the cytoplasmic domain of gp4l also plays a role in other steps of virus replication. Retrovirus .... cysteine-free RPMI 1640 medium containing 10% fetal calf.
Vol. 67, No. 1
JOURNAL OF VIROLOGY, Jan. 1993, p. 213-221
0022-538X/93/010213-09$02.00/0 Copyright ©D 1993, American Society for Microbiology
Mutations in the Cytoplasmic Domain of Human Immunodeficiency Virus Type 1 Transmembrane Protein Impair the Incorporation of Env Proteins into Mature Virions XIAOFANG YU, XIN YUAN, MARY FRAN McLANE, TUN-HOU LEE, AND MAX ESSEX* Department of Cancer Biology, Harvard School of Public Health, Boston, Massachusetts 02115 Received 16 July 1992/Accepted 20 October 1992
In-frame stop codons were introduced into the coding region of human immunodeficiency virus type 1 (HIV-1) transmembrane protein (gp4l). Truncation of 147 amino acids from the carboxyl terminus of gp4l (TM709) significantly decreased the stability and cell surface expression of the viral Env proteins, while truncation of 104 amino acids (TM752) did not. Truncation of 43 or more amino acids from the carboxyl terminus of gp4l generated mutant viruses which were noninfectious in several human CD4+ T lymphoid cell lines and fresh peripheral blood mononuclear cells. Analysis of the noninfectious mutant virions revealed significantly reduced incorporation of the Env proteins compared with the wild-type virions. Comparable amounts of Env proteins were detected on the surfaces of wild-type- and TM752-transfected cells, suggesting that the structures of gp4l required for efficient incorporation of Env proteins were disrupted in mutant TM752. Truncation of the last 12 amino acids (TM844) from the carboxyl terminus of gp4l did not significantly affect the assembly and release of virions or the incorporation of Env proteins into mature virions. However, the TM844 virus had dramatically decreased infectivity compared with the wild-type virus. This suggests that the cytoplasmic domain of gp4l also plays a role in other steps of virus replication.
Retrovirus transmembrane (TM) proteins contain extracellular, transmembrane, and cytoplasmic domains (22). The transmembrane domain serves as a stop translocation signal during the insertion of Env protein into the cell membrane and anchors the TM protein within the cell membrane and viral membrane (22). For most retroviruses, such as the B-, C-, and D-type viruses and the bovine leukemia virus/human T-cell lymphotropic virus group of viruses, the cytoplasmic domain consists of fewer than 50 amino acids (12, 22). Little is known about the function of the TM protein cytoplasmic domain in retrovirus replication. Truncation of the entire cytoplasmic domain of a Rous sarcoma virus (RSV) TM protein did not affect the biosynthesis, processing, transport, or surface expression of Env proteins (35). Mutant RSV virions incorporated mutant viral Env proteins efficiently and were as infectious as the wild-type virions (35), suggesting that the cytoplasmic domain of RSV TM protein is dispensable for viral replication in vitro. In several groups of retroviruses, including murine leukemia virus (19, 24), equine infectious anemia virus (40), and Mason-Pfizer monkey virus (1), part of the cytoplasmic domain is cleaved from the mature TM protein in released virions. This cleavage apparently occurs after the virus is released from the cells since processed TM proteins are not detected in infected cells, only in the released virions (1, 40). Viral protease is presumably involved since mutations in the viral protease gene blocked TM protein processing (1, 6, 25). The functional consequence of this TM protein processing is still not clear. One possibility is that processing removes sequences whose functions are required only before and during virus assembly. Another possibility is that processing of the TM protein induces conformational changes in viral glycoproteins which may play an important role in virus entry. The latter possibility is supported by the observation
*
that truncations in the cytoplasmic domain of human immunodeficiency virus type 1 (HIV-1) and HIV-2 TM proteins increase the fusogenic ability of the viral Env proteins (8, 27, 32). The TM protein of lentiviruses extends further downstream from the transmembrane domain (12, 22), resulting in a long cytoplasmic domain of more than 100 amino acids. For example, the cytoplasmic domain of HIV-1, HIV-2, and simian immunodeficiency virus (SIV) TM proteins consists of approximately 150 amino acids (12, 22), while the cytoplasmic domain of the TM protein of equine infectious anemia virus consists of more than 200 amino acids (40). It seems that a large portion of the cytoplasmic domain of the SIV and HIV-2 TM protein is dispensable for viral replication in certain