Recruitment of the Adaptor Protein 2 Complex by th
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病菌学杂志 2006年第6期
Department of Biochemistry and Molecular Biology
Department of Pediatrics, University of Southern California Keck School of Medicine, Los Angeles, California
Saban Research Institute of Childrens Hospital Los Angeles, Los Angeles, California
ABSTRACT
The envelope (Env) protein of human immunodeficiency virus type 2 (HIV-2) and the HIV-1 Vpu protein stimulate the release of retroviral particles from human cells that restrict virus production, an activity that we call the enhancement of virus release (EVR). We have previously shown that two separate domains in the HIV-2 envelope protein are required for this activity: a glycine-tyrosine-x-x-hydrophobic (GYxx) motif in the cytoplasmic tail and an unmapped region in the ectodomain of the protein. We here report that the cellular partner of the GYxx motif is the adaptor protein complex AP-2. The mutation of this motif or the depletion of AP-2 by RNA interference abrogated EVR activity and changed the cellular distribution of the Env from a predominantly punctate pattern to a more diffuse distribution. Since the L domain of equine infectious anemia virus (EIAV) contains a Yxx motif that interacts with AP-2, we used both wild-type and L domain-defective particles of HIV-1 and EIAV to examine whether the HIV-2 Env EVR function was analogous to L domain activity. We observed that the production of all particles was stimulated by HIV-2 Env or Vpu, suggesting that the L domain and EVR activities play independent roles in the release of retroviruses. Interestingly, we found that the cytoplasmic tail of the murine leukemia virus (MLV) Env could functionally substitute for the HIV-2 Env tail, but it did so in a manner that did not require a Yxx motif or AP-2. The cellular distribution of the chimeric HIV-2/MLV Env was significantly less punctate than the wild-type Env, although confocal analysis revealed an overlap in the steady-state locations of the two proteins. Taken together, these data suggest that the essential GYxx motif in the HIV-2 Env tail recruits AP-2 in order to direct Env to a cellular pathway or location that is necessary for its ability to enhance virus release but that an alternate mechanism provided by the MLV Env tail can functionally substitute.
INTRODUCTION
The assembly and budding of C-type retroviruses are primarily driven by the Gag protein, and virus-like particles form efficiently in the absence of any other retroviral gene products or the RNA genome (20). Several distinct regions in Gag are necessary for virus-like particle formation, including a membrane-targeting domain at the N terminus of MA, a multimerization domain present in CA, and a late or L domain that is necessary for "pinching off" budding viral particles. L domains are located at various positions within Gag and have also been found in other enveloped RNA viruses, where they function as protein interaction motifs that recruit components of the cell's class E vacuolar protein synthesis (Vps) and endocytic trafficking pathways (for reviews, see references 12 and 29). In mammalian cells, the class E Vps pathway is involved in the export of cargo and the budding of exosomes into multivesicular bodies (MVBs). The process of exosome formation is similar to that of retrovirus budding since it involves the outward curvature of a membrane away from the cytosol and a membrane fusion event to allow release of the vesicle into the lumen of the MVB.
Although the retroviral Env protein is not absolutely required for the assembly and release of viral particles, it is now increasingly appreciated that Env plays an active role in these events. For example, the human immunodeficiency virus type 1 (HIV-1) Env is able to target virus assembly to the basolateral membrane of polarized epithelial cells (26, 37) and to sites of cell-cell contact in infected monocytes and lymphocytes (9, 15). In both cases, a conserved Y-x-x-hydrophobic (Yxx) motif in the cytoplasmic tail of Env is necessary for this redirection (15, 27). The clustering of HIV-1 assembly at cell-cell junctions has previously been documented (18, 40), and recent evidence suggests that the interaction of cell-associated HIV-1 Env with its cellular receptor(s) on target cells leads to the formation of a synapse that recruits the host cell proteins that are necessary for HIV entry as well as the viral Gag and Env proteins (24).
In HIV-2, the Env protein has an even more dramatic effect on virus assembly and release through its ability to boost production from certain human cell types (6, 44). We refer to this activity as the enhancement of virus release (EVR). In HIV-1, the Env protein does not generally possess EVR activity and this function is provided, instead, by the accessory protein Vpu (52, 53). Like HIV-2, most simian immunodeficiency virus (SIV) strains do not code for Vpu (notable exceptions being HIV-1-like monkey isolates, such as SIVcpz) and the Env proteins from SIVmac239 and SIVmnd have also been reported to have enhancing activity (7, 23). These observations lead us to suggest that Vpu evolved in HIV-1 to take over the EVR function from the Env protein.
In vitro, EVRs typically boost steady-state levels of virus production from cell lines by 4- to 10-fold and this activity has been observed in various different human cell types, including HeLa and HEp-2 cells, various T-cell lines, primary blood mononuclear cells, and macrophages (2, 8, 6, 7, 10, 14, 17, 21, 39, 44, 45, 46, 54). EVRs are able to stimulate the budding of heterologous viral particles from human cells (1), and it has been suggested that they counteract a natural human restriction factor that acts to inhibit retrovirus budding (21, 47). In contrast, simian cells do not appear to restrict HIV budding and do not support EVR activity (19, 47). Analysis of the heterokaryons formed between human and simian cells suggests that human cells contain a dominant factor that restricts virus production and which the EVRs counteract (1, 54).
