,809 168.0 78 ND

--

--

--

--

++

++

--

--

++

++

--

--

++

++

--

--

a HIV-treated HeLa and HeLa CD4 derivative culture supernatants were assayed weekly for 4 weeks for RT activity (counts per minute) and for HIV-1 p24 core protein (nanograms per milliliter). The cytopathic effects (CPE) of the primary infection of HIV in HeLa and HeLa cell derivatives were subjectively evaluated during the course of the experiment. Week 4 HIV-treated HeLa culture supernatants were used to recover HIV-l11IB in H9 cells, which were assayed for RT activity on day 7 postinfection. + +, H9 cultures RT positive by day 7 (virus isolated); - -, negative cultures; ND, not determined.

The determinants for the high-affinity binding of gpl20 have been shown to reside within the first 106 amino acids of CD4, and a discrete binding domain has been further localized into a region encompassing amino acid residues 41 to 56 (1, 3, 17, 31, 34, 36). Although the binding of gpl20 to CD4 has been clearly defined, the mechanism of virus entry into the cell is unclear. It appears that the virus enters the cell directly by fusing with the cell membrane (27). However, evidence also exists supporting a receptor-mediated endocytosis pathway as the means of virus entry (33). The role of the cytoplasmic domain of CD4 in the virus internalization process has been investigated by expressing derivatives of CD4 with various point and deletion mutations in the cytoplasmic domain (2). These studies have indicated that an intact cytoplasmic domain is not required for a successful infection. Replacement of the transmembrane domain of CD4 with the transmembrane domain of the related CD8 molecule also had no detectable effect on virus infection (2). However, the absolute requirement for an intact transmembrane domain was not addressed by these studies. In this study, to further characterize the role of the transmembrane and cytoplasmic domains of CD4 in the HIV-1 infection process, a glycophospholipid-anchored form of CD4 lacking a 61-amino-acid segment encoding the entire transmembrane and cytoplasmic regions has been expressed in HeLa cells. This was accomplished by expressing a fusion protein consisting of the extracellular domain of CD4 fused to the carboxy-terminal 37 amino acids of DAF, one of a group of proteins known to be linked to the cell membrane via a glycophospholipid anchor (24, 29). In the prototype glycophospholipid-anchored protein, the a-carboxy group of the carboxy-terminal amino acid of the protein is linked to an ethanolamine phosphate. This is attached to a glycan group of varying structure and ends in an inositol phospholipid, which is inserted into the cell membrane (for reviews, see references 10, 14, 23, and 24). Previous studies have shown that the linkage of the carboxy-terminal segment of DAF to transmembrane-minus forms of the herpesvirus glycoprotein D (6) and the CD8 cell surface glycoprotein (47)

results in glycophospholipid-anchored forms of these proteins. The specificity of this linkage is demonstrated by the cleavage of the glycophospholipid-anchored protein from the cell membrane following treatment with PI-PLC. In the case of DAF, the actual carboxy-terminal amino acid which links to the glycophospholipid anchor has yet to be determined. The CD4-DAF fusion protein expressed by HeLa cells has a molecular mass of approximately 56 to 62 kDa. Immunoblot analysis of whole cells with an anti-CD4 antibody revealed two major forms of the fusion protein. These may result from the processing events involved in the generation of the glycophospholipid-anchored form of the protein, since the visualized protein bands represent the forms of CD4 present in the cytoplasm and on the cell surface. Further experiments aimed at characterizing the protein associated with just the cell membrane may help to clarify this point. Treatment of the HeLa CD4-DAF cells with PI-PLC resulted in the removal of approximately 90% of the membraneassociated CD4, as judged by the FACS analysis. This is in agreement with previous results reported for the PI-PLCspecific cleavage of glycophospholipid-anchored proteins (12). In contrast to the HeLa CD4-DAF cells, treatment of the HeLa CD4 cells with PI-PLC resulted in no detectable release of CD4. Infection of the HeLa CD4-DAF cells with HIV-1 resulted in a productive infection, as evidenced by an increase in virus RT activity and p24 core protein synthesis. In addition, the infected cells supported a productive infection, as shown by the presence of infectious virus in culture supernatants. At present, the virus infectivity studies do not allow specific conclusions to be made regarding the relative infectability of the HeLa CD4-DAF cells compared with the HeLa CD4 cells. To draw a valid conclusion, it would be necessary to quantitate the level of CD4 and CD4-DAF expressed on the respective cell types. Syncytia appeared to develop following the infection of the HeLa CD4-DAF cells with HIV-1. The ability of these cells to undergo fusion was substantiated by the development of syncytia in cultures transiently transfected with plasmids that direct the expression of HIV-1

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t'hr k

FIG. 4. Syncytium formation following transfection of HeLa CD4 and HeLa CD4-DAF cells with pLVWT. HeLa CD4 and HeLa CD4-DAF cells were transfected with pLVWT, which expresses HIV-1 envelope proteins, or pUC19 and observed for syncytium formation. (A) HeLa CD4-DAF plus pUC19, (B) HeLa CD4-DAF plus pLVWT, (C) HeLa CD4 plus pUC19, (D) HeLa CD4 plus pLVWT. Magnification, x125.

envelope proteins and by cocultivation with HeLa cells expressing HIV-1 envelope proteins. The treatment of HeLa CD4-DAF cells with PI-PLC resulted in the release of approximately 90% of the cellassociated CD4. PI-PLC treatment of HeLa cells expressing DAF has also been shown to result in the release of approximately 80 to 85% of the membrane-bound DAF (50). Analysis of the remaining PI-PLC-insensitive DAF indicated that this small fraction of DAF molecules was anchored to the membrane via an acylated inositol linkage. Similar to the HeLa cell DAF, it is likely that the small fraction of CD4-DAF molecules that we have reported to be resistant to PI-PLC treatment is also anchored to the membrane in this manner.

Recent studies have indicated that the cytoplasmic domain of CD4 can interact with the N-terminal region of the

lymphocyte-specific receptor p56Ick (38, 46, 48, 49). This interaction has been postulated to be involved in the signal transduction process required for T-cell activation. Our results indicate that such an interaction is not required for CD4 to act as a competent receptor for HIV-1 infection in HeLa cells. In summary, our results indicate that the cytoplasmic and transmembrane domains of CD4 are not required to support a productive HIV-1 infection. Furthermore, these regions of the CD4 molecule are not required for HIV-1-induced syncytium formation. During the course of these studies, it was reported (13) that T cells expressing a glycophospholipidanchored form of CD4 could be infected with HIV-1. Our findings are consistent with these results. Therefore, the major role of CD4 as the receptor for HIV appears to be the targeted localization of the virus to cells expressing CD4 on

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