established human cell lines (4, 10, 16, 20). This region of the TM protein may even inhibit viral replication in some human cells. SIV and HIV-2 with truncated TM proteins propagated better in the HUT-78 cell line than viruses with full-length TM proteins (3, 21, 26). This may be explained by the increased fusogenic ability of the truncated TM protein (32). In vitro selection of a truncated TM protein has also been observed in equine infectious anemia virus (40). The truncated TM proteins were quickly converted to full-length TM proteins when SIV from macaques (SIVmac) were allowed to grow in rhesus macaque peripheral blood mononuclear cells (PBMC) (21, 26), suggesting that the whole cytoplasmic domain of the SIV TM protein is required for viral replication in its natural host. The long cytoplasmic domain of lentivirus TM proteins may have a unique function in lentivirus replication. In contrast to SIV and HIV-2, all of the infectious HIV-1 clones that have been sequenced contain a full-length TM protein (gp4l), with only one exception. Thus, the full-length TM protein appears to be critical for HIV-1 replication. Consistent with these observations, small deletions introduced in the cytoplasmic domain of HIV-1 gp4l dramatically reduced virus infectivity (9, 28, 48). Computer analysis
Corresponding author. 213
214
YU ET AL.
indicates that the cytoplasmic domain of gp4l contains two amphipathic a-helical structures (50) which can form a secondary association with the membrane bilayer (17). However, the function of these structures in virus replication is still not clear. Large truncations that deleted both of the a helices in the cytoplasmic domain of gp4l did not significantly affect viral Env protein biosynthesis, processing, transport, or surface expression (8). Syncytium formation (8, 27) and oligomerization (7) of the mutant Env protein were also not impaired. To study the functional role of the gp4l cytoplasmic domain in HIV-1 replication, a series of inframe stop codons were introduced in the coding region of the gp4l cytoplasmic domain. The effect of these mutations on HIV-1 replication was investigated.
MATERUILS AND METHODS DNA constructs. The 2.75-kb EcoRI-BamHI fragment (for generating TM709) and the 0.4-kb BamHI-XhoI fragment (for generating TM752, TM775, TM795, TM812, and TM844) from HXB2R3 (54) were subcloned into pGEM7Zf(+) (Promega, Madison, Wis.). Single-stranded uracil-containing DNA was prepared and used for site-directed mutagenesis according to the protocol of the manufacturer (Bio-Rad, Richmond, Calif.). The sequences of primers used for mutagenesis were as follows: TM709, 5'-GAA TAG AGT TAG CTA GCG ATA TTC ACC AT-3'; TM752, 5'-GTC CCA GAT AAG TGC CTA GGA TCC-3'; TM775, 5'-GTT ACA ATC TAG AGT AAG TC-3'; TM795, 5'-TAG GAG ATT CCA CTA AAA lTT GAG GGC TTC-3'; TM812, 5'-GTG GCA TTG AGC TAG CTA ACA GCA C-3'; TM844, 5'-GCC CTG TCT TAT TCC TTA AGG TAT GTG GCG AA-3'. Mutants were screened by restriction enzyme digestion and DNA sequencing. The 2.7-kb SalI-BamHI fragments and the 0.4-kb BamHI-XhoI fragments that contained the gp4l mutations were cloned back into the vectors of HXB2R3. Cells and sera. COS-7 cells were maintained in Dulbecco's modified Eagle's medium (containing L-glutamine and D-glucose)-10% fetal calf serum. SupTl and H9 cells were maintained in RPMI 1640 medium-10% fetal calf serum. MT-2 cells were maintained in RPMI 1640 medium-15% fetal calf serum. PBMC were isolated by density-gradient centrifugation on lymphocyte separation medium (Organon Teknika Corp., Durham, N.C.) and maintained in RPMI 1640 medium-20% fetal calf serum-20 U of interleukin-2 per ml (Becton Dickinson Labware, Bedford, Mass.). PBMC were treated with 5 jig of phytohemagglutinin per ml (Sigma, St. Louis, Mo.) for two days before they were used for the infection assay. The HIV-1-positive serum and the sheep anti-gpl20 serum have been previously described (54). For immunoblotting using sucrose gradient-purified virions, another pooled HIV-1-positive serum was used. Mouse monoclonal anti-gpl20 antibody was obtained from Du Pont (Wilmington, Del.). Transfection, infection, and reverse transcriptase assay. Transfection and the reverse transcriptase assay were performed as previously described (54). Briefly, subconfluent COS-7 cells (5 x 10 ) were trypsinized and transfected with 2 sLg of wild-type or mutant plasmid DNA by the DEAEdextran method as previously described (54). Culture supernatants from transfected or infected cells were used to concentrate virus pellets by the polyethylene glycol method and subjected to reverse transcriptase assay as previously described (54). Radioimmunoprecipitation analysis of viral proteins. At 60 h posttransfection, COS-7 cells were incubated for 12 h in