Our previous studies of the HIV-2 Env defined two functional domains in the protein that are required for EVR activity, a conserved GYxx motif in the membrane-proximal part of the cytoplasmic tail (GYPRV) and an unidentified region in the ectodomain of the protein (1). Yxx motifs are found in the cytosolic domains of a variety of transmembrane proteins, where they play central roles in protein sorting (for a review, see reference 5). In mammalian cells, they act predominately to signal rapid internalization from the cell surface through interactions with components of the cellular trafficking machinery, most notably the adaptor protein (AP) complexes. Although the Yxx tetrapeptide is the minimal motif conferring sorting information, the presence of a conserved upstream glycine residue is characteristic of a lysosomal targeting motif.
The analogous membrane-proximal GYxx motif in the HIV-1 Env cytoplasmic tail, which we have previously shown to be functionally equivalent to the HIV-2 sequence (1), promotes endocytosis of HIV-1 Env from the cell surface and interacts specifically with the AP-2 complex (4). We therefore hypothesized that such an interaction could function to target the HIV-2 Env to an intracellular location that was important for its EVR activity. In the present study, we determined that AP-2 is indeed necessary for the EVR activity of the HIV-2 Env. Furthermore, by analyzing the cellular distribution of both a nonfunctional mutant of Env and a non-AP-2-requiring chimeric protein containing the cytoplasmic domain of the murine leukemia virus (MLV) Env, we have begun to examine whether targeting to a distinct cellular location is necessary for EVR activity.
MATERIALS AND METHODS
Cell lines and plasmid constructs. HeLa cells (obtained from the American Type Culture Collection) were maintained in Dulbecco's modified Eagle's medium (Cellgro, Herndon,VA) supplemented with 10% fetal bovine serum (U.S. Bio-Technologies, Pottstown, PA).
The HIV-1 Gag-Pol-Rev expression plasmid pHIV-1-pack, the vector genome plasmid pSMPU-MND-nlacZ, and the expression plasmids for the HIV-2ROD10 Env and HIV-1NL4-3 Vpu have been previously described (1). Chimeric proteins containing the ectodomain and membrane-spanning regions of the HIV-2 Env fused to the cytoplasmic domains of either CD8 or the MLV Env were generated by splice overlap PCR. The equine infectious anemia virus (EIAV) proviral clone, EIAVUK, and an L domain mutant containing the substitution YPDLSRSA were kindly provided by Ron Montelaro (25). An L domain mutant of pHIV-1-pack (PTAPLTAL) was generated by PCR mutagenesis and splice overlap PCR. This mutation was identical to a previously reported mutation that does not affect the function of the pol gene, which overlaps this region of gag in a different reading frame (13).
Generation of virus particles and Western blot analysis. HeLa cells grown to 80 to 90% confluence were transiently transfected with the appropriate plasmids using Lipofectamine 2000 (Invitrogen, Carlsbad, Calif.) as previously described (1). Cell lysates were harvested, and viral particles were collected from the supernatant by centrifugation through 2 ml of 20% (mass/vol) sucrose at 4°C for 2 h at 25,000 rpm using an SW40 rotor (Beckman Instruments, Inc., Palo Alto, Calif.). HIV-1 CA proteins were detected by using mouse anti-p24 monoclonal antibody 183-H12-5C (Bruce Chesebro and Kathy Wehrly, AIDS Research and Reference Reagent Program) at a 1:3,000 dilution. EIAV CA was detected by using a monoclonal antibody against p26 at a 1:1,000 dilution (11). The secondary antibody was horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G (1:10,000) (Pierce, Rockford, IL). Specific proteins were visualized using the enhanced chemiluminescence detection system (Amersham International, Arlington Heights, IL). Exposed and developed films were scanned with an HP Scanjet 4570c scanner, and bands were quantified by using the public domain NIH ImageJ software. Protein standards were run to ensure that the quantified gel bands remained within the linear range for analysis. The intensity of the CA-reacting bands on the Western blots was measured, and the ratio of the signal in virions to lysates was obtained. The enhancement of budding was calculated by normalizing all values to the Gag-Pol-only controls.
siRNA knockdowns. The 50-kDa μ2 subunit of the AP-2 complex was targeted by using an oligonucleotide with the sequence 5'-AAGUGGAUGCCUUUCGGGUCA-3' and a control nonfunctional oligonucleotide with the sequence 5'-AACACAGCAACCUCUACUUGG-3' (30). Both short interfering RNAs (siRNAs) were synthesized as option C by Dharmacon, Inc. (Lafayette, CO).
HeLa cells were seeded in 10-cm dishes at a density of 106 cells per dish 24 h before transfection. Forty microliters of Lipofectamine 2000 (Invitrogen) was added to 110 μl Opti-MEM I (Invitrogen) and incubated at room temperature for 10 min. This was added to a second solution of 800 μl Opti-MEM I plus 50 μl of a 20-μM solution of siRNA and incubated at room temperature for a further 20 min. Four milliliters of Opti-MEM I was added to the siRNA mixture for a final volume of 5 ml, and this was added to the cells, which were first rinsed with Opti-MEM I. The cells were incubated with the transfection mixture for 4 h, which was then replaced with Dulbecco's modified Eagle's medium plus 20% fetal bovine serum for overnight incubation. On the following day, the cells were trypsinized and reseeded into 10-cm dishes. Twenty-four hours later, a second transfection with siRNA was performed. Twenty-four hours after the second transfection, the cells were trypsinized and samples were taken from both the control and anti-AP-2 siRNA cells to assay the efficiency of AP-2 knockdown by using a mouse monoclonal antibody against the μ2 subunit of AP-2 (BD-Transduction Laboratories, San Jose, Calif.). The remaining cells were transfected 24 h later for a third time by using plasmids for virus release analysis as described above.
