Functional Domains within the Human Immunodeficien
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病菌学杂志 2005年第6期
Department of Biochemistry and Molecular Biology
Department of Pediatrics, University of Southern California Keck School of Medicine
Saban Research Institute of Childrens Hospital Los Angeles, Los Angeles, California
ABSTRACT
Primate lentiviruses code for a protein that stimulates virus production. In human immunodeficiency virus type 1 (HIV-1), the activity is provided by the accessory protein, Vpu, while in HIV-2 and simian immunodeficiency virus it is a property of the envelope (Env) glycoprotein. Using a group of diverse retroviruses and cell types, we have confirmed the functional equivalence of the two proteins. However, despite these similarities, the two proteins have markedly different functional domains. While the Vpu activity is associated primarily with its membrane-spanning region, we have determined that the HIV-2 Env activity requires both the cytoplasmic tail and ectodomain of the protein, with the membrane-spanning domain being less important. Within the Env cytoplasmic tail, we further defined the necessary sequence as a membrane-proximal tyrosine-based motif. Providing the two Env regions separately as distinct CD8 chimeric proteins did not increase virus release. This suggests that the two domains must be either contained within a single protein or closely associated within a multiprotein oligomer, such as the Env trimer, in order to function. Finally, we observed that wild-type levels of incorporation of the HIV-2 Env into budding viruses were not required for this activity.
INTRODUCTION
It is widely acknowledged that human immunodeficiency virus (HIV) pathology results from the continuous, high-level production of viral particles over an extended period. Consequently, therapeutic interventions that reduce virus yield or infectivity could prolong, perhaps indefinitely, the period before AIDS develops. Recently, there has been considerable progress in our understanding of how HIV assembles and is released from host cells, and in particular the role played by the late (L)-domain sequences in Gag (25, 72). These regions contain protein recognition motifs that enable viruses to recruit components of the cellular vacuolar protein sorting (VPS) pathway and thereby mediate release. In the absence of an L domain, viral particles appear to be defective at "pinching off" and remain tethered to the cell surface (17).
Primate lentiviruses also require the action of an additional protein to ensure high-level production of viral particles. For HIV type 1 (HIV-1), this function is provided by the accessory protein Vpu (69), while for HIV-2 and simian immunodeficiency virus (SIV), which in general do not code for Vpu, the functional homologue is the Env glycoprotein (9, 35, 57). Although both L domains and Vpu/HIV-2 Env contribute to increased virus production, cell type differences in their activity suggest that the two processes are independent (66).
Vpu is an 81-residue class I integral membrane protein that can homo-oligomerize. Its N-terminal hydrophobic domain provides a membrane anchor, while the C terminus is a hydrophilic cytoplasmic tail. In common with the other HIV-1 accessory proteins, Vpu exhibits more than one activity (reviewed in reference 11). In addition to augmenting virus release, it also plays a role in removing CD4 from the surface of infected cells by degrading CD4-gp160 complexes that form in the endoplasmic reticulum, promotes apoptosis of HIV-1-infected cells, and increases VCAM-1 expression on endothelial cells. Recently, it has been shown to have homology to the ion channel-forming protein TASK-1 and to disrupt the function of this protein through hetero-oligomer formation (34).
Mutational studies have shown that the different functions of Vpu segregate between the two domains of the protein. CD4 degradation is a property of the cytoplasmic tail of the protein, while enhancement of virus production maps to the membrane-spanning domain (62). There is speculation that the ability of the membrane-spanning region to form ion channels (65) or, alternatively, to disrupt the action of TASK-1 (34) may be important for its ability to stimulate virus release. However, despite a number of studies characterizing the conductive properties of this putative viral ion channel (23, 24, 41, 45, 65), the mechanism by which Vpu enhances virus production is largely unknown.
Still less is known about how the HIV-2 Env stimulates virus production, although it has been suggested that Vpu and HIV-2 Env may work by a similar mechanism (9). In support of this hypothesis, both HIV-2 Env and Vpu form homo-oligomeric structures (43, 56), and the functional domain of the HIV-2 Env appears to be the membrane-anchored TM subunit (8, 9). However, the more precise location of the functional domain(s) within the HIV-2 TM is unclear. One study identified a critical role for residue A598 in the extracellular domain of the TM of the HIV-2ROD10 isolate (8), but the mechanism by which this residue could influence the budding enhancing phenotype is unknown. Furthermore, there are conflicting reports on whether the cytoplasmic tail of HIV-2 Env is required (7, 57).
Interestingly, the expression of Vpu is known to augment the release of widely divergent retroviruses, including HIV-2, SIV, visna virus, and murine leukemia virus (MLV) (10, 29). This suggests that its target is a broadly acting cellular factor. It has been proposed that a dominant factor is present in certain human cell lines which restricts HIV-1 budding and which Vpu counteracts (29, 66), and recent studies with heterokaryons formed between simian and human cell lines support this hypothesis (71). However, despite the identification of at least two Vpu-interacting proteins (13, 34), the nature of this putative restriction factor remains elusive.
We have begun to analyze how the HIV-2 Env enhances virus production. Our studies reveal that two separate domains of Env are required, a conserved Y-X-X-hydrophobic (YXX) motif in the membrane proximal part of the cytoplasmic tail, and a less well defined region in the ectodomain of the protein. The YXX motif is necessary, but not sufficient, to stimulate budding and can also be provided by the HIV-1 Env tail or an unrelated cytoplasmic domain engineered to contain such a motif. Finally, complementation studies demonstrated that these two regions could not be provided in trans by two separate molecules, suggesting a functional interaction.
MATERIALS AND METHODS
Cell lines. HeLa, Cos-7, and 293T cells were obtained from the American Type Culture Collection and maintained in Dulbecco's modified Eagle medium (DMEM; Cellgro, Herndon, Va.) supplemented with 10% fetal calf serum (FCS) (Gemini, Woodland, Calif.). 3T3-T4-CXCR4 cells, Ghost [3] X4/R5 cells and HeLa-CD4+ cells were obtained from the National Institutes of Health AIDS Research and Reference Reagent Program (ARRRP) from reagents deposited by Dan Littman, Vineet KewalRamani, and Bruce Chesebro. 3T3-T4-CXCR4 cells were maintained in DMEM plus 10% FCS, Ghost [3] X4/R5 cells were maintained in DMEM plus 10% FCS supplemented with 1 μg of puromycin/ml, 100 μg of hygromycin/ml, and 500 μg of G418/ml (Sigma) and HeLa-CD4+ cells were maintained in RPMI (Cellgro) supplemented with 10% FCS and 1 mg of G418/ml.
Plasmids. pHIV-1-pack expresses HIV-1 Gag-Pol and Rev. It was derived from construct pCMVR8.91 (74) by mutation of the Tat start codon and replacement of the plasmid backbone sequences with those in plasmid pMDLg/p (21). Plasmid pCgp expresses the Moloney MLV Gag-Pol (31), and plasmid EIAVUK is a proviral clone of equine infectious anemia virus (EIAV) (38).
Env expression plasmids were generated by cloning the coding sequence of the protein into the multiple cloning site of the immediate-early cytomegalovirus (CMV) promoter expression plasmid pSA91. The HIV Env proteins used were derived from proviral clones of HIV-1BH10, HIV-2ROD10 (provided by Mike Malim), HIV-2ST (obtained from the ARRRP, deposited by Beatrice Hahn and George Shaw), and HIV-2ROD14 (kindly provided by Stephan Bour).
The Vpu expression plasmid, pCMV-Vpu, was generated by PCR cloning the Vpu gene from HIV-1NL4-3 into the EcoRI site within the CMV expression plasmid CEEEnv (1). Plasmid pCMV-CD8 contains human CD8 derived from plasmid pT8F1 (ARRRP, from Richard Axel) cloned into plasmid pSA91.
Generation of vector particles and Western blot analysis. HeLa cells plated in a 10-cm dish and grown to 80 to 90% confluence were transiently transfected with the appropriate plasmids by using 30 μl of Lipofectamine 2000 (Invitrogen, Carlsbad, Calif.) in serum-free DMEM and incubated for 4 h, and then the medium was replaced with DMEM plus 10% FCS. For retroviral vector particles, the plasmids used were 10 μg of pCgp and 1 μg of the retroviral vector pCnBg (30), which expresses lacZ and neo; for lentiviral vectors, the plasmids used were 10 μg of pHIV-1-pack and 1 μg of the lentiviral vector pSMPU-MND-nlacZ, which expresses a nucleus-localized form of LacZ (M. Barcova and P. M. Cannon, unpublished data). EIAV particles were generated using 10 μg of pEIAVUK. Expression plasmids for the various Env proteins or Vpu were used at 2.5 μg per plate.
Vector particles were harvested 36 h posttransfection from the supernatants of the transfected HeLa cells and filtered through 0.45-μm-pore-size filters. The filtrate was overlaid on a cushion of 2 ml of 20% (mass/vol) sucrose and centrifuged at 4°C for 2 h at 30,000 rpm with an SW41 rotor (Beckman Instruments, Inc., Palo Alto, Calif.). The remaining HeLa cells were washed with phosphate-buffered saline (PBS), dissociated with trypsin-EDTA (Sigma, St. Louis, Mo.), pelleted by a brief centrifugation in an Eppendorf Microfuge, and washed with PBS, and the cell pellets were lysed in 100 μl of lysis buffer (20 mM Tris-HCl [pH 7.5], 1% Triton X-100, 0.05% sodium dodecyl sulfate, 5 mg of sodium deoxycholate/ml, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride [Sigma]) at 4°C for 10 min. Following centrifugation in an Eppendorf Microfuge at 16,000 x g for 10 min, the cleared supernatants were collected. Viral pellets and lysate samples were diluted 1:1 in 2 x sodium dodecyl sulfate gel loading buffer (Novex, San Diego, Calif.) plus 10% 2-mercaptoethanol, boiled for 10 min, and electrophoresed in 14% polyacrylamide Tris-glycine gels (Novex). The proteins were transferred to an Immobilon P polyvinylidene fluoride transfer membrane (Millipore Corp., Bedford, Mass.) and blocked overnight at 4°C with blocking buffer (5% dried milk in 0.01 M PBS [pH 7.4]-0.24% Tween 20).