J. VIROL.
cysteine-free RPMI 1640 medium containing 10% fetal calf serum and [35S]cysteine (0.1 mCi/ml; Du Pont, NEN Research Products, Boston, Mass.). Cells were then lysed with lysis buffer (0.15 M NaCl, 0.05 M Tris-HCl, pH 7.2, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate) and centrifuged at 40,000 rpm (Beckman 70 Ti rotor) for 1 h to remove cell debris. Culture supernatants were precleared by centrifugation at 1,000 x g for 30 min and then centrifuged at 40,000 rpm (Beckman 70 Ti rotor) for 1 h to remove virus pellets. Cell lysates and culture supernatants were reacted with HIV-1-positive serum that had been preabsorbed with protein A-Sepharose CL-4B (Sigma) for 12 h at 4°C. Samples were then washed three times with lysis buffer (without sodium deoxycholate) and once with washing buffer (0.15 M NaCl, 0.05 M Tris-HCl, pH 7.2). Sixty microliters of sample buffer (0.08 M Tris-HCI, pH 6.8, 0.1 M dithiothreitol, 2% sodium dodecyl sulfate, 10% glycerol, 0.2% bromophenol blue) was added to each sample tube. Samples were boiled for 2 min before loading and separated by sodium dodecyl sulfate-12% polyacrylamide gel electrophoresis. Purification of virions and analysis of viral proteins by immunoblotting. At 72 h posttransfection, culture media from transfected COS-7 cells were centrifuged at 1,000 x g for 30 min. Supernatants were filtered through 0.2-,um-poresize filtration units (Nalge Company, Rochester, N.Y.) and centrifuged for 2 h through 3 ml of a 20% sucrose cushion at 20,000 rpm (Beckman SW28 rotor). Virus-free supernatants were discarded, and residual liquid was removed from the centrifuge tubes with dry swabs. Virus pellets were dissolved in sample buffer and separated by sodium dodecyl sulfate-12% polyacrylamide gel electrophoresis. For further purification of the virions in a sucrose gradient, virus pellets were dissolved in TNE buffer (0.01 M Tris-HCl, pH 7.2, 0.1 M NaCl, 0.001 M EDTA) and this solution was overlaid on the sucrose gradients. Sucrose gradients were prepared by a stepwise overlay of 2 ml of sucrose in TNE buffer that decreased in sucrose concentration from 60% to 20% in 2.5% increments. Samples were centrifuged at 20,000 rpm (Beckman SW28 rotor) for 20 h, and 18 fractions were collected dropwise from the bottom of the centrifuge tubes. The fractions with the highest reverse transcriptase activity were used to isolate the virions and analyzed by immunoblotting as described above. Indirect immunofluorescence assay. Cells were washed twice with phosphate-buffered saline 60 h posttransfection and incubated with 1:10-diluted mouse monoclonal antibody against gpl20 (Du Pont, Wilmington, Del.) for 60 min at room temperature. Cells were then washed three times with phosphate-buffered saline and incubated with a 1:25 dilution of fluorescein isothiocyanate-conjugated goat anti-mouse antibody (Becton Dickinson, San Jose, Calif.) for 45 min at room temperature. Cells were washed three times with phosphate-buffered saline and fixed with 1% paraformaldehyde before examination under a fluorescence microscope. RESULTS Construction of gp4l mutants. The wild-type provirus clone, HXB2R3, has been described before (54). In-frame stop codons were generated at different positions in the gp4l coding region (Fig. 1). The stop codon in TM709 is two amino acids downstream from the putative transmembrane region. Two positively charged arginine residues at positions 707 and 709, which are presumably important for the stop transfer signal, were preserved. TM752 and TM775 retained