Immunofluorescence and confocal microscopy. HeLa cells were transfected with pHIV-1-pack, pSMPU-MND-nlacZ, and the appropriate HIV-2 Env expression plasmid in 10-cm dishes and then, 12 h later, were seeded on coverslips that were coated with poly-L-lysine (Sigma). The cells were incubated for an additional 48 h and then processed for antibody staining. Cells were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde for 20 min at room temperature, washed three times in PBS, permeabilized for 10 min in 0.1% Triton X-100 at room temperature, washed three times in PBS, and then blocked for 1 hour in 1% bovine serum albumin. HIV-2 Env proteins were stained by using a rabbit polyclonal serum against the HIV-2ST SU protein (Raymond Sweet, AIDS Research and Reference Reagent Program) at a 1:5,000 dilution. Various cellular organelles were costained as follows: Golgi, by using anti-human Golgin97 mouse monoclonal antibody (Molecular Probes, Eugene, OR) at 1:100 dilution; trans-Golgi network, by using sheep anti-TGN46 (Serotec, Oxford, United Kingdom) at 1:1,000 dilution; early endosomes, by using anti-EEA1 mouse monoclonal antibody (Abcam, Cambridge, MA) at 1:200 dilution; lysosomal associated protein-3/CD63, by using anti-LAMP-3 mouse monoclonal antibody (Santa Cruz Biotechnologies, Santa Cruz, CA) at a 1:500 dilution; and lysosomal-associated membrane protein-2/CD107b, by using mouse monoclonal antibody (BD Pharmingen, San Diego, CA) at 1:100 dilution. The secondary antibodies used were either donkey anti-rabbit AlexaFluor 488-conjugated, donkey anti-mouse AlexaFluor 594-conjugated, or donkey anti-sheep AlexaFluor 594-conjugated secondary antibodies (Molecular Probes). Nuclei were stained with DAPI (4',6'-diamidino-2-phenylindole) and To-Pro-3 (Molecular Probes). Images were acquired with a Leica TCS-SP1 spectral confocal microscope (Leica Microsystems, Bannockburn, IL) and processed with LCS Lite software.
RESULTS
AP-2 is needed for the HIV-2 Env-mediated enhancement of virus release. Our previous studies identified a membrane-proximal GYxx motif, centered at tyrosine-707 in the HIV-2 Env cytoplasmic tail, that was absolutely essential for the protein's EVR activity (1). The analogous motif in the HIV-1 Env has been shown to promote the internalization of Env through an interaction with AP-2 (4). We therefore examined whether the recruitment of AP-2 was necessary for the HIV-2 Env EVR activity by using RNA interference targeting the μ2 subunit of the AP-2 complex as described previously (30). Two hundred nanomolar siRNA was transfected into HeLa cells on days 1 and 3, and the cells were transfected a third time on day 5 with an HIV-1-based vector system comprising a plasmid expressing the HIV-1 Gag-Pol and Rev proteins and a plasmid transcribing a packageable vector genome. In addition, we cotransfected expression vectors for either the HIV-2ROD10 Env or the HIV-1NL4-3 Vpu protein (Fig. 1A). An inactive siRNA was included as a control, and AP-2 depletion was confirmed by Western blotting (Fig. 1B).
The levels of p24 (CA)-reacting bands in the cell lysates and viral particles that were harvested from culture supernatants were measured by Western blot analysis, and the enhancement of virus release resulting from the HIV-2 Env or Vpu proteins was determined (Fig. 1C). This revealed that, in the HeLa cells that received the control siRNA, the cotransfection of either HIV-2 Env or Vpu resulted in an approximately fivefold increase in virus production. However, the depletion of AP-2 completely abrogated the EVR activity of the HIV-2 Env, while having no effect on the Vpu activity. Interestingly, we also observed that the loss of AP-2 increased the baseline level of HIV-1 release in the absence of any EVR proteins by an average of 1.6-fold (n = 3), which is in agreement with a recent report (3). However, this increase was considerably lower than the stimulation achieved by Vpu or HIV-2 Env, suggesting that the inability of the HIV-2 Env to enhance release in the absence of AP-2 is a direct effect on the HIV-2 Env rather than resulting from abrogation of the budding restriction.
Position dependence of the GYRPV motif in the HIV-2 Env tail. We next asked whether the GYxx motif was the only contribution of the HIV-2 Env tail to EVR activity and, therefore, whether the motif could function in the context of a heterologous cytoplasmic domain. We have previously described a nonfunctional chimeric protein comprising the ectodomain and a membrane-spanning domain of the HIV-2 Env fused to the cytoplasmic domain of CD8 (protein E2M2T8) (1). The insertion of the HIV-2 GYRPV motif into this protein at the same location as that in the native Env protein (5 to 9 amino acids from the membrane-spanning domain) resulted in a fully functional protein (Fig. 2).
It has been previously noted that the distance of a Yxx motif from the membrane can influence both the cellular partner and function of the motif. For example, purely endocytic signals are typically situated 10 to 40 residues from the membrane, while lysosomal targeting motifs are present 6 to 9 residues from the membrane (5). We therefore examined whether the HIV-2 Env motif was position dependent by inserting the GYRPV motif at various sites within the CD8 tail and assessing the ability of the chimeric proteins to enhance virus release (Fig. 2). Interestingly, we obtained only a fully functional protein when the motif was placed in the analogous position to its native location in the HIV-2 Env, i.e., at 5 to 9 amino acids downstream from the membrane-spanning region. The insertion of the motif at more distal sites did not result in any EVR activity. These data are in agreement with a model whereby the GYRPV motif acts to recruit AP-2 and is a sorting signal for both endocytosis and subsequent trafficking towards lysosomes. Furthermore, they indicate that that the only contribution of the HIV-2 Env cytoplasmic tail to EVR activity is to provide a correctly positioned GYRPV sequence.