For the detection of specific proteins, the membranes were cut into two strips that separately contained the HIV Env glycoproteins and the vector Gag proteins. The capsid (CA) protein from the MLV-based retrovirus vectors was detected by using a goat anti-Rauscher MLV p30 antiserum (Quality Biotech, Camden, N.J.) at a 1:10,000 dilution, and the HIV-1 CA protein was detected by using mouse anti-p24 monoclonal antibody 183-H12-5C (ARRRP, from Bruce Chesebro and Kathy Wehrly) at a 1:3,000 dilution. EIAV CA was detected with a monoclonal antibody against p26 at a 1:1,000 dilution (15). The surface (SU) subunit of the HIV Env proteins was detected by using a rabbit polyclonal serum against the HIV-2ST gp120 (ARRRP, from Raymond Sweet) at a 1:5,000 dilution. The secondary antibodies used were horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G (IgG) (1:10,000) (Pierce, Rockford, Ill.) and horseradish peroxidase-conjugated goat anti-rabbit IgG (1:10,000) (Pierce). Specific proteins were visualized with the enhanced chemiluminescence detection system (Amersham International plc., Arlington Heights, Ill.).
Exposed and developed films were scanned with a UMAX Powerlook II scanner, and bands were quantified with the public-domain image analysis software NIH ImageJ. A dilution series of protein standards was also analyzed to ensure that the bands that were quantified remained within the linear range for analysis.
Heterokaryon analysis. Cos-7 and HeLa cells plated in 10-cm culture dishes at 80 to 90% confluence were transiently transfected with 10 μg of pHIV-1-pack, together with either 2.5 μg of an HIV-2 Env plasmid or 5 μg of pCMV-Vpu, using 30 μl of Lipofectamine 2000. Twenty-four hours posttransfection, the medium was replaced and cells were stained and processed for cell fusion assays, essentially as described elsewhere (71). Briefly, cells were stained by incubation for 45 min at 37°C in growth medium containing 1 μM CellTracker green CMFDA (5-chloromethylfluorecein diacetate; Molecular Probes, Eugene, Oreg.) for Cos-7 cells or 10 μM CellTracker orange CMTMR [5-(and 6)-(4-chloromethyl(benzoyl)amino} tetramethylrhodamine; Molecular Probes] for HeLa cells. The cells were then washed with PBS and incubated for an additional 30 min to 4 h in DMEM plus 10% FCS at 37°C.
The labeled Cos-7 and HeLa cells were harvested by cell dissociation buffer and fused by the addition of polyethylene glycol (PEG) with a molecular mass of 3,000 Da (Sigma). One x 107 cells of each type were mixed and pelleted by low-speed centrifugation (1,000 x g), the supernatants were removed, and the pellets were loosened by tapping. One milliliter of 50% PEG in PBS plus 2% glucose was added dropwise, with gentle mixing, and the mixed cells were incubated for 90 s at room temperature, followed by slowly adding 1 ml of PBS and then incubating for an additional 60 s. Three milliliters of PBS plus 2% FCS was then added slowly, and the cells were pelleted by low-speed centrifugation. The cells were then washed twice with PBS plus 2% FCS to remove the PEG, resuspended in DMEM plus 20% FCS, and incubated overnight at 37°C prior to sorting for the doubly labeled population. Cell sorting was performed in the Childrens Hospital Los Angeles fluorescence-activated cell sorting (FACS) facility using a FACS DIVA (Becton Dickinson, San Jose, Calif.) with an excitation laser frequency of 488 nm and emission detected at 525 nm. The sorted cells were plated in 10-cm dishes, and 24 to 48 h later, both cell lysates and supernatants were harvested and processed for Western blot analysis.
Cell surface expression. HeLa cells plated in 10-cm tissue culture dishes and grown to 80 to 90% confluence were transfected with 2.5 μg of an Env expression plasmid, using 30 μl of Lipofectamine 2000. Twenty-four hours posttransfection, the cells were harvested with enzyme-free cell dissociation buffer (Sigma) and washed with PBS. The cells were incubated with a 1:500 dilution of the rabbit polyclonal anti-gp120 serum or a 1:250 dilution of monoclonal antibody against CD8 (Beckman Coulter, Miami, Fla.) in PBS supplemented with 10% goat serum (PBSG) for 1 h at 4°C. The cells were washed in PBSG and then incubated at 4°C for 1 h in a 1:200 dilution of fluorescein isothiocyanate-labeled goat anti-rabbit immunoglobulin G (IgG) (Pierce) or a 1:200 dilution of fluorescein isothiocyanate-labeled goat anti-mouse IgG (Pierce) diluted in PBSG. Following a final wash with PBSG, the cells were resuspended in 4% paraformaldehyde in PBS, and samples were analyzed by flow cytometry using a FACScan (Becton Dickinson).
Cell-cell fusion assay. 293T cells (2 x 106) were plated in a 60-mm tissue culture dish marked with 2- by 2-mm grids (Corning Glass Works, Corning, N.Y.) and transfected with 1 μg of Env expression plasmid by calcium phosphate transfection as described previously (67). Twenty-four hours later, 4 x 106 Ghost [3] X4/R5 cells were added to the transfected 293T cells. After incubation for an additional 18 h, the cells were fixed and stained with 1% methylene blue in methanol. The number of nuclei recruited into syncytia (fused cell masses containing three or more nuclei) in a 12-mm2 area was determined under a light microscope.
Lentiviral vector titer determination. Lentiviral vector particles were produced by transient transfection of 293T cells by calcium phosphate precipitation, essentially as described previously (16). The plasmids used were 10 μg of pHIV-1-pack and 10 μg of the transfer vector pSMPU-MND-nlacZ cotransfected with 10 μg of the appropriate HIV Env expression plasmid. Vector supernatants were collected 48 h posttransfection and filtered through a 0.45-μm filter. Titers were determined on Ghost [3] X4/X5 or HeLa-CD4+ cells by staining for ?-galactosidase expression as previously described (42).
RESULTS
Vpu and HIV-2 Env enhance the production of diverse retrovirus particles. It was previously reported that both Vpu and the HIV-2 Env can stimulate the production of HIV-1 particles (9, 35, 57, 69). For our studies, we used a minimal HIV-1 construct that expresses only HIV-1 Gag-Pol and Rev (pHIV-1-pack). HeLa cells were transfected with pHIV-1-pack, together with expression vectors for either Vpu, HIV-2ROD10 Env, or HIV-1BH10 Env (as a negative control). Western blot analysis confirmed that virus production was increased about four- to sixfold by either Vpu or HIV-2 Env, in agreement with previous reports, and that the effect was at the level of virus budding and/or release (Fig. 1).
It has also been reported that Vpu can stimulate the production of heterologous particles from MLV and visna virus (29). We therefore examined whether Vpu or HIV-2 Env could increase the production of MLV-based retroviral vector particles or an EIAV proviral clone. Western analysis revealed that both proteins could enhance the production of MLV and EIAV particles to the same extent as HIV-1 production, while the HIV-1 Env did not and actually inhibited budding for EIAV (Fig. 1). Taken together, these results demonstrate that both Vpu and HIV-2 Env can enhance the production of diverse retroviruses and lentiviruses, suggesting that these proteins are influencing a general cellular pathway and not acting through a specific HIV target.
Common cell type specificity in the action of Vpu and HIV-2 Env. Cell type differences in the ability of Vpu to stimulate virus production have been reported. For example, Vpu increases HIV-1 production from HeLa and Hep-2 cells, various T-cell lines, primary blood mononuclear cells, and macrophages (2, 7, 9, 10, 14, 18, 23, 29, 51, 57, 60, 64, 71) while having no effect on virus yield from various simian cell lines (27, 66, 71). To address whether Vpu and HIV-2 Env act on a common pathway, we examined their ability to stimulate virus budding from HeLa and Cos-7 cells, which are examples of Vpu-responsive and nonresponsive cell lines, respectively. Similar to Vpu, we observed that the HIV-2 Env stimulated production only from HeLa cells (Fig. 2A). Furthermore, we noted that HeLa cells had lower baseline levels of virus production than Cos-7 cells (Fig. 2A [compare levels of virus particles released into supernatant with those in cell lysates]). These observations are in agreement with the hypothesis that certain human cell lines can restrict HIV-1 production by a mechanism that is counteracted by Vpu (29, 66, 71).
To test this hypothesis further, we labeled HeLa and Cos-7 cells with different vital dyes, generated both homologous and heterologous PEG-induced heterokaryons, and examined virus production from FACS-sorted populations of heterokaryons. As before, we measured both baseline levels of virus production (Fig. 2B) and responsiveness to Vpu or HIV-2 Env (Fig. 2C). In agreement with the model of a dominant restriction factor in HeLa cells, we found that the HeLa-Cos-7 fusions exhibited a HeLa-like phenotype, with lower baseline levels of virus production that could be increased by either Vpu or HIV-2 Env. These data therefore suggest that both Vpu and HIV-2 Env can counteract a putative HeLa cell restriction factor.
Essential role for a membrane-proximal YXX in the cytoplasmic tail of HIV-2 Env. Since the functional domains of the HIV-2 Env required for enhanced virus production have not been fully determined, we began a systematic analysis of the protein. Conflicting reports have appeared in the literature regarding the importance of the cytoplasmic tail in this process. For example, Ritter et al. (57) saw no enhancement with a truncated form of the HIV-2ST Env that contained 17 amino acids in its cytoplasmic tail and concluded therefore that the tail was essential. In contrast, a later study by Bour et al. (7) reported activity from a version of the HIV-2ROD10 Env with 17 amino acids, leading those authors to conclude that the cytoplasmic tail was not required.