VOL. 67, 1993
HIV-1 TM MUTATIONS AND ENV PROTEIN INCORPORATION
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752 v TM709 v 709 FIG. 1. Construction of gp4l mutants. The HIV- 1 Env precursor protein contains 856 amino acids in HXB2R3. The s;olid black areas represent hydrophilic regions. The arrow indicatets the cleavage site between gp120 and gp4l. The numbers at tlhe end of each diagram indicate the last amino acid that can be syntihesized in gpl60 by each construct, except for TM812, which termin;ates after amino TM752
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cytoplasmic domain and deleted the predicted distal amphipathic a helix (50). The stop codon in TM844 is just upstream from the putative Vif cleavage site (15) and truncates the last 12 amino acids of gp41. The amino acids of the overlapping rev open reading frame were not affected by the nucleotide substitutions in any of the mutants. Infectivity of gp4l mutant viruses. Viruses were generated from COS-7 cells after transfection with wild-type and mutant plasmid DNA (data not shown), suggesting that mutations in the cytoplasmic domain of gp4l did not block virus assembly and release. The infectivity of mutant viruses was compared with that of the wild-type virus in several T-lymphoid cell lines and PBMC. Non-cell-associated wildtype and mutant viruses with comparable amounts of reverse transcriptase activity were prepared and analyzed for infectivity as previously described (54). Except for mutant TM844, none of the mutants established a productive infection in SupTl, MT-2, or H9 cells or in fresh PBMC (Fig. 2). Although virus production was detected in TM844-infected MT-2 and SupTl cells, the kinetics of TM844 replication in these cells was dramatically slower than that of the wild-type virus (Fig. 2). Cytopathic effects and syncytium formation
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helices (50) were deleted from the cytoplasmic domain of TM752 and TM775 TM proteins. Mutations in TM795 and TM812 preserved the first 88 and 106 amino acids of the gp4l
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FIG. 2. Virus infectivity assay. Non-cell-associated wild-type and mutant viruses were prepared from the supernatants of transfected COS-7 cells and tested on SupTl, MT-2, H9, and fresh PBMC as previously described (54). Reverse transcriptase values represent samples from 0.1-ml culture supernatants. cpm, counts per minute.
216
YU ET AL. anti-gpl 20 14 a
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J. VIROL.
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FIG. 3. Analysis of viral proteins by immunoblotting. (a) Virions were purified and analyzed as described in Materials and Methods. Samples were transferred onto two nitrocellulose filters. One was blotted with sheep anti-gpl20 of HIV-1 (anti-gpl20). Another was blotted with HIV-1-positive human serum (anti-HIV-1). (b) Cell lysates (cell) and sucrose gradient-purified virions (virus) were blotted with another HIV-1-positive human serum. The position of TM752 gp4l is indicated by a bracket.