HIV-2 Env and Vpu EVR activities are independent from L domain activity. It is increasingly clear that retrovirus assembly and budding use the cell's protein trafficking and sorting machinery. L domains interact with various members of the class E Vps pathway, and both AP-3 and AP-2 have been implicated in HIV-1 Gag trafficking and release (3, 16). Several different classes of L domains have been identified, including Yxx motifs in EIAV and influenza virus. Interestingly, the EIAV L domain has been reported to interact with both AP-2 (41) and AIP1/ALIX (28, 51) and EIAV is unusual among the lentiviruses in that its cytoplasmic tail does not contain a membrane-proximal Yxx motif. We therefore considered the possibility that the membrane-proximal GYxx motif of the HIV-2 Env and the Yxx motif in the EIAV L domain could both be functioning to enhance virus release through similar mechanisms involving the recruitment of AP-2 and that the HIV-2 Env EVR activity was therefore analogous to L domain activity.
To test this hypothesis, we examined whether the HIV-2 Env or Vpu could compensate for the loss of L domain activity in either HIV-1 or EIAV particles and, conversely, whether L domain mutants of these two viruses were still responsive to HIV-2 Env or Vpu. HeLa cells were transfected with either wild-type or L domain mutants of HIV-1 and EIAV and cotransfected with expression plasmids for either Vpu or HIV-2 Env. For the HIV-1 particles, we used a previously reported specific mutation of the PTAP L domain sequence (13), while for EIAV, we used proviral clones of either the wild-type virus or a YPDLSRSA L domain mutant (25). For both HIV-1 and EIAV, as was expected, the loss of the L domain significantly reduced overall virus production, giving only 14 and 12%, respectively, of the amount of particles released when the L domain sequences were present. Despite the lower levels of release, both mutant viruses remained fully responsive to the enhancing effects of Vpu and HIV-2 Env (Fig. 3). However, even when stimulated by the presence of the EVRs, the level of release of the mutant constructs was increased to only 37 to 49% of the level produced by the wild-type constructs in the absence of EVRs, indicating that the loss of an L domain cannot be fully compensated for by EVR activity. In addition, the characteristic processing defect in the HIV-1 L domain mutant that leads to an increase in the level of p25 was not corrected by coexpression of an EVR (Fig. 3A). Taken together, these data suggest that L domain and EVR activities are independent.
Mutation of Y707 or depletion of AP-2 alters the cellular distribution of the HIV-2 Env. We speculated that the interaction between the GYPRV motif and AP-2 contributed to HIV-2 Env EVR function by targeting the protein to a specific cellular pathway or location necessary for its activity. Our previously reported finding of a requirement for the upstream glycine residue (1), combined with the position dependence we observed in the present study, suggests that this motif acts to target Env towards lysosomal compartments, possibly including the late endosomes/MVBs that can serve as sites of retrovirus budding (31, 33, 35, 49).
As a first analysis, we examined the overall pattern of Env distribution in HeLa cells and compared the wild-type protein to a defective tyrosine mutant, HIV-2Y707A (1). For each Env protein, we saw both cells showing a predominantly punctate pattern of Env distribution throughout the cell and others with a mostly diffuse distribution. However, the relative amounts of each pattern differed considerably; the wild-type Env was observed in a punctate distribution in 87% of the cells examined, while only 41% of the cells expressing the Y707A mutant showed this pattern (Fig. 4A). In addition, we noted that the punctate spots for the Y707A mutant were considerably larger than those observed for the wild-type Env (Fig. 4C).
We also analyzed the pattern of staining for the wild-type HIV-2 Env protein when AP-2 was depleted by RNA interference. Similar to the pattern resulting from the Y707A mutation, we observed that the loss of AP-2 caused a significant decrease in the percentage of cells showing a punctate pattern (Fig. 4D and E).
The MLV Env tail can substitute for the HIV-2 tail, despite not using its Yxx motif or requiring AP-2. We next asked whether the presumed trafficking function of the HIV-2 Env cytoplasmic tail could be substituted by the cytoplasmic domains of another retroviral Env protein. Accordingly, we constructed a chimeric protein containing the MLV Env tail [protein E2M2T(MLV)] and observed that this protein also supported wild-type levels of EVR activity (Fig. 5).
The MLV Env cytoplasmic tail contains a Yxx motif, located in the C-terminal half of the tail, which is referred to as the R peptide. This peptide is proteolytically cleaved from the Env protein during virion maturation, an event that increases the fusogenic potential of the Env protein (42, 43). Although the MLV motif is positioned 22 to 25 residues from the membrane and therefore does not fit the criteria for functionality that we had established with the HIV-2 Env/CD8 chimeras, we examined whether it played a role in the EVR activity of the chimeric protein. However, the mutation of the tyrosine had no effect on EVR activity (Fig. 5). This finding suggested that the MLV tail may not have an AP-2 interacting motif and therefore that AP-2 might not be required for the activity of the chimeric protein. In order to address this possibility, we examined whether the E2M2T(MLV) Env was affected by the loss of AP-2 by RNA interference and found that the chimeric Env was insensitive to the loss of AP-2 (Fig. 5).
Finally, we examined the overall pattern of cellular distribution of E2M2T(MLV) Env and found that it differed from the wild-type Env, as only 33% of the cells exhibited a punctate staining pattern, with spots that were often smaller and less distinct than those we observed with the wild-type Env (see the supplemental material). Together, these results indicate that, while the HIV-2 Env tail provides an essential function that utilizes AP-2, the same activity can be provided by the MLV Env tail through a different mechanism that results in a different gross cellular distribution.