To address these discrepancies, we constructed a series of derivatives of the HIV-2ROD10 Env with either a full-length tail or truncated to 16, 9, or 6 amino acids (Fig. 3A). All of the truncation mutants were expressed normally in HeLa cells, as detected by Western blot analysis, and were detected on the cell surface by FACS analysis. In addition, the mutants were fully functional in cell-cell fusion assays and were capable of transducing HeLa-CD4 cells when incorporated into lentiviral vector particles (data not shown). By examining the abilities of these proteins to stimulate both lentiviral and retroviral vector particle production, we identified an essential role for the residues between position 6 and 9 of the tail (Fig. 3B). Examination of this region revealed a YXX motif (YRPV). Such motifs play many roles in intracellular signaling and trafficking events, with the tyrosine residue being absolutely required (5). Subsequent mutation of the tyrosine to an alanine (Y707A) demonstrated that this single substitution was sufficient to abolish the effect on budding for both HIV-1 and MLV particles. Our data therefore demonstrate that the membrane-proximal YXX motif of the HIV-2 Env cytoplasmic tail is essential for this process.
Several studies have demonstrated that the activity of YXX motifs can be influenced by the identity of other amino acids, both within and upstream of the motif (4, 5, 6). We noted that the glycine residue upstream of the tyrosine is highly conserved among lentiviral Env glycoproteins, and therefore we generated point mutants of this residue and examined their properties. Although these substitutions had no effects on Env expression, fusion ability, incorporation into HIV-1 particles, or lentiviral vector titer (data not shown), the mutants had a significantly diminished ability to stimulate budding (Fig. 3B). Finally, we observed the same pattern of activity when coexpressing this panel of Env proteins with MLV vector particles.
HIV-2ST Env enhances the release of HIV-1 particles and is similarly dependent on the cytoplasmic YXX motif. Previously, Ritter et al. (57) reported that an HIV-2ST Env protein with a tail truncated to 17 amino acids (16 native residues plus an additional C-terminal serine) was unable to enhance budding. Since the HIV-2ST Env used in their studies contains the same GYRPV motif as the HIV-2ROD10 Env used in our analyses, and this truncation leaves the motif intact, we were surprised by this finding. However, it is possible that other regions of the HIV-2ST protein account for these discrepancies. Accordingly, we repeated our analyses using both the full-length HIV-2ST Env and the same set of truncation mutants that we used to study the HIV-2ROD10 Env (Fig. 4A).
We first confirmed that all the HIV-2ST Env constructs were expressed normally, were competent in cell-cell fusion assays, and were capable of producing titers when incorporated into lentiviral vectors (data not shown). Coexpression of the full-length HIV-2ST Env with pHIV-1-pack resulted in an enhancement of particle budding, as expected from previous studies (57) (Fig. 4B). However, in contrast to these previous studies, we observed that Env proteins with a 16-residue cytoplasmic domain retained the ability to enhance viral particle release, recapitulating our observations with HIV-2ROD10 Env. To rule out the possibility that there were unexpected differences between the 16-residue tail we had generated and the 16+serine tail described by Ritter et al. (57), we also constructed this mutant and found it to be indistinguishable from the 16-residue tail with regard to budding enhancement (data not shown). Only a further truncation that removed a portion of the YXX motif (HIV-2ST 6ct) prevented the HIV-2ST Env from enhancing HIV-1 particle release (Fig. 4B). These experiments demonstrate that in our system, HIV-2ST Env enhances HIV-1 particle release with the same characteristics as HIV-2ROD10 Env.
An additional functional domain is present in the HIV-2 Env ectodomain. The finding that the YXX motif in the HIV-2 tail is an important determinant for stimulating virus production is at odds with the fact that the nonfunctional HIV-1 Env also has a similar motif at the same position (GYRPV in HIV-2ROD10 Env compared to GYSPL in HIV-1BH10 Env). To date, only one HIV-1 isolate, AD8, has been reported to contain an Env protein that can enhance viral budding (63). This suggests that either the HIV-1 motif is indeed nonfunctional or other determinants for budding enhancement are present within the HIV-2 Env.
To explore the possibility that a second functional domain exists within the HIV-2 Env, we made chimeric proteins between the HIV-1BH10 and HIV-2ROD10 Env proteins and examined their properties. Junctions were chosen at the interface between the cytoplasmic and membrane-spanning domains: E1M1T2 contains the extracellular and membrane-spanning domains of HIV-1BH10 Env and the cytoplasmic tail of HIV-2ROD10 Env, with E2M2T1 being the reciprocal chimera (Fig. 5A). We also made chimeras with junctions at the juxtaposition of the ectodomain and the membrane-spanning region (Fig. 5A). All chimeric proteins were expressed normally and functioned in cell-cell fusion assays and titer assays (data not shown).
Analysis of the properties of the chimeric proteins revealed that only E2M2T1 and E2M1T1 were able to enhance budding (Fig. 5B). These two proteins both contain an HIV-1 tail. A requirement for the YXX motif within the HIV-1 Env tail in these chimeras was determined by examining the properties of the truncation mutants E2M2T1-16ct and E2M2T1-6ct, which contain 16- and 6-residue cytoplasmic domains, respectively (Fig. 5B). Taken together, these data reveal that the HIV-1BH10 Env cytoplasmic tail is just as functional as the HIV-2 tail when fused to the rest of the HIV-2 Env protein but that an additional necessary functional domain(s) is present within the ectodomain of the HIV-2 Env. The requirement for the ectodomain agrees with observations by Bour et al. (8), who determined that the difference in activity between the functional HIV-2ROD10 Env and the related but nonfunctional HIV-2ROD14 protein was caused by a single substitution, A598T, in the ectodomain of gp41.
Role of the HIV-2 Env membrane-spanning domain. The functional domain of the Vpu protein that is important for enhancing virus release is the membrane-spanning region (62, 65). However, our studies with chimeric Env proteins suggested that the HIV-2 membrane-spanning region might not be critical, since the HIV-1 domain could substitute. We further examined the contribution of this region by replacing the HIV-2ROD10 domain with that of CD8, to create chimera E2M8T2 (Fig. 6A). Western analysis determined that this construct was expressed, although not as efficiently as HIV-2ROD10 Env (data not shown). In addition, E2M8T2 was found to be nonfunctional in cell-cell fusion assays and was unable to give rise to functional lentiviral vectors, which is in agreement with previous reports of the importance of the membrane-spanning region of HIV-1 Env for fusion activity (32, 50) (data not shown). Despite this defect, E2M8T2 retained the ability to enhance the release of HIV-1 particles (Fig. 6B), albeit only about half as efficiently as the full-length HIV-2ROD10 Env. Taken together, these findings support the notion that the specific HIV-2 membrane-spanning domain is not absolutely required to enhance particle budding, which suggests that the mechanism by which HIV-2 Env enhances virus release may be different from that of Vpu.
The cytoplasmic and extracellular domains of HIV-2 Env cannot function in trans. So far, our data have shown that both the YXX-containing cytoplasmic tail and the extracellular domain of the HIV-2 Env are required in order to enhance virus production. Next, we asked whether providing these two domains on separate molecules could still stimulate virus production, which would indicate independent activities. For these studies we used the cell surface antigen CD8, which does not affect HIV-1 budding and assembly and which has been used in chimeric proteins to study aspects of retroviral Env function (3). We constructed chimeric proteins between HIV-2ROD10 Env and CD8 that separated the two Env domains between two proteins (Fig. 6A). Both E2M2T8 and E8M8T2 were expressed at the cell surface, as determined by FACS analysis using specific antibodies against the HIV-2 Env or CD8 ectodomain (data not shown). In addition, E2M2T8 was functional in cell-cell fusion assays and could be incorporated into lentiviral vectors to give transduction (data not shown). As expected, neither E2M2T8 nor E8M2T2 alone could increase the release of virus particles when coexpressed with pHIV-1-pack (Fig. 6B and 6C). Interestingly, coexpression of the two chimeric proteins also did not result in enhancement (Fig. 6C).
A limitation of the CD8-chimera experiments is that we cannot rule out the possibility that the chimeric proteins do not properly present the individual Env functional domains. In addition, we are not able to distinguish between the possibilities that the two functional domains need to be present in cis on the same molecule in order to function or that they simply need to be present within a higher-order complex, such as an Env oligomer, which the two CD8 chimeras may not reproduce. To address these issues, we also attempted to coexpress Env mutants that are defective in each of the two domains, in order to examine their ability to complement in trans. In a similar approach, we have previously analyzed mutations in distinct functional domains in the MLV Env protein that are essential for virus-cell fusion. These studies revealed that while it is possible for certain combinations of mutations to restore activity, not all combinations can, leading us to suggest that certain domains of the protein needed to be physically linked in cis on the same molecule in order to function (73).
First, we examined the effects of expression of the panel of mutants on wild-type HIV-2ROD10 Env activity. We noted that over the range of DNA concentrations used in these analyses, the stimulation of virus release by the HIV-2ROD10 Env was somewhat dose responsive (Fig. 7). Next, we observed that while coexpression of HIV-2ROD10 Y707A, HIV-2ROD14, or E2M2T8 with the wild-type Env decreased the extent of particle budding, the effects were not strongly trans dominant. When excess wild-type Env was present at a ratio of 4:1, no significant inhibition was observed, and even at a 1:1 ratio, partial activity was retained. Only with a 4:1 excess of the mutant proteins was budding enhancement abolished. These results indicate that although these mutations are inhibitory, they do not exhibit a strong trans-dominant effect, as has been seen with certain other Env mutants (73) and which would be expected if a single defective monomer in an Env trimer could inactive the whole oligomer.