observed for TM844 in MT-2 and SupTl cells (data not shown), suggesting that mutation in TM844 did not significantly affect the fusion ability of the Env proteins. The TM844 virus was at least two orders of magnitude less infectious than the wild-type virus in SupTl cells (data not shown). TM844 virus replication was even more dramatically impaired in H9 cells and PBMC, so that mutant virus production never reached the level of wild-type virus production during the 30-day follow-up period (Fig. 2). Analysis of virion proteins by immunoblotting. To study the defect in the mutant viruses, virions harvested from culture supematants of wild-type- and mutant-transfected COS-7 cells were analyzed by immunoblotting. As was the case with the wild-type virions, the gag gene-encoded proteins, p24 and p17, and the pol gene-encoded proteins, p66, pSi, and p34, were readily detected in all of the mutant virions by using a pooled HIV-1-positive human serum (Fig. 3a). However, in sharp contrast to the wild-type virions, the env gene-encoded protein (gpl20) was dramatically decreased in mutant virions TM709, TM752, TM775, and TM795 as detected by sheep anti-gpl20 serum (Fig. 3a, upper panel) or the pooled HIV-1-positive serum (lower panel). Mutant gp4l was not detected in these mutant virions by HIV-1-positive serum (Fig. 3a, lower panel). For mutant TM812, no gp4l and a lesser amount of gpl20 compared with wild-type virions were detected by the HIV-1-positive serum (Fig. 3a, lower panel). Comparable amounts of gpi20 and gp4l were detected in wild-type and mutant TM844 virions (Fig. 3a). The TM844 gp4l migrated slightly more quickly than the wild-type gp4l (Fig. 3a). Wild-type, TM844, and TM752 virions were also purified in sucrose gradients and analyzed by immunoblotting using another pooled HIV-1-positive serum. This pooled HIV-1positive serum reacted to gpi20 better than the one described above under our assay conditions. The amount of gpi20 and gp41 detected in mutant TM844 virions remained
were
comparable to that in the wild-type virions after purification in a sucrose gradient (Fig. 3b). In contrast, the amount of gpl20 detected in mutant TM752 virions was significantly less than that seen in the wild-type virions (Fig. 3b). The amount of gpl20 detected in TM752 virions was estimated to be less than 20% of that detected in the wild-type virions when compared with a fourfold dilution of the wild-type virion sample (Fig. 3b). gp4l was not detected in the mutant TM752 virions (Fig. 3b). When cell lysates were analyzed with the same HIV-1-positive serum, comparable amounts of gp120 and of the wild-type and truncated gp41 were detected in wild-type- and TM752-transfected COS-7 cells (Fig. 3b). This suggests that the decreased detection of Env proteins in mutant TM752 virions probably did not result from a decreased immunoreactivity of the HIV-1-positive serum.
Analysis of viral Env protein synthesis, processing, transport, and surface expression. To address the question of whether the decreased incorporation of Env proteins in mutant TM709 and TM752 virions was due to decreased expression of mutant Env proteins in transfected cells, the expression of viral proteins in wild-type-transfected COS-7 cells and in COS-7 cells transfected with mutants TM709, TM752, and TM844 was analyzed by radioimmunoprecipitation. After transfection of the COS-7 cells, gpl60 and gpl20 were detected in the wild-type-transfected cells (Fig. 4). Similar amounts of gpl60 and gpl20, when adjusted for similar amounts of the Gag polyprotein (p55), were also detected in TM844- and TM752-transfected cells (Fig. 4). The migration of TM844 and TM752 gpl20 was similar to that of the wild-type gpl20 (Fig. 4), suggesting that mutations in TM844 and TM752 did not affect the expression and processing of mutant Env proteins. The transport of TM844 and TM752 Env proteins was not significantly affected since gpl20 could be detected in the supernatants of TM844- and TM752-transfected cells (Fig. 4). The amount of gpl60 and
HIV-1 TM MUTATIONS AND ENV PROTEIN INCORPORATION
VOL. 67, 1993
sup oamgWSbqum