Do EVR-active proteins [HIV-2 Env and E2M2T(MLV)] share common cellular locations Despite the fact that the wild-type Env and the functional E2M2T(MLV) chimera gave distinct staining patterns when expressed in HeLa cells, it remained possible that they were present in similar subcellular locations that are important for EVR activity. To address this question, we examined the extent of colocalization of each of the Env proteins used in this study with subcellular markers. Cells exhibiting both punctate and diffuse staining patterns were examined separately (Table 1; see the supplemental material). This analysis revealed that a significant portion of the wild-type HIV-2 Env was found to colocalize with markers for the Golgi apparatus and the trans-Golgi network (TGN), especially in the punctate-staining cells. We also saw some costaining with the early endosome marker, EEA1, and with LAMP-2 (found in early endosomes, the plasma membrane, late endosomes/MVBs, and lysosomes). HIV-1 budding has been reported to occur at late endosomes/MVBs and tetraspanin-enriched microdomains at the cell surface (34), both of which contain CD63, and the HIV-1 Env has been shown to colocalize with surface CD63 (34). However, we observed no significant overlap with CD63, although it is possible that the sensitivity of our assay is not sufficient to detect such an interaction.
The Y707A mutant of the HIV-2 Env displayed a markedly different distribution than the wild-type Env. In addition to having a more diffuse overall staining pattern, we noticed that, in those cells that did display a punctate staining pattern, the spots appeared larger than those we observed with either the wild-type Env or the E2M2T(MLV) chimera. The Y707A protein exhibited reduced colocalization with the Golgi and the TGN and gave no overlap with the marker EEA1, CD63, or LAMP-2. The marked decrease in punctate staining for the Y707A mutant, together with the loss of EEA1 costaining, is in agreement with the GYxx motif of the HIV-2 Env functioning as an endocytosis signal. It also suggests that endocytosis from the cell surface is a prerequisite for entry of the HIV-2 Env into internal compartments containing EEA1, CD63, or LAMP-2.
As noted above, although the substitution of the MLV tail in chimera E2M2T(MLV) resulted in a fully functional protein, this chimera did not exhibit the same overall staining pattern as the wild-type Env. Only 33% of cells expressing the chimera displayed a punctate distribution, and the spots we observed were often smaller and less distinct than those observed for the wild-type Env. However, despite these gross differences in distribution, the costaining studies with subcellular markers revealed a distribution that resembled that of wild-type Env, with colocalization with markers for the Golgi apparatus, TGN, early endosomes, and LAMP-2 but not with CD63. It is therefore possible that both the HIV-2 and MLV Env cytoplasmic tails contain signals that direct the HIV-2 Env to a common compartment that is necessary for its EVR activity but that, while the HIV-2 tail uses AP-2-mediated endocytosis, the MLV tail uses a different route. Our ongoing studies are aimed at elucidating the pathway used by the MLV cytoplasmic domain.
DISCUSSION
The HIV-2 Env and HIV-1 Vpu proteins provide analogous functions in the HIV life cycle that we refer to as the enhancement of virus release. They act to increase the production of viral particles from various human cell types, including primary lymphocytes and macrophages, and can also stimulate the release of heterologous retroviral particles. The importance of this activity is suggested by the fact that intact Vpu open reading frames are maintained in AIDS patients, and studies of chimeric SIV/HIV infections in macaques have demonstrated that Vpu is essential for pathogenicity (22, 50).
A current model of EVR activity is that human cells contain a restriction factor that suppresses retroviral budding and whose activity is counteracted by the EVRs. This is supported by analyses of the heterokaryons formed between restrictive (human) and nonrestrictive (simian) cell types, where the human cell phenotype of restricted budding/EVR responsiveness is dominant (1, 54). It is something of a philosophical question as to whether EVR activity is viewed as counteracting a host defense mechanism that has evolved to restrict retrovirus budding or as simply reflecting a viral mechanism to override normal cellular checkpoints. Either way, the cellular proteins or pathways that the EVRs target are presently unknown. As part of our investigations into these activities, we are mapping functional domains within the EVR proteins and elucidating their contribution to the activity. In the present study, we show that the cytoplasmic tail of the HIV-2 Env contributes a membrane-proximal GYRPV motif that interacts with AP-2 and which, by its sequence and position dependence characteristics, shares properties with lysosomal targeting motifs.
We speculated that two possible mechanisms could account for the requirement for the GYRPV motif and AP-2 in the HIV-2 Env EVR activity. First, this sequence could be recruiting AP-2 as part of an activity that is similar to L domain function. This hypothesis was based on the fact that the L domain in the EIAV p9 protein contains a Yxx sequence that is capable of interacting with both AIPI/ALIX and AP-2 (41, 51), with both interactions being critical for L domain activity (32). Intriguingly, the EIAV L domain has characteristics that are distinct from other L domains (36, 38, 48) and the cytoplasmic tail of the EIAV Env is unusual among the lentiviruses in having no membrane-proximal Yxx motif. However, our analysis of the response of both wild-type and L domain mutants of EIAV and HIV-1 revealed that all virions were stimulated by both HIV-2 Env and Vpu. Furthermore, although virus production was significantly decreased for both viruses when the L domains were mutated, this could not be fully restored by the addition of an EVR. Taken together, these findings indicate that L domain activity is a distinct process from that of the EVR activity of either HIV-2 Env or Vpu.