Finally, we examined the abilities of various mutants to trans complement when coexpressed with other defective proteins. The combinations of tail-defective and ectodomain-defective proteins that we examined are listed in Table 1. Interestingly, none of the combinations we tested restored function. This suggests either that three wild-type copies of each domain must be present in an Env trimer for activity (which would contradict the finding that none of the mutants are strongly trans dominant) or that the two separate regions of the HIV-2 Env need to be present in cis within a single monomer. Another possibility that we cannot rule out is that the tail-defective mutants without a YXX motif may not be trafficked to the same place as the wild-type protein and are therefore unable to complement.
Enhancement of particle release by HIV-2 Env does not require efficient Env incorporation into virions. We have already shown that the fusion activity of the HIV-2 Env is not necessary for the budding enhancement function, since the E2M8T2 chimera is unable to promote cell-cell fusion or transduction, yet it increases virus production. We next asked whether wild-type levels of incorporation of the HIV-2 Env into viral particles were required in order to enhance release. Of note, it is known that Vpu is not incorporated into HIV-1 particles.
To perform these studies, we took advantage of the fact that full-length HIV Env proteins are inhibited in their ability to be incorporated into MLV particles, presumably because their large cytoplasmic tail cannot be accommodated by the physical constraints of an MLV particle, while truncating the tail promotes incorporation (33, 36, 37, 44, 61). Using virions produced in 293T cells (where the higher levels of particles make it easier to detect even low levels of HIV-2 Env incorporation into MLV particles), we determined that this is indeed the case, that the 6ct, 9ct, and 16ct truncated Env proteins are incorporated at much higher levels than full-length HIV-2 Env (Fig. 8), and that these relationships are also preserved in HeLa cells (data not shown). However, comparison with the budding enhancement data (Fig. 3B) suggests that Env incorporation levels do not correlate with the degree of budding enhancement observed, suggesting that either only low levels of Env are sufficient to induce budding or incorporation is not required at all for this activity. Interestingly, we also observed that neither HIV-2ROD10 Env nor Vpu is capable of influencing particle release from 293T cells (Fig. 8), suggesting that these cells are more like Cos-7 cells than HeLa cells and demonstrating that not all human cells are HIV-2 Env/Vpu responsive.
DISCUSSION
Using HIV-1, MLV, and EIAV particles, we have demonstrated the equivalent roles played by the HIV-1 Vpu protein and the HIV-2 Env protein in stimulating virus release. We also observed a common cell type specificity in their activities, since both proteins functioned in HeLa cells but not in Cos-7 or 293T cells and both were able to rescue virus release from heterokaryons formed between HeLa and Cos-7 cells. Together with the finding that neither protein needs to be incorporated into viral particles in order to perform this function, our observations suggest a cellular target for their action.
Despite these similarities, we have shown that markedly different functional domains are responsible for this activity in the Vpu and HIV-2 Env proteins. In Vpu, this function has been mapped to the membrane-spanning region (62). This domain is known to have pore-forming activity (65), which may directly relate to its ability to enhance virus release. In support of this hypothesis, several other viral proteins that promote virus release also contain ion channel activity in their membrane-spanning regions, including influenza virus M2 and the alphavirus 6K protein (reviewed in reference 28). Alternatively, it has been suggested that Vpu's activity may derive from its ability to interfere with the action of a cellular ion channel protein, TASK-1, which is postulated to inhibit HIV release (34). How Vpu's inherent ion channel activity or its ability to inhibit TASK-1 could lead to increased virus release is unclear. It is possible that these activities induce a local effect at the site of virus budding, affecting either membrane fluidity and curvature or the activity of a host protein (11, 70). Alternatively, the mostly intracellular location of these proteins suggests that they could be acting to promote the release of a signal from an intracellular compartment to the site of viral assembly (28).
In marked contrast, our analysis of the HIV-2 Env protein revealed that the membrane-spanning region was not critical for its activity. Instead, two separate functional domains were identified, being present in the ectodomain and the cytoplasmic tail of the protein. In the cytoplasmic tail, we further defined the essential region as a membrane-proximal YXX motif. This sequence was previously assigned several functions in the HIV-1 life cycle. For example, it has been shown to interact with the medium chains of adaptor protein complexes, in particular AP-2 (4, 49), and to be responsible for the targeting of virus budding to basolateral membranes (39) or sites of cell-cell contact (19). In addition, studies with SIV have demonstrated that this motif is essential for in vivo pathogenicity (26).
Several possible mechanisms could account for the importance of the YXX motif in the enhancement of virus production. First, in common with the L domains present in retroviral Gag proteins, this motif could be acting to recruit cellular proteins important for virus budding. It is now well established that the VPS machinery drives retrovirus budding, with L domains from different viruses interacting with different VPS components (25, 46, 68, 72). It is therefore possible that the YXX motif also recruits VPS or endosomal components. In this regard, it is noteworthy that the L domain of the EIAV p9 also contains a YXX motif which has been shown to interact with AP-2 at sites of virus release (53), and this interaction is critical for its L-domain activity (B. Noble and P. M. Cannon, unpublished data). Alternatively, the tyrosine motif could act to displace an inhibitor of budding that is present in restrictive cell types such as HeLa but not in 293T or Cos-7 cells. Neither of these explanations alone predicts the lack of activity that was observed for the HIV-1 Env protein, which also contains a fully active cytoplasmic domain.
An alternate, although not mutually exclusive, explanation is that the YXX motif contributes indirectly to the HIV-2 Env activity by targeting the protein to a specific cellular location where it can perform this function. The site of HIV-1 assembly is known to be influenced by the Env protein, whose membrane localization is, in turn, determined by this tyrosine motif (39). Although the primary reason for such localized assembly has been suggested to be increased cell-to-cell spread of the virus (19, 52), these observations do not rule out the possibility that Env instead targets the viral core proteins to sites where assembly and release can occur more efficiently. The existence of specific "budding platforms" is an attractive idea, and it has been suggested that HIV-1 assembly occurs at discrete sites on the cell membrane that are derived from late endosomes (48) or that contain lipid rafts (47, 59) or other detergent-resistant membrane fractions (20).
Although the role of the YXX motif remains to be fully elucidated, our studies of HIV-2 Env clearly identified an additional functional region in the extracellular domain of the protein. The involvement of ectodomains of viral fusion proteins in virus assembly and release is not without precedent. Work on the vesicular stomatitis virus G protein (VSV-G) has shown that its membrane-proximal extracellular "stem" domain augments the production of VSV particles (58). The authors of that study speculated that the VSV-G stem might assist with the membrane curvature and fission events that are part of the budding process. Similar to VSV-G, the membrane-proximal regions of HIV Envs contain several bulky aromatics, and it is possible that this region in the HIV-2 protein helps to perturb the membrane at sites of budding.
Previously, Bour et al. (8) reported that an alanine at position 598 in the ectodomain of gp41 was critical for HIV-2 Env activity and accounted for the differences in activity between the functional (HIV-2ROD10) and nonfunctional (HIV-2ROD14) Env proteins. We have also observed a lack of activity with HIV-2ROD14 Env (Fig. 7) and have confirmed that substitution of residue 598 alone is sufficient to alter the phenotype of both the ROD10 and ROD14 isolates (data not shown). Since alanine is almost always conserved at this position in HIV-2 and SIV proteins, while not being present in HIV-1, it is tempting to assign this residue a direct role in the stimulation of virus budding. However, structural studies of part of the SIV TM protein have shown that A598 is located within a presumed cysteine-constrained loop at one end of the triple-stranded coiled-coil structure predicted to form upon fusion activation, in a region implicated in gp120-gp41 interactions (12). Consequently, it is also possible that this residue is indirectly involved in the enhancing activity of the HIV-2 and SIV Env proteins, through a global effect on the conformation of the protein.
An interesting question to address in our studies was whether the two separate Env determinants needed to be present on the same molecule in order to function. We approached this by coexpressing various molecules that only contained either a functional ectodomain region or a YXX-containing tail and looking for evidence of trans complementation. However, such studies can be difficult to interpret because mutant proteins can also have trans-dominant negative effects on the wild-type partner and thereby prevent complementation. Consequently, it will not be possible to achieve complementation between two proteins carrying separate mutations if all three of the individual Env monomers within a trimer are required to constitute an active domain. Despite these considerations, we were struck by the fact that we did not observe complementation between any of the combinations of mutants that we tested, even those we predicted to be fully capable of forming hetero-oligomers. This leads us to speculate that both domains may indeed be required to be present on the same monomer, which in turn suggests long-range influences within the Env protein and cross talk between its different domains. Such a finding is reminiscent of the integrin family, where interactions between the cytoplasmic and external domains of the proteins are involved in both outside-in and inside-out signaling events (reviewed in reference 40). In addition, the ability of the cytoplasmic tail of retroviral Env proteins to regulate the structure and function of the protein's ectodomain has been clearly demonstrated by our lab and others (1, 22, 54, 55, 73).
Despite the differences we have uncovered in the location of the functional domains of the HIV-2 Env and Vpu proteins, similarities in the size of the observed effect on virus budding and the cell type specificities of their activities remain. Interestingly, both proteins were able to overcome the dominant inhibitory factor that restricts budding from HeLa cells and HeLa/Cos-7 heterokaryons. Although the nature of this putative restriction factor is currently unknown, it is possible that Vpu and the HIV-2 Env have evolved to counteract this factor by using different strategies.
ACKNOWLEDGMENTS
We acknowledge the NIH AIDS Reference Reagent Program for several reagents that were vital to this project. We thank Stephan Bour and Klaus Strebel for providing reagents and generously communicating unpublished data, French Anderson and Marcus Thali for helpful discussion, and our colleagues at USC and CHLA.