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gpl20 detected in the TM709-transfected cells was much less than that detected in the wild-type-transfected cells (Fig. 4). In addition, TM709 gpl20 migrated slightly more slowly than the wild-type gpl20 (Fig. 4). This was not due to second-site mutations in gpl20 as the entire env coding region of TM709 was sequenced. TM709 gpl20 was detected in the culture supernatant of TM709-transfected COS-7 cells (Fig. 4). This suggests that Env protein transport to the cell surface could still occur in TM709-transfected cells. Surface expression of the gpl20 in wild-type-, TM752-, and TM709-transfected COS-7 cells was also studied by indirect immunofluorescence staining. Comparable levels of gpl20 were detected on the surfaces of wild-type- and TM752-transfected cells by mouse anti-gpl20 monoclonal antibody (Fig. 5). This indicates that the surface anchorage of gpl20 was not affected by the truncation of the cytoplasmic domain in TM752. This observation is in agreement with previous reports in which truncation of the last 104 amino acids from the C terminus of gp4l (same as TM752) did not affect the surface expression of the viral Env protein (8). In contrast to TM752, the ability of mutant TM709 to express Env proteins on the cell surface was greatly diminished, as is indicated by the lack of immunofluorescence staining of gpl20 on the surfaces of the TM709-transfected COS-7 cells (Fig. 5). DISCUSSION The results presented here suggest that the cytoplasmic domain of gp4l plays a critical role in HIV-1 replication. Truncation of 43 (TM812), 61 (TM795), 81 (TM775), 104 (TM752), and 147 (TM709) amino acids from the cytoplasmic
217
domain of gp4l generated noninfectious virions (Fig. 2). Analysis of these mutant virions indicated that the incorporation of viral Env proteins was significantly impaired, although other viral structural proteins were present at normal ratios compared with the wild-type virions (Fig. 3). Truncation of the last 12 amino acids from gp4l did not significantly decrease virus assembly and release or the incorporation of viral Env proteins (Fig. 3). However, the infectivity of mutant TM844 virus was dramatically decreased compared with that of the wild-type virus (Fig. 2). This observation suggests that the cytoplasmic domain of gp4l may also function in viral replication steps other than assembly. Recently, Gabuzda et al. also reported that deletion of residues 840 to 856 of HIV-1 gp4l blocked viral replication at a step prior to the formation of provirus DNA (11). Since the defect of TM844 virus was more severe in H9 cells and fresh PBMC than in SupTl and MT-2 cells (Fig. 2), it appears that cellular factors could also influence the function of the gp4l cytoplasmic domain in viral replication. It has been suggested that the cytoplasmic domain of gp4l is cleaved by Vif, a potential cysteine protease (15). The putative cleavage site is very close to the stop codon introduced in the mutant TM844 (15). The observation that the wild-type gp4l migrated more slowly than the TM844 gp4l (Fig. 3) suggests that, at least at the virion purification stage in our study, the cleavage of the cytoplasmic domain of gp4l had not occurred. Truncation of 147 amino acids from the C terminus of gp4l dramatically decreased the steady-state level of viral Env proteins in COS-7 cells (Fig. 4 and 5). Mutant Env protein synthesis was not significantly affected, as shown by pulsechase experiments (data not shown). Thus, the stability of TM709 Env proteins was apparently reduced compared with the stability of the wild-type Env proteins. It was reported recently that mutations in the cytoplasmic domain of gp4l could decrease the stability of mutant Env proteins (11). The TM709 gpl20 migrated slightly more slowly than the wildtype gpl20 (Fig. 4 and 5), suggesting that the modification of this mutant gpl20 might be different from that of the wildtype gpl20. The decreased stability of the mutant Env proteins and the aberrant modification of mutant gpl20 may indicate that the cytoplasmic domain of gp4l is important for mediating Env protein intracellular transport. It has been reported that the cytoplasmic domain of HIV-1 gp4l modulates the intracellular transport of viral Env proteins in certain cells (18). The cytoplasmic domains of many viral glycoproteins have been shown to modulate intracellular transport. Mutations in the cytoplasmic domain of vesicular stomatitis virus G glycoprotein (36, 41) and herpes simplex virus glycoprotein gB-1 (39) dramatically decreased the transport of the glycoprotein from the endoplasmic reticulum to the Golgi apparatus and to the cell surface. On the other hand, a short sequence in the cytoplasmic domain of adenovirus E19 glycoprotein contains a retention signal that makes the glycoprotein a resident of the endoplasmic reticulum (37). Deletion of seven amino acids from the cytoplasmic domain allowed mutant E19 proteins to be transported to the cell surface (37). The lack of surface staining of gpl20 in TM709-transfected COS-7 cells (Fig. 4 and 5) argues that part of the cytoplasmic domain of gp4l may be required for the stable anchorage of Env proteins on the cell surface. Alternatively, the mutation in TM709 may disturb the association of gpl20 and gp4l. The most intriguing observation in our study is that mutations in the cytoplasmic domain of gp4l significantly impaired the incorporation of viral Env proteins into mature
218
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YU ET AL.