The second hypothesis that we explored was that the GYRPV/AP-2 interaction was critical for HIV-2 Env EVR activity by virtue of targeting the protein to a specific cellular location or pathway that was necessary for its activity. In particular, the lysosome-targeting characteristics of the GYPRV sequence are consistent with a model whereby Env is directed to an endosomal/lysosomal compartment, where it may interact with other components involved in virus assembly that are also targeted towards the late endosome/MVB that is proposed to be a site of virus assembly (31, 33, 35, 49). We explored the possibility that a distinct subcellular location is correlated with HIV-2 Env EVR activity by comparing the distributions of both EVR-active proteins [wild-type HIV-2 Env and the E2M2T(MLV) chimera] and the nonfunctional HIV-2Y707A mutant. We observed that the mutation Y707A in the HIV-2 tail as well the loss of AP-2 by RNA interference altered the distribution of Env from a predominantly punctate to a more diffuse staining pattern. Furthermore, the Y707A mutant had significantly less colocalization with markers of the endocytic pathway such as EEA1, which is in keeping with a role for the GYPRV sequence in promoting endocytosis through an AP-2 interaction. However, the finding that the MLV tail could substitute for this activity in a manner that did not use AP-2, that did not depend on a Yxx motif, and that resulted in a cellular distribution that was more diffuse than punctate, suggests that AP-2-mediated endocytosis is not the only route whereby the HIV-2 Env can be directed to such an active cellular compartment. Identification of the cellular partner(s) used by the MLV tail may shed light on the mechanism involved. Finally, it remains a possibility that only a fraction of the Env needs to be localized to a specific site for activity and sufficient protein is correctly targeted by either Env tail. Such an active cellular location may be obscured by the background distribution that we observe for different constructs, especially when we consider that the EVR proteins have other functions in the HIV life cycle. Our ongoing analyses are aimed at elucidating both the functional subcellular compartment for EVR activities and the cellular partners that are involved.
ACKNOWLEDGMENTS
We acknowledge the NIH AIDS Research and Reference Reagent Program for several reagents that were vital to this project. We thank Chaoping Chen and Ronald Montelaro for the EIAV constructs, Markus Thali for helpful discussions, and George McNamara at the CHLA Imaging Core for technical assistance with the confocal microscopy.
This work was supported by Public Health Service grant CA-59318 and the Universitywide AIDS Research Program, ID03-CHLA-036.
Supplemental material for this article may be found at http://jvi.asm.org/.
REFERENCES
Abada, P., B. Noble, and P. M. Cannon. 2005. Functional domains within the human immunodeficiency virus type 2 envelope protein required to enhance virus production. J. Virol. 79:3627-3638.
Balliet, J. W., D. L. Kolson, G. Eiger, F. M. Kim, K. A. McGann, A. Srinivasan, and R. Collman. 1994. Distinct effects in primary macrophages and lymphocytes of the human immunodeficiency virus type 1 accessory genes vpr, vpu, and nef: mutational analysis of a primary HIV-1 isolate. Virology 200:623-631.
Batonick, M., M. Favre, M. Boge, P. Spearman, S. Honing, and M. Thali. 2005. Interaction of HIV-1 Gag with the clathrin-associated adaptor AP-2. Virology 342:190-200.
Boge, M., S. Wyss, J. S. Bonifacino, and M. Thali. 1998. A membrane-proximal tyrosine-based signal mediates internalization of the HIV-1 envelope glycoprotein via interaction with the AP-2 clathrin adaptor. J. Biol. Chem. 273:15773-15778.
Bonifacino, J. S., and L. M. Traub. 2003. Signals for sorting of transmembrane proteins to endosomes and lysosomes. Annu. Rev. Biochem. 72:395-447.
Bour, S., U. Schubert, K. Peden, and K. Strebel. 1996. The envelope glycoprotein of human immunodeficiency virus type 2 enhances viral particle release: a Vpu-like factor J. Virol. 70:820-829.
Bour, S., and K. Strebel. 1996. The human immunodeficiency virus (HIV) type 2 envelope protein is a functional complement to HIV type 1 Vpu that enhances particle release of heterologous retroviruses. J. Virol. 70:8285-8300.
Bour, S. P., C. Aberham, C. Perrin, and K. Strebel. 1999. Lack of effect of cytoplasmic tail truncations on human immunodeficiency virus type 2 ROD Env particle release activity. J. Virol. 73:778-782.
Carr, J. M., H. Hocking, P. Li, and C. J. Burrell. 1999. Rapid and efficient cell-to-cell transmission of human immunodeficiency virus infection from monocyte-derived macrophages to peripheral blood lymphocytes. Virology 265:319-329.
Cervantes-Acosta, G., R. Lodge, G. Lemay, and E. A. Cohen. 2001. Influence of human immunodeficiency virus type 1 envelope glycoprotein YXXL endocytosis/polarization signal on viral accessory protein functions. J. Hum. Virol. 4:249-259.
Chong, Y. H., S. L. Payne, C. J. Issel, R. C. Montelaro, and K. E. Rushlow. 1991. Characterization of the antigenic domains of the major core protein (p26) of equine infectious anemia virus. J. Virol. 65:1007-1012.
Demirov, D. G., and E. O. Freed. 2004. Retrovirus budding. Virus Res. 106:87-102.
Demirov, D. G., J. M. Orenstein, and E. O. Freed. 2002. The late domain of human immunodeficiency virus type 1 p6 promotes virus release in a cell type-dependent manner. J. Virol. 76:105-117.
Deora, A., and L. Ratner. 2001. Viral protein U (Vpu)-mediated enhancement of human immunodeficiency virus type 1 particle release depends on the rate of cellular proliferation. J. Virol. 75:6714-6718.