This work was supported by Public Health Service grant CA-59318 and the Universitywide AIDS Research Program, ID03-CHLA-036.
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Department of Pediatrics, University of Southern California Keck School of Medicine
Saban Research Institute of Childrens Hospital Los Angeles, Los Angeles, California
ABSTRACT
Primate lentiviruses code for a protein that stimulates virus production. In human immunodeficiency virus type 1 (HIV-1), the activity is provided by the accessory protein, Vpu, while in HIV-2 and simian immunodeficiency virus it is a property of the envelope (Env) glycoprotein. Using a group of diverse retroviruses and cell types, we have confirmed the functional equivalence of the two proteins. However, despite these similarities, the two proteins have markedly different functional domains. While the Vpu activity is associated primarily with its membrane-spanning region, we have determined that the HIV-2 Env activity requires both the cytoplasmic tail and ectodomain of the protein, with the membrane-spanning domain being less important. Within the Env cytoplasmic tail, we further defined the necessary sequence as a membrane-proximal tyrosine-based motif. Providing the two Env regions separately as distinct CD8 chimeric proteins did not increase virus release. This suggests that the two domains must be either contained within a single protein or closely associated within a multiprotein oligomer, such as the Env trimer, in order to function. Finally, we observed that wild-type levels of incorporation of the HIV-2 Env into budding viruses were not required for this activity.
INTRODUCTION
It is widely acknowledged that human immunodeficiency virus (HIV) pathology results from the continuous, high-level production of viral particles over an extended period. Consequently, therapeutic interventions that reduce virus yield or infectivity could prolong, perhaps indefinitely, the period before AIDS develops. Recently, there has been considerable progress in our understanding of how HIV assembles and is released from host cells, and in particular the role played by the late (L)-domain sequences in Gag (25, 72). These regions contain protein recognition motifs that enable viruses to recruit components of the cellular vacuolar protein sorting (VPS) pathway and thereby mediate release. In the absence of an L domain, viral particles appear to be defective at "pinching off" and remain tethered to the cell surface (17).
Primate lentiviruses also require the action of an additional protein to ensure high-level production of viral particles. For HIV type 1 (HIV-1), this function is provided by the accessory protein Vpu (69), while for HIV-2 and simian immunodeficiency virus (SIV), which in general do not code for Vpu, the functional homologue is the Env glycoprotein (9, 35, 57). Although both L domains and Vpu/HIV-2 Env contribute to increased virus production, cell type differences in their activity suggest that the two processes are independent (66).
Vpu is an 81-residue class I integral membrane protein that can homo-oligomerize. Its N-terminal hydrophobic domain provides a membrane anchor, while the C terminus is a hydrophilic cytoplasmic tail. In common with the other HIV-1 accessory proteins, Vpu exhibits more than one activity (reviewed in reference 11). In addition to augmenting virus release, it also plays a role in removing CD4 from the surface of infected cells by degrading CD4-gp160 complexes that form in the endoplasmic reticulum, promotes apoptosis of HIV-1-infected cells, and increases VCAM-1 expression on endothelial cells. Recently, it has been shown to have homology to the ion channel-forming protein TASK-1 and to disrupt the function of this protein through hetero-oligomer formation (34).
Mutational studies have shown that the different functions of Vpu segregate between the two domains of the protein. CD4 degradation is a property of the cytoplasmic tail of the protein, while enhancement of virus production maps to the membrane-spanning domain (62). There is speculation that the ability of the membrane-spanning region to form ion channels (65) or, alternatively, to disrupt the action of TASK-1 (34) may be important for its ability to stimulate virus release. However, despite a number of studies characterizing the conductive properties of this putative viral ion channel (23, 24, 41, 45, 65), the mechanism by which Vpu enhances virus production is largely unknown.
Still less is known about how the HIV-2 Env stimulates virus production, although it has been suggested that Vpu and HIV-2 Env may work by a similar mechanism (9). In support of this hypothesis, both HIV-2 Env and Vpu form homo-oligomeric structures (43, 56), and the functional domain of the HIV-2 Env appears to be the membrane-anchored TM subunit (8, 9). However, the more precise location of the functional domain(s) within the HIV-2 TM is unclear. One study identified a critical role for residue A598 in the extracellular domain of the TM of the HIV-2ROD10 isolate (8), but the mechanism by which this residue could influence the budding enhancing phenotype is unknown. Furthermore, there are conflicting reports on whether the cytoplasmic tail of HIV-2 Env is required (7, 57).
Interestingly, the expression of Vpu is known to augment the release of widely divergent retroviruses, including HIV-2, SIV, visna virus, and murine leukemia virus (MLV) (10, 29). This suggests that its target is a broadly acting cellular factor. It has been proposed that a dominant factor is present in certain human cell lines which restricts HIV-1 budding and which Vpu counteracts (29, 66), and recent studies with heterokaryons formed between simian and human cell lines support this hypothesis (71). However, despite the identification of at least two Vpu-interacting proteins (13, 34), the nature of this putative restriction factor remains elusive.
We have begun to analyze how the HIV-2 Env enhances virus production. Our studies reveal that two separate domains of Env are required, a conserved Y-X-X-hydrophobic (YXX) motif in the membrane proximal part of the cytoplasmic tail, and a less well defined region in the ectodomain of the protein. The YXX motif is necessary, but not sufficient, to stimulate budding and can also be provided by the HIV-1 Env tail or an unrelated cytoplasmic domain engineered to contain such a motif. Finally, complementation studies demonstrated that these two regions could not be provided in trans by two separate molecules, suggesting a functional interaction.
MATERIALS AND METHODS
Cell lines. HeLa, Cos-7, and 293T cells were obtained from the American Type Culture Collection and maintained in Dulbecco's modified Eagle medium (DMEM; Cellgro, Herndon, Va.) supplemented with 10% fetal calf serum (FCS) (Gemini, Woodland, Calif.). 3T3-T4-CXCR4 cells, Ghost [3] X4/R5 cells and HeLa-CD4+ cells were obtained from the National Institutes of Health AIDS Research and Reference Reagent Program (ARRRP) from reagents deposited by Dan Littman, Vineet KewalRamani, and Bruce Chesebro. 3T3-T4-CXCR4 cells were maintained in DMEM plus 10% FCS, Ghost [3] X4/R5 cells were maintained in DMEM plus 10% FCS supplemented with 1 μg of puromycin/ml, 100 μg of hygromycin/ml, and 500 μg of G418/ml (Sigma) and HeLa-CD4+ cells were maintained in RPMI (Cellgro) supplemented with 10% FCS and 1 mg of G418/ml.
Plasmids. pHIV-1-pack expresses HIV-1 Gag-Pol and Rev. It was derived from construct pCMVR8.91 (74) by mutation of the Tat start codon and replacement of the plasmid backbone sequences with those in plasmid pMDLg/p (21). Plasmid pCgp expresses the Moloney MLV Gag-Pol (31), and plasmid EIAVUK is a proviral clone of equine infectious anemia virus (EIAV) (38).
Env expression plasmids were generated by cloning the coding sequence of the protein into the multiple cloning site of the immediate-early cytomegalovirus (CMV) promoter expression plasmid pSA91. The HIV Env proteins used were derived from proviral clones of HIV-1BH10, HIV-2ROD10 (provided by Mike Malim), HIV-2ST (obtained from the ARRRP, deposited by Beatrice Hahn and George Shaw), and HIV-2ROD14 (kindly provided by Stephan Bour).
The Vpu expression plasmid, pCMV-Vpu, was generated by PCR cloning the Vpu gene from HIV-1NL4-3 into the EcoRI site within the CMV expression plasmid CEEEnv (1). Plasmid pCMV-CD8 contains human CD8 derived from plasmid pT8F1 (ARRRP, from Richard Axel) cloned into plasmid pSA91.
Generation of vector particles and Western blot analysis. HeLa cells plated in a 10-cm dish and grown to 80 to 90% confluence were transiently transfected with the appropriate plasmids by using 30 μl of Lipofectamine 2000 (Invitrogen, Carlsbad, Calif.) in serum-free DMEM and incubated for 4 h, and then the medium was replaced with DMEM plus 10% FCS. For retroviral vector particles, the plasmids used were 10 μg of pCgp and 1 μg of the retroviral vector pCnBg (30), which expresses lacZ and neo; for lentiviral vectors, the plasmids used were 10 μg of pHIV-1-pack and 1 μg of the lentiviral vector pSMPU-MND-nlacZ, which expresses a nucleus-localized form of LacZ (M. Barcova and P. M. Cannon, unpublished data). EIAV particles were generated using 10 μg of pEIAVUK. Expression plasmids for the various Env proteins or Vpu were used at 2.5 μg per plate.
Vector particles were harvested 36 h posttransfection from the supernatants of the transfected HeLa cells and filtered through 0.45-μm-pore-size filters. The filtrate was overlaid on a cushion of 2 ml of 20% (mass/vol) sucrose and centrifuged at 4°C for 2 h at 30,000 rpm with an SW41 rotor (Beckman Instruments, Inc., Palo Alto, Calif.). The remaining HeLa cells were washed with phosphate-buffered saline (PBS), dissociated with trypsin-EDTA (Sigma, St. Louis, Mo.), pelleted by a brief centrifugation in an Eppendorf Microfuge, and washed with PBS, and the cell pellets were lysed in 100 μl of lysis buffer (20 mM Tris-HCl [pH 7.5], 1% Triton X-100, 0.05% sodium dodecyl sulfate, 5 mg of sodium deoxycholate/ml, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride [Sigma]) at 4°C for 10 min. Following centrifugation in an Eppendorf Microfuge at 16,000 x g for 10 min, the cleared supernatants were collected. Viral pellets and lysate samples were diluted 1:1 in 2 x sodium dodecyl sulfate gel loading buffer (Novex, San Diego, Calif.) plus 10% 2-mercaptoethanol, boiled for 10 min, and electrophoresed in 14% polyacrylamide Tris-glycine gels (Novex). The proteins were transferred to an Immobilon P polyvinylidene fluoride transfer membrane (Millipore Corp., Bedford, Mass.) and blocked overnight at 4°C with blocking buffer (5% dried milk in 0.01 M PBS [pH 7.4]-0.24% Tween 20).