.4
FIG. 5. Surface expression of HIV-1 Env proteins detected by indirect immunofluorescence using mouse monoclonal antibody against gp120. (a and b) Mock-transfected cells; (c and d) wild-type-virus-transfected cells; (e and f) TM752-transfected cells; (g and h) TM709-transfected cells. The corresponding Nomarski pictures (panels a, c, e, and g) are adjacent to the fluorescence pictures (panels b, d, f, and h).
virions (Fig. 3). This effect could apparently be separated from the effect of the cytoplasmic domain of gp4l on the stability of viral Env proteins as previously suggested (11). For mutant TM752, Env protein synthesis, processing, transport, and surface expression were not significantly altered compared with those of the wild-type Env proteins (Fig. 3b, 4, and 5); however, the incorporation of viral Env proteins was less than 20% of that of the wild-type virions
(Fig. 3b).
The mechanism by which retrovirus Env proteins are incorporated into virions is poorly understood. Several lines
of evidence have suggested that there is a specific interaction between retrovirus Env and Gag proteins. The MA protein of RSV (p19) could be coimmunoprecipitated with the SU protein (gp85) and TM protein (gp35/gp37) complex by anti-gp85 antibodies, suggesting that there is an interaction between the MA protein and the Env proteins in RSV virions (31, 44). This interaction was further demonstrated by the detection of gp35 and the p19 complex in RSV virions by using the chemical cross-linking method (13). Heterodimers of the MA protein (plO) and TM protein (gp36) were also detected in murine mammary tumor virus virions by chem-
VOL. 67, 1993
HIV-1 TM MUTATIONS AND ENV PROTEIN INCORPORATION
ical cross-linking (38). A double immunofluorescence study showed that the staining patterns of Moloney murine leukemia virus TM protein (pl5E) and MA protein (p15) were superimposable at the same loci on infected cells (43). This suggests an association between pl5E and p15 at the murine leukemia virus-infected cell membrane. A possible interaction between the Mason-Pfizer monkey virus MA protein and the cytoplasmic domain of the TM protein was recently reported (1). Mutations in the MA protein inhibited the processing of the cytoplasmic domain of the Mason-Pfizer monkey virus TM protein in released virions (1). HIV-1 Env proteins have been described as localizing almost exclusively at the basolateral surface of polarized epithelial cells (33). In the absence of viral Env protein expression, virus particles formed by the Gag proteins were released from the apical or basolateral surface at approximately equal ratios (34). However, in the presence of viral Env proteins, 94% of the virus particles were released from the basolateral surface (34). Clearly, the Env proteins of HIV-1 determine the site of virus release via a specific interaction with the Gag proteins in polarized epithelial cells. It has been speculated that specific interactions between the retrovirus Env and Gag proteins provide the basis for the selective incorporation of viral Env proteins into virions. We have previously reported that mutations in the MA protein of HIV-1 inhibited the incorporation of viral Env proteins into mature virions although virus assembly was not blocked (54). Combined with the results presented here, our data provide strong evidence that a specific interaction between HIV-1 Env and Gag proteins is required for efficient incorporation of Env proteins into mature virions. It is still not clear how mutations in the cytoplasmic domain of HIV-1 gp4i affected incorporation of the viral Env proteins. Mutations in the cytoplasmic domain of Moloney murine leukemia virus TM protein also impaired the incorporation of Env proteins into virions (14). Interactions between the cytoplasmic domains of viral glycoproteins of other enveloped viruses, such as Semliki Forest virus (23, 49) and vesicular stomatitis virus (51), and the viral capsid proteins have been implicated in the selective incorporation of viral Env proteins into virions. Vaux et al. used internal image anti-idiotype antibodies to show that there is a specific interaction between the cytoplasmic domain of the E2 glycoprotein and the nucleocapsid of Semliki Forest virus (49). Synthetic peptides corresponding to sequences of the E2 cytoplasmic domain inhibited virus budding when they were microinjected with infectious viral RNA into cells (23). These synthetic peptides were able to bind specifically to nucleocapsids in infected cells (23). It appears that oligomers of the cytoplasmic tail peptides bind