Deschambeault, J., J. P. Lalonde, G. Cervantes-Acosta, R. Lodge, E. A. Cohen, and G. Lemay. 1999. Polarized human immunodeficiency virus budding in lymphocytes involves a tyrosine-based signal and favors cell-to-cell viral transmission. J. Virol. 73:5010-5017.
Dong, X., H. Li, A. Derdowski, L. Ding, A. Burnett, X. Chen, T. R. Peters, T. S. Dermody, E. Woodruff, J. J. Wang, and P. Spearman. 2005. AP-3 directs the intracellular trafficking of HIV-1 Gag and plays a key role in particle assembly. Cell 120:663-674.
Ewart, G. D., K. Mills, G. B. Cox, and P. W. Gage. 2002. Amiloride derivatives block ion channel activity and enhancement of virus-like particle budding caused by HIV-1 protein Vpu. Eur. Biophys. J. 31:26-35.
Fais, S., M. R. Capobianchi, I. Abbate, C. Castilletti, M. Gentile, P. Cordiali Fei, F. Ameglio, and F. Dianzani. 1995. Unidirectional budding of HIV-1 at the site of cell-to-cell contact is associated with co-polarization of intercellular adhesion molecules and HIV-1 viral matrix protein. AIDS 9:329-335.
Geraghty, R. J., K. J. Talbot, M. Callahan, W. Harper, and A. T. Panganiban. 1994. Cell type-dependence for Vpu function. J. Med. Primatol. 23:146-150.
Gheysen, D., E. Jacobs, F. de Foresta, C. Thiriart, M. Francotte, D. Thines, and M. De Wilde. 1989. Assembly and release of HIV-1 precursor Pr55gag virus-like particles from recombinant baculovirus-infected insect cells. Cell 59:103-112.
Gottlinger, H. G., T. Dorfman, E. A. Cohen, and W. A. Haseltine. 1993. Vpu protein of human immunodeficiency virus type 1 enhances the release of capsids produced by gag gene constructs of widely divergent retroviruses. Proc. Natl. Acad. Sci. USA 90:7381-7385.
Hout, D. R., E. R. Mulcahy, E. Pacyniak, L. M. Gomez, M. L. Gomez, and E. B. Stephens. 2004. Vpu: a multifunctional protein that enhances the pathogenesis of human immunodeficiency virus type 1. Curr. HIV Res. 2:255-270.
Iida, S., T. Fukumori, Y. Oshima, H. Akari, A. H. Koyama, and A. Adachi. 1999. Compatibility of Vpu-like activity in the four groups of primate immunodeficiency viruses. Virus Gene 18:183-187.
Jolly, C., K. Kashefi, M. Hollinshead, and Q. J. Sattentau. 2004. HIV-1 cell to cell transfer across an Env-induced, actin-dependent synapse. J. Exp. Med. 199:283-293.
Li, F., C. Chen, B. A. Puffer, and R. C. Montelaro. 2002. Functional replacement and positional dependence of homologous and heterologous L domains in equine infectious anemia virus replication. J. Virol. 76:1569-1577.
Lodge, R., H. Gottlinger, D. Gabuzda, E. A. Cohen, and G. Lemay. 1994. The intracytoplasmic domain of gp41 mediates polarized budding of human immunodeficiency virus type 1 in MDCK cells. J. Virol. 68:4857-4861.
Lodge, R., J. P. Lalonde, G. Lemay, and E. A. Cohen. 1997. The membrane-proximal intracytoplasmic tyrosine residue of HIV-1 envelope glycoprotein is critical for basolateral targeting of viral budding in MDCK cells. EMBO J. 16:695-705.
Martin-Serrano, J., A. Yarovoy, D. Perez-Caballero, and P. D. Bieniasz. 2003. Divergent retroviral late-budding domains recruit vacuolar protein sorting factors by using alternative adaptor proteins. Proc. Natl. Acad. Sci. USA 100:12414-12419.
Morita, E., and W. I. Sundquist. 2004. Retrovirus budding. Annu. Rev. Cell Dev. Biol. 20:395-425.
Motley, A., N. A. Bright, M. N. Seaman, and M. S. Robinson. 2003. Clathrin-mediated endocytosis in AP-2-depleted cells. J. Cell Biol. 162:909-918.
Nguyen, D. G., A. Booth, S. J. Gould, and J. E. Hildreth. 2003. Evidence that HIV budding in primary macrophages occurs through the exosome release pathway. J. Biol. Chem. 278:52347-52354.
Noble, B., and P. M. Cannon. Unpublished observations.
Nydegger, S., M. Foti, A. Derdowski, P. Spearman, and M. Thali. 2003. HIV-1 egress is gated through late endosomal membranes. Traffic 4:902-910.
Nydegger, S., S. Khurana, M. Foti, and M. Thali. High resolution mapping of tetraspanin-enriched microdomains (TEMs) that function as gateways for HIV-1. Submitted for publication.
Ono, A., and E. O. Freed. 2004. Cell-type-dependent targeting of human immunodeficiency virus type 1 assembly to the plasma membrane and the multivesicular body. J. Virol. 78:1552-1563.
Ott, D. E., L. V. Coren, R. C. Sowder, II, J. Adams, K. Nagashima, and U. Schubert. 2002. Equine infectious anemia virus and the ubiquitin-proteasome system. J. Virol. 76:3038-3044.
Owens, R. J., J. W. Dubay, E. Hunter, and R. W. Compans. 1991. Human immunodeficiency virus envelope protein determines the site of virus release in polarized epithelial cells. Proc. Natl. Acad. Sci. USA 88:3987-3991.