For the detection of specific proteins, the membranes were cut into two strips that separately contained the HIV Env glycoproteins and the vector Gag proteins. The capsid (CA) protein from the MLV-based retrovirus vectors was detected by using a goat anti-Rauscher MLV p30 antiserum (Quality Biotech, Camden, N.J.) at a 1:10,000 dilution, and the HIV-1 CA protein was detected by using mouse anti-p24 monoclonal antibody 183-H12-5C (ARRRP, from Bruce Chesebro and Kathy Wehrly) at a 1:3,000 dilution. EIAV CA was detected with a monoclonal antibody against p26 at a 1:1,000 dilution (15). The surface (SU) subunit of the HIV Env proteins was detected by using a rabbit polyclonal serum against the HIV-2ST gp120 (ARRRP, from Raymond Sweet) at a 1:5,000 dilution. The secondary antibodies used were horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G (IgG) (1:10,000) (Pierce, Rockford, Ill.) and horseradish peroxidase-conjugated goat anti-rabbit IgG (1:10,000) (Pierce). Specific proteins were visualized with the enhanced chemiluminescence detection system (Amersham International plc., Arlington Heights, Ill.).
Exposed and developed films were scanned with a UMAX Powerlook II scanner, and bands were quantified with the public-domain image analysis software NIH ImageJ. A dilution series of protein standards was also analyzed to ensure that the bands that were quantified remained within the linear range for analysis.
Heterokaryon analysis. Cos-7 and HeLa cells plated in 10-cm culture dishes at 80 to 90% confluence were transiently transfected with 10 μg of pHIV-1-pack, together with either 2.5 μg of an HIV-2 Env plasmid or 5 μg of pCMV-Vpu, using 30 μl of Lipofectamine 2000. Twenty-four hours posttransfection, the medium was replaced and cells were stained and processed for cell fusion assays, essentially as described elsewhere (71). Briefly, cells were stained by incubation for 45 min at 37°C in growth medium containing 1 μM CellTracker green CMFDA (5-chloromethylfluorecein diacetate; Molecular Probes, Eugene, Oreg.) for Cos-7 cells or 10 μM CellTracker orange CMTMR [5-(and 6)-(4-chloromethyl(benzoyl)amino} tetramethylrhodamine; Molecular Probes] for HeLa cells. The cells were then washed with PBS and incubated for an additional 30 min to 4 h in DMEM plus 10% FCS at 37°C.
The labeled Cos-7 and HeLa cells were harvested by cell dissociation buffer and fused by the addition of polyethylene glycol (PEG) with a molecular mass of 3,000 Da (Sigma). One x 107 cells of each type were mixed and pelleted by low-speed centrifugation (1,000 x g), the supernatants were removed, and the pellets were loosened by tapping. One milliliter of 50% PEG in PBS plus 2% glucose was added dropwise, with gentle mixing, and the mixed cells were incubated for 90 s at room temperature, followed by slowly adding 1 ml of PBS and then incubating for an additional 60 s. Three milliliters of PBS plus 2% FCS was then added slowly, and the cells were pelleted by low-speed centrifugation. The cells were then washed twice with PBS plus 2% FCS to remove the PEG, resuspended in DMEM plus 20% FCS, and incubated overnight at 37°C prior to sorting for the doubly labeled population. Cell sorting was performed in the Childrens Hospital Los Angeles fluorescence-activated cell sorting (FACS) facility using a FACS DIVA (Becton Dickinson, San Jose, Calif.) with an excitation laser frequency of 488 nm and emission detected at 525 nm. The sorted cells were plated in 10-cm dishes, and 24 to 48 h later, both cell lysates and supernatants were harvested and processed for Western blot analysis.
Cell surface expression. HeLa cells plated in 10-cm tissue culture dishes and grown to 80 to 90% confluence were transfected with 2.5 μg of an Env expression plasmid, using 30 μl of Lipofectamine 2000. Twenty-four hours posttransfection, the cells were harvested with enzyme-free cell dissociation buffer (Sigma) and washed with PBS. The cells were incubated with a 1:500 dilution of the rabbit polyclonal anti-gp120 serum or a 1:250 dilution of monoclonal antibody against CD8 (Beckman Coulter, Miami, Fla.) in PBS supplemented with 10% goat serum (PBSG) for 1 h at 4°C. The cells were washed in PBSG and then incubated at 4°C for 1 h in a 1:200 dilution of fluorescein isothiocyanate-labeled goat anti-rabbit immunoglobulin G (IgG) (Pierce) or a 1:200 dilution of fluorescein isothiocyanate-labeled goat anti-mouse IgG (Pierce) diluted in PBSG. Following a final wash with PBSG, the cells were resuspended in 4% paraformaldehyde in PBS, and samples were analyzed by flow cytometry using a FACScan (Becton Dickinson).
Cell-cell fusion assay. 293T cells (2 x 106) were plated in a 60-mm tissue culture dish marked with 2- by 2-mm grids (Corning Glass Works, Corning, N.Y.) and transfected with 1 μg of Env expression plasmid by calcium phosphate transfection as described previously (67). Twenty-four hours later, 4 x 106 Ghost [3] X4/R5 cells were added to the transfected 293T cells. After incubation for an additional 18 h, the cells were fixed and stained with 1% methylene blue in methanol. The number of nuclei recruited into syncytia (fused cell masses containing three or more nuclei) in a 12-mm2 area was determined under a light microscope.
Lentiviral vector titer determination. Lentiviral vector particles were produced by transient transfection of 293T cells by calcium phosphate precipitation, essentially as described previously (16). The plasmids used were 10 μg of pHIV-1-pack and 10 μg of the transfer vector pSMPU-MND-nlacZ cotransfected with 10 μg of the appropriate HIV Env expression plasmid. Vector supernatants were collected 48 h posttransfection and filtered through a 0.45-μm filter. Titers were determined on Ghost [3] X4/X5 or HeLa-CD4+ cells by staining for ?-galactosidase expression as previously described (42).
RESULTS
Vpu and HIV-2 Env enhance the production of diverse retrovirus particles. It was previously reported that both Vpu and the HIV-2 Env can stimulate the production of HIV-1 particles (9, 35, 57, 69). For our studies, we used a minimal HIV-1 construct that expresses only HIV-1 Gag-Pol and Rev (pHIV-1-pack). HeLa cells were transfected with pHIV-1-pack, together with expression vectors for either Vpu, HIV-2ROD10 Env, or HIV-1BH10 Env (as a negative control). Western blot analysis confirmed that virus production was increased about four- to sixfold by either Vpu or HIV-2 Env, in agreement with previous reports, and that the effect was at the level of virus budding and/or release (Fig. 1).
It has also been reported that Vpu can stimulate the production of heterologous particles from MLV and visna virus (29). We therefore examined whether Vpu or HIV-2 Env could increase the production of MLV-based retroviral vector particles or an EIAV proviral clone. Western analysis revealed that both proteins could enhance the production of MLV and EIAV particles to the same extent as HIV-1 production, while the HIV-1 Env did not and actually inhibited budding for EIAV (Fig. 1). Taken together, these results demonstrate that both Vpu and HIV-2 Env can enhance the production of diverse retroviruses and lentiviruses, suggesting that these proteins are influencing a general cellular pathway and not acting through a specific HIV target.
Common cell type specificity in the action of Vpu and HIV-2 Env. Cell type differences in the ability of Vpu to stimulate virus production have been reported. For example, Vpu increases HIV-1 production from HeLa and Hep-2 cells, various T-cell lines, primary blood mononuclear cells, and macrophages (2, 7, 9, 10, 14, 18, 23, 29, 51, 57, 60, 64, 71) while having no effect on virus yield from various simian cell lines (27, 66, 71). To address whether Vpu and HIV-2 Env act on a common pathway, we examined their ability to stimulate virus budding from HeLa and Cos-7 cells, which are examples of Vpu-responsive and nonresponsive cell lines, respectively. Similar to Vpu, we observed that the HIV-2 Env stimulated production only from HeLa cells (Fig. 2A). Furthermore, we noted that HeLa cells had lower baseline levels of virus production than Cos-7 cells (Fig. 2A [compare levels of virus particles released into supernatant with those in cell lysates]). These observations are in agreement with the hypothesis that certain human cell lines can restrict HIV-1 production by a mechanism that is counteracted by Vpu (29, 66, 71).
To test this hypothesis further, we labeled HeLa and Cos-7 cells with different vital dyes, generated both homologous and heterologous PEG-induced heterokaryons, and examined virus production from FACS-sorted populations of heterokaryons. As before, we measured both baseline levels of virus production (Fig. 2B) and responsiveness to Vpu or HIV-2 Env (Fig. 2C). In agreement with the model of a dominant restriction factor in HeLa cells, we found that the HeLa-Cos-7 fusions exhibited a HeLa-like phenotype, with lower baseline levels of virus production that could be increased by either Vpu or HIV-2 Env. These data therefore suggest that both Vpu and HIV-2 Env can counteract a putative HeLa cell restriction factor.
Essential role for a membrane-proximal YXX in the cytoplasmic tail of HIV-2 Env. Since the functional domains of the HIV-2 Env required for enhanced virus production have not been fully determined, we began a systematic analysis of the protein. Conflicting reports have appeared in the literature regarding the importance of the cytoplasmic tail in this process. For example, Ritter et al. (57) saw no enhancement with a truncated form of the HIV-2ST Env that contained 17 amino acids in its cytoplasmic tail and concluded therefore that the tail was essential. In contrast, a later study by Bour et al. (7) reported activity from a version of the HIV-2ROD10 Env with 17 amino acids, leading those authors to conclude that the cytoplasmic tail was not required.