to the nucleocapsids more efficiently than the monomer does (30). However, a recent report challenged the theory of specific interaction between the cytoplasmic domain of the E2 glycoprotein and the nucleocapsid of Semliki Forest virus (47). The cytoplasmic domain of the G protein is required to rescue a temperature-sensitive G protein mutant of vesicular stomatitis virus at nonpermissive temperatures (51). Deletions within the cytoplasmic domain of G proteins or G proteins with a cytoplasmic domain derived from other cellular or viral glycoproteins did not rescue the mutant (51). Rescue of virus infectivity correlated directly with the G protein's ability to be incorporated into virus particles (51), suggesting that the cytoplasmic domain is required for efficient assembly of the vesicular stomatitis virus G protein into virions. It is possible that the cytoplasmic domain of HIV-1 gp41 interacts with the MA domain of the Gag
219
polyprotein, and it is therefore important for the incorporation of viral Env protein into virions. Several lines of evidence suggest that the cytoplasmic domain of HIV-1 gp4i may not directly interact with Gag proteins. A mutant that contained only seven amino acids in the cytoplasmic domain of gp4i retained its ability to incorporate Env proteins into virions and its infectivity in MT-4 cells (52). However, a mutant that contained 25 amino acids in the cytoplasmic domain of gp4i had a severe defect in Env protein incorporation and was not infectious in MT-4 cells (52). When adapted to TALL-1 cells, an HIV-1 strain (KB-i 32) carrying a truncated TM protein was selected (46). The truncated TM protein contained 18 amino acids in the cytoplasmic domain and was presumably competent for incorporation into virions because the viruses were infectious in TALL-1 cells (46). However, when the same mutation was introduced into another HIV-1 clone (pNL-432), the viruses were not infectious in TALL-1 cells. This suggests that other regions in KB-lgp32 Env were compensating for the cytoplasmic domain truncation (46). It seems that mutations in the cytoplasmic domain of gp4i can change the overall structure of gp4i. This could impair the Env and Gag protein interaction that is required for the efficient incorporation of Env proteins into mature virions. Further study will be required to identify the structure(s) that is required for Env protein incorporation. Incorporation of viral glycoproteins into enveloped virus particles is one of the key steps in generating infectious virions. For enveloped viruses, selective incorporation of viral glycoproteins into mature virions is a highly specific process since cellular surface proteins are largely excluded from the released virions (2, 29, 42, 53). The specificity is apparently mediated through specific interaction between viral glycoprotein and capsid proteins. Strategies designed to block this interaction have inhibited Semliki Forest virus (23), Sindbis virus (45), and influenza virus (5) replication. Provided that there is also a specific interaction between the retrovirus TM protein and the MA protein, such interaction could be a potential target for the design of anti-HIV inhibitors. ACKNOWLEDGMENTS We thank Z. Matsuda, Q. C. Yu, K. Chow, and W. K. Wang for helpful discussions and technical assistance and E. Conway for editorial assistance. This work was supported by Public Health Service grants CA39805 and HL-33774 from the National Institutes of Health. REFERENCES 1. Brody, B. A., S. S. Rhee, M. A. Sommerfelt, and E. Hunter. 1992. A viral protease-mediated cleavage of the transmembrane glycoprotein of Mason-Pfizer monkey virus can be suppressed by mutations within the matrix protein. Proc. Natl. Acad. Sci. USA 89:3443-3447. 2. Calafat, J., H. Janssen, P. Demant, J. Hilgers, and J. Zavada. 1983. Specific selection of host cell glycoproteins during assembly of murine leukaemia virus and vesicular stomatitis virus: presence of thy-1 glycoprotein and absence of H-2, Pgp-1 and T-200 glycoproteins on the envelopes of these virus particles. J. Gen. Virol. 64:1241-1253. 3. Chakrabarti, L., M. Emerman, P. Tiollais, and P. Sonigo. 1989. The cytoplasmic domain of simian immunodeficiency virus transmembrane protein modulates infectivity. J. Virol. 63:43954403. 4. Chakrabarti, L., M. Guyader, M. Alizon, M. D. Daniel, R. C. Desrosiers, P. Tiollais, and P. Sonigo. 1987. Sequence of simian immunodeficiency virus from macaque and its relationship to other human and simian retroviruses. Nature (London) 328:543-
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