Patnaik, A., V. Chau, F. Li, R. C. Montelaro, and J. W. Wills. 2002. Budding of equine infectious anemia virus is insensitive to proteasome inhibitors. J. Virol. 76:2641-2647.
Paul, M., S. Mazumder, N. Raja, and M. A. Jabbar. 1998. Mutational analysis of the human immunodeficiency virus type 1 Vpu transmembrane domain that promotes the enhanced release of virus-like particles from the plasma membrane of mammalian cells. J. Virol. 72:1270-1279.
Phillips, D. M. 1994. The role of cell-to-cell transmission in HIV infection. AIDS 8:719-731.
Puffer, B. A., S. C. Watkins, and R. C. Montelaro. 1998. Equine infectious anemia virus Gag polyprotein late domain specifically recruits cellular AP-2 adapter protein complexes during virion assembly. J. Virol. 72:10218-10221.
Ragheb, J. A., and W. F. Anderson. 1994. pH-independent murine leukemia virus ecotropic envelope-mediated cell fusion: implications for the role of the R peptide and p12E TM in viral entry. J. Virol. 68:3220-3231.
Rein, A., J. Mirro, J. G. Haynes, S. M. Ernst, and K. Nagashima. 1994. Function of the cytoplasmic domain of a retroviral transmembrane protein: p15E-p2E cleavage activates the membrane fusion capability of the murine leukemia virus Env protein. J. Virol. 68:1773-1781.
Ritter, G. D., Jr., G. Yamshchikov, S. J. Cohen, and M. J. Mulligan. 1996. Human immunodeficiency virus type 2 glycoprotein enhancement of particle budding: role of the cytoplasmic domain. J. Virol. 70:2669-2673.
Sakai, H., K. Tokunaga, M. Kawamura, and A. Adachi. 1995. Function of human immunodeficiency virus type 1 Vpu protein in various cell types. J. Gen. Virol. 76:2717-2722.
Schubert, U., K. A. Clouse, and K. Strebel. 1995. Augmentation of virus secretion by the human immunodeficiency virus type 1 Vpu protein is cell type independent and occurs in cultured human primary macrophages and lymphocytes. J. Virol. 69:7699-7711.
Schwartz, M. D., R. J. Geraghty, and A. T. Panganiban. 1996. HIV-1 particle release mediated by Vpu is distinct from that mediated by p6. Virology 224:302-309.
Shehu-Xhilaga, M., S. Ablan, D. G. Demirov, C. Chen, R. C. Montelaro, and E. O. Freed. 2004. Late domain-dependent inhibition of equine infectious anemia virus budding. J. Virol. 78:724-732.
Sherer, N. M., M. J. Lehmann, L. F. Jimenez-Soto, A. Ingmundson, S. M. Horner, G. Cicchetti, P. G. Allen, M. Pypaert, J. M. Cunningham, and W. Mothes. 2003. Visualization of retroviral replication in living cells reveals budding into multivesicular bodies. Traffic 4:785-801.
Stephens, E. B., C. McCormick, E. Pacyniak, D. Griffin, D. M. Pinson, F. Sun, W. Nothnick, S. W. Wong, R. Gunderson, N. E. Berman, and D. K. Singh. 2002. Deletion of the vpu sequences prior to the env in a simian-human immunodeficiency virus results in enhanced Env precursor synthesis but is less pathogenic for pig-tailed macaques. Virology 293:252-261.
Strack, B., A. Calistri, S. Craig, W. Popova, and H. G. Gottlinger. 2003. AIP1/ALIX is a binding partner for HIV-1 p6 and EIAV p9 functioning in virus budding. Cell 114:689-699.
Strebel, K., T. Klimkait, F. Maldarelli, and M. A. Martin. 1989. Molecular and biochemical analyses of human immunodeficiency virus type 1 vpu protein. J. Virol. 63:3784-3791.
Terwilliger, E. F., E. A. Cohen, Y. C. Lu, J. G. Sodroski, and W. A. Haseltine. 1989. Functional role of human immunodeficiency virus type 1 vpu. Proc. Natl. Acad. Sci. USA 86:5163-5167.
Varthakavi, V., R. M. Smith, S. P. Bour, K. Strebel, and P. Spearman. 2003. Viral protein U counteracts a human host cell restriction that inhibits HIV-1 particle production. Proc. Natl. Acad. Sci. USA 100:15154-15159.(Beth Noble, Paolo Abada, )
Department of Pediatrics, University of Southern California Keck School of Medicine, Los Angeles, California
Saban Research Institute of Childrens Hospital Los Angeles, Los Angeles, California
ABSTRACT
The envelope (Env) protein of human immunodeficiency virus type 2 (HIV-2) and the HIV-1 Vpu protein stimulate the release of retroviral particles from human cells that restrict virus production, an activity that we call the enhancement of virus release (EVR). We have previously shown that two separate domains in the HIV-2 envelope protein are required for this activity: a glycine-tyrosine-x-x-hydrophobic (GYxx) motif in the cytoplasmic tail and an unmapped region in the ectodomain of the protein. We here report that the cellular partner of the GYxx motif is the adaptor protein complex AP-2. The mutation of this motif or the depletion of AP-2 by RNA interference abrogated EVR activity and changed the cellular distribution of the Env from a predominantly punctate pattern to a more diffuse distribution. Since the L domain of equine infectious anemia virus (EIAV) contains a Yxx motif that interacts with AP-2, we used both wild-type and L domain-defective particles of HIV-1 and EIAV to examine