To address these discrepancies, we constructed a series of derivatives of the HIV-2ROD10 Env with either a full-length tail or truncated to 16, 9, or 6 amino acids (Fig. 3A). All of the truncation mutants were expressed normally in HeLa cells, as detected by Western blot analysis, and were detected on the cell surface by FACS analysis. In addition, the mutants were fully functional in cell-cell fusion assays and were capable of transducing HeLa-CD4 cells when incorporated into lentiviral vector particles (data not shown). By examining the abilities of these proteins to stimulate both lentiviral and retroviral vector particle production, we identified an essential role for the residues between position 6 and 9 of the tail (Fig. 3B). Examination of this region revealed a YXX motif (YRPV). Such motifs play many roles in intracellular signaling and trafficking events, with the tyrosine residue being absolutely required (5). Subsequent mutation of the tyrosine to an alanine (Y707A) demonstrated that this single substitution was sufficient to abolish the effect on budding for both HIV-1 and MLV particles. Our data therefore demonstrate that the membrane-proximal YXX motif of the HIV-2 Env cytoplasmic tail is essential for this process.
Several studies have demonstrated that the activity of YXX motifs can be influenced by the identity of other amino acids, both within and upstream of the motif (4, 5, 6). We noted that the glycine residue upstream of the tyrosine is highly conserved among lentiviral Env glycoproteins, and therefore we generated point mutants of this residue and examined their properties. Although these substitutions had no effects on Env expression, fusion ability, incorporation into HIV-1 particles, or lentiviral vector titer (data not shown), the mutants had a significantly diminished ability to stimulate budding (Fig. 3B). Finally, we observed the same pattern of activity when coexpressing this panel of Env proteins with MLV vector particles.
HIV-2ST Env enhances the release of HIV-1 particles and is similarly dependent on the cytoplasmic YXX motif. Previously, Ritter et al. (57) reported that an HIV-2ST Env protein with a tail truncated to 17 amino acids (16 native residues plus an additional C-terminal serine) was unable to enhance budding. Since the HIV-2ST Env used in their studies contains the same GYRPV motif as the HIV-2ROD10 Env used in our analyses, and this truncation leaves the motif intact, we were surprised by this finding. However, it is possible that other regions of the HIV-2ST protein account for these discrepancies. Accordingly, we repeated our analyses using both the full-length HIV-2ST Env and the same set of truncation mutants that we used to study the HIV-2ROD10 Env (Fig. 4A).
We first confirmed that all the HIV-2ST Env constructs were expressed normally, were competent in cell-cell fusion assays, and were capable of producing titers when incorporated into lentiviral vectors (data not shown). Coexpression of the full-length HIV-2ST Env with pHIV-1-pack resulted in an enhancement of particle budding, as expected from previous studies (57) (Fig. 4B). However, in contrast to these previous studies, we observed that Env proteins with a 16-residue cytoplasmic domain retained the ability to enhance viral particle release, recapitulating our observations with HIV-2ROD10 Env. To rule out the possibility that there were unexpected differences between the 16-residue tail we had generated and the 16+serine tail described by Ritter et al. (57), we also constructed this mutant and found it to be indistinguishable from the 16-residue tail with regard to budding enhancement (data not shown). Only a further truncation that removed a portion of the YXX motif (HIV-2ST 6ct) prevented the HIV-2ST Env from enhancing HIV-1 particle release (Fig. 4B). These experiments demonstrate that in our system, HIV-2ST Env enhances HIV-1 particle release with the same characteristics as HIV-2ROD10 Env.
An additional functional domain is present in the HIV-2 Env ectodomain. The finding that the YXX motif in the HIV-2 tail is an important determinant for stimulating virus production is at odds with the fact that the nonfunctional HIV-1 Env also has a similar motif at the same position (GYRPV in HIV-2ROD10 Env compared to GYSPL in HIV-1BH10 Env). To date, only one HIV-1 isolate, AD8, has been reported to contain an Env protein that can enhance viral budding (63). This suggests that either the HIV-1 motif is indeed nonfunctional or other determinants for budding enhancement are present within the HIV-2 Env.
To explore the possibility that a second functional domain exists within the HIV-2 Env, we made chimeric proteins between the HIV-1BH10 and HIV-2ROD10 Env proteins and examined their properties. Junctions were chosen at the interface between the cytoplasmic and membrane-spanning domains: E1M1T2 contains the extracellular and membrane-spanning domains of HIV-1BH10 Env and the cytoplasmic tail of HIV-2ROD10 Env, with E2M2T1 being the reciprocal chimera (Fig. 5A). We also made chimeras with junctions at the juxtaposition of the ectodomain and the membrane-spanning region (Fig. 5A). All chimeric proteins were expressed normally and functioned in cell-cell fusion assays and titer assays (data not shown).
Analysis of the properties of the chimeric proteins revealed that only E2M2T1 and E2M1T1 were able to enhance budding (Fig. 5B). These two proteins both contain an HIV-1 tail. A requirement for the YXX motif within the HIV-1 Env tail in these chimeras was determined by examining the properties of the truncation mutants E2M2T1-16ct and E2M2T1-6ct, which contain 16- and 6-residue cytoplasmic domains, respectively (Fig. 5B). Taken together, these data reveal that the HIV-1BH10 Env cytoplasmic tail is just as functional as the HIV-2 tail when fused to the rest of the HIV-2 Env protein but that an additional necessary functional domain(s) is present within the ectodomain of the HIV-2 Env. The requirement for the ectodomain agrees with observations by Bour et al. (8), who determined that the difference in activity between the functional HIV-2ROD10 Env and the related but nonfunctional HIV-2ROD14 protein was caused by a single substitution, A598T, in the ectodomain of gp41.
Role of the HIV-2 Env membrane-spanning domain. The functional domain of the Vpu protein that is important for enhancing virus release is the membrane-spanning region (62, 65). However, our studies with chimeric Env proteins suggested that the HIV-2 membrane-spanning region might not be critical, since the HIV-1 domain could substitute. We further examined the contribution of this region by replacing the HIV-2ROD10 domain with that of CD8, to create chimera E2M8T2 (Fig. 6A). Western analysis determined that this construct was expressed, although not as efficiently as HIV-2ROD10 Env (data not shown). In addition, E2M8T2 was found to be nonfunctional in cell-cell fusion assays and was unable to give rise to functional lentiviral vectors, which is in agreement with previous reports of the importance of the membrane-spanning region of HIV-1 Env for fusion activity (32, 50) (data not shown). Despite this defect, E2M8T2 retained the ability to enhance the release of HIV-1 particles (Fig. 6B), albeit only about half as efficiently as the full-length HIV-2ROD10 Env. Taken together, these findings support the notion that the specific HIV-2 membrane-spanning domain is not absolutely required to enhance particle budding, which suggests that the mechanism by which HIV-2 Env enhances virus release may be different from that of Vpu.
The cytoplasmic and extracellular domains of HIV-2 Env cannot function in trans. So far, our data have shown that both the YXX-containing cytoplasmic tail and the extracellular domain of the HIV-2 Env are required in order to enhance virus production. Next, we asked whether providing these two domains on separate molecules could still stimulate virus production, which would indicate independent activities. For these studies we used the cell surface antigen CD8, which does not affect HIV-1 budding and assembly and which has been used in chimeric proteins to study aspects of retroviral Env function (3). We constructed chimeric proteins between HIV-2ROD10 Env and CD8 that separated the two Env domains between two proteins (Fig. 6A). Both E2M2T8 and E8M8T2 were expressed at the cell surface, as determined by FACS analysis using specific antibodies against the HIV-2 Env or CD8 ectodomain (data not shown). In addition, E2M2T8 was functional in cell-cell fusion assays and could be incorporated into lentiviral vectors to give transduction (data not shown). As expected, neither E2M2T8 nor E8M2T2 alone could increase the release of virus particles when coexpressed with pHIV-1-pack (Fig. 6B and 6C). Interestingly, coexpression of the two chimeric proteins also did not result in enhancement (Fig. 6C).
A limitation of the CD8-chimera experiments is that we cannot rule out the possibility that the chimeric proteins do not properly present the individual Env functional domains. In addition, we are not able to distinguish between the possibilities that the two functional domains need to be present in cis on the same molecule in order to function or that they simply need to be present within a higher-order complex, such as an Env oligomer, which the two CD8 chimeras may not reproduce. To address these issues, we also attempted to coexpress Env mutants that are defective in each of the two domains, in order to examine their ability to complement in trans. In a similar approach, we have previously analyzed mutations in distinct functional domains in the MLV Env protein that are essential for virus-cell fusion. These studies revealed that while it is possible for certain combinations of mutations to restore activity, not all combinations can, leading us to suggest that certain domains of the protein needed to be physically linked in cis on the same molecule in order to function (73).
First, we examined the effects of expression of the panel of mutants on wild-type HIV-2ROD10 Env activity. We noted that over the range of DNA concentrations used in these analyses, the stimulation of virus release by the HIV-2ROD10 Env was somewhat dose responsive (Fig. 7). Next, we observed that while coexpression of HIV-2ROD10 Y707A, HIV-2ROD14, or E2M2T8 with the wild-type Env decreased the extent of particle budding, the effects were not strongly trans dominant. When excess wild-type Env was present at a ratio of 4:1, no significant inhibition was observed, and even at a 1:1 ratio, partial activity was retained. Only with a 4:1 excess of the mutant proteins was budding enhancement abolished. These results indicate that although these mutations are inhibitory, they do not exhibit a strong trans-dominant effect, as has been seen with certain other Env mutants (73) and which would be expected if a single defective monomer in an Env trimer could inactive the whole oligomer.
Finally, we examined the abilities of various mutants to trans complement when coexpressed with other defective proteins. The combinations of tail-defective and ectodomain-defective proteins that we examined are listed in Table 1. Interestingly, none of the combinations we tested restored function. This suggests either that three wild-type copies of each domain must be present in an Env trimer for activity (which would contradict the finding that none of the mutants are strongly trans dominant) or that the two separate regions of the HIV-2 Env need to be present in cis within a single monomer. Another possibility that we cannot rule out is that the tail-defective mutants without a YXX motif may not be trafficked to the same place as the wild-type protein and are therefore unable to complement.
Enhancement of particle release by HIV-2 Env does not require efficient Env incorporation into virions. We have already shown that the fusion activity of the HIV-2 Env is not necessary for the budding enhancement function, since the E2M8T2 chimera is unable to promote cell-cell fusion or transduction, yet it increases virus production. We next asked whether wild-type levels of incorporation of the HIV-2 Env into viral particles were required in order to enhance release. Of note, it is known that Vpu is not incorporated into HIV-1 particles.
To perform these studies, we took advantage of the fact that full-length HIV Env proteins are inhibited in their ability to be incorporated into MLV particles, presumably because their large cytoplasmic tail cannot be accommodated by the physical constraints of an MLV particle, while truncating the tail promotes incorporation (33, 36, 37, 44, 61). Using virions produced in 293T cells (where the higher levels of particles make it easier to detect even low levels of HIV-2 Env incorporation into MLV particles), we determined that this is indeed the case, that the 6ct, 9ct, and 16ct truncated Env proteins are incorporated at much higher levels than full-length HIV-2 Env (Fig. 8), and that these relationships are also preserved in HeLa cells (data not shown). However, comparison with the budding enhancement data (Fig. 3B) suggests that Env incorporation levels do not correlate with the degree of budding enhancement observed, suggesting that either only low levels of Env are sufficient to induce budding or incorporation is not required at all for this activity. Interestingly, we also observed that neither HIV-2ROD10 Env nor Vpu is capable of influencing particle release from 293T cells (Fig. 8), suggesting that these cells are more like Cos-7 cells than HeLa cells and demonstrating that not all human cells are HIV-2 Env/Vpu responsive.
DISCUSSION
Using HIV-1, MLV, and EIAV particles, we have demonstrated the equivalent roles played by the HIV-1 Vpu protein and the HIV-2 Env protein in stimulating virus release. We also observed a common cell type specificity in their activities, since both proteins functioned in HeLa cells but not in Cos-7 or 293T cells and both were able to rescue virus release from heterokaryons formed between HeLa and Cos-7 cells. Together with the finding that neither protein needs to be incorporated into viral particles in order to perform this function, our observations suggest a cellular target for their action.
Despite these similarities, we have shown that markedly different functional domains are responsible for this activity in the Vpu and HIV-2 Env proteins. In Vpu, this function has been mapped to the membrane-spanning region (62). This domain is known to have pore-forming activity (65), which may directly relate to its ability to enhance virus release. In support of this hypothesis, several other viral proteins that promote virus release also contain ion channel activity in their membrane-spanning regions, including influenza virus M2 and the alphavirus 6K protein (reviewed in reference 28). Alternatively, it has been suggested that Vpu's activity may derive from its ability to interfere with the action of a cellular ion channel protein, TASK-1, which is postulated to inhibit HIV release (34). How Vpu's inherent ion channel activity or its ability to inhibit TASK-1 could lead to increased virus release is unclear. It is possible that these activities induce a local effect at the site of virus budding, affecting either membrane fluidity and curvature or the activity of a host protein (11, 70). Alternatively, the mostly intracellular location of these proteins suggests that they could be acting to promote the release of a signal from an intracellular compartment to the site of viral assembly (28).
In marked contrast, our analysis of the HIV-2 Env protein revealed that the membrane-spanning region was not critical for its activity. Instead, two separate functional domains were identified, being present in the ectodomain and the cytoplasmic tail of the protein. In the cytoplasmic tail, we further defined the essential region as a membrane-proximal YXX motif. This sequence was previously assigned several functions in the HIV-1 life cycle. For example, it has been shown to interact with the medium chains of adaptor protein complexes, in particular AP-2 (4, 49), and to be responsible for the targeting of virus budding to basolateral membranes (39) or sites of cell-cell contact (19). In addition, studies with SIV have demonstrated that this motif is essential for in vivo pathogenicity (26).
Several possible mechanisms could account for the importance of the YXX motif in the enhancement of virus production. First, in common with the L domains present in retroviral Gag proteins, this motif could be acting to recruit cellular proteins important for virus budding. It is now well established that the VPS machinery drives retrovirus budding, with L domains from different viruses interacting with different VPS components (25, 46, 68, 72). It is therefore possible that the YXX motif also recruits VPS or endosomal components. In this regard, it is noteworthy that the L domain of the EIAV p9 also contains a YXX motif which has been shown to interact with AP-2 at sites of virus release (53), and this interaction is critical for its L-domain activity (B. Noble and P. M. Cannon, unpublished data). Alternatively, the tyrosine motif could act to displace an inhibitor of budding that is present in restrictive cell types such as HeLa but not in 293T or Cos-7 cells. Neither of these explanations alone predicts the lack of activity that was observed for the HIV-1 Env protein, which also contains a fully active cytoplasmic domain.
An alternate, although not mutually exclusive, explanation is that the YXX motif contributes indirectly to the HIV-2 Env activity by targeting the protein to a specific cellular location where it can perform this function. The site of HIV-1 assembly is known to be influenced by the Env protein, whose membrane localization is, in turn, determined by this tyrosine motif (39). Although the primary reason for such localized assembly has been suggested to be increased cell-to-cell spread of the virus (19, 52), these observations do not rule out the possibility that Env instead targets the viral core proteins to sites where assembly and release can occur more efficiently. The existence of specific "budding platforms" is an attractive idea, and it has been suggested that HIV-1 assembly occurs at discrete sites on the cell membrane that are derived from late endosomes (48) or that contain lipid rafts (47, 59) or other detergent-resistant membrane fractions (20).
Although the role of the YXX motif remains to be fully elucidated, our studies of HIV-2 Env clearly identified an additional functional region in the extracellular domain of the protein. The involvement of ectodomains of viral fusion proteins in virus assembly and release is not without precedent. Work on the vesicular stomatitis virus G protein (VSV-G) has shown that its membrane-proximal extracellular "stem" domain augments the production of VSV particles (58). The authors of that study speculated that the VSV-G stem might assist with the membrane curvature and fission events that are part of the budding process. Similar to VSV-G, the membrane-proximal regions of HIV Envs contain several bulky aromatics, and it is possible that this region in the HIV-2 protein helps to perturb the membrane at sites of budding.
Previously, Bour et al. (8) reported that an alanine at position 598 in the ectodomain of gp41 was critical for HIV-2 Env activity and accounted for the differences in activity between the functional (HIV-2ROD10) and nonfunctional (HIV-2ROD14) Env proteins. We have also observed a lack of activity with HIV-2ROD14 Env (Fig. 7) and have confirmed that substitution of residue 598 alone is sufficient to alter the phenotype of both the ROD10 and ROD14 isolates (data not shown). Since alanine is almost always conserved at this position in HIV-2 and SIV proteins, while not being present in HIV-1, it is tempting to assign this residue a direct role in the stimulation of virus budding. However, structural studies of part of the SIV TM protein have shown that A598 is located within a presumed cysteine-constrained loop at one end of the triple-stranded coiled-coil structure predicted to form upon fusion activation, in a region implicated in gp120-gp41 interactions (12). Consequently, it is also possible that this residue is indirectly involved in the enhancing activity of the HIV-2 and SIV Env proteins, through a global effect on the conformation of the protein.
An interesting question to address in our studies was whether the two separate Env determinants needed to be present on the same molecule in order to function. We approached this by coexpressing various molecules that only contained either a functional ectodomain region or a YXX-containing tail and looking for evidence of trans complementation. However, such studies can be difficult to interpret because mutant proteins can also have trans-dominant negative effects on the wild-type partner and thereby prevent complementation. Consequently, it will not be possible to achieve complementation between two proteins carrying separate mutations if all three of the individual Env monomers within a trimer are required to constitute an active domain. Despite these considerations, we were struck by the fact that we did not observe complementation between any of the combinations of mutants that we tested, even those we predicted to be fully capable of forming hetero-oligomers. This leads us to speculate that both domains may indeed be required to be present on the same monomer, which in turn suggests long-range influences within the Env protein and cross talk between its different domains. Such a finding is reminiscent of the integrin family, where interactions between the cytoplasmic and external domains of the proteins are involved in both outside-in and inside-out signaling events (reviewed in reference 40). In addition, the ability of the cytoplasmic tail of retroviral Env proteins to regulate the structure and function of the protein's ectodomain has been clearly demonstrated by our lab and others (1, 22, 54, 55, 73).
Despite the differences we have uncovered in the location of the functional domains of the HIV-2 Env and Vpu proteins, similarities in the size of the observed effect on virus budding and the cell type specificities of their activities remain. Interestingly, both proteins were able to overcome the dominant inhibitory factor that restricts budding from HeLa cells and HeLa/Cos-7 heterokaryons. Although the nature of this putative restriction factor is currently unknown, it is possible that Vpu and the HIV-2 Env have evolved to counteract this factor by using different strategies.
ACKNOWLEDGMENTS
We acknowledge the NIH AIDS Reference Reagent Program for several reagents that were vital to this project. We thank Stephan Bour and Klaus Strebel for providing reagents and generously communicating unpublished data, French Anderson and Marcus Thali for helpful discussion, and our colleagues at USC and CHLA.
This work was supported by Public Health Service grant CA-59318 and the Universitywide AIDS Research Program, ID03-CHLA-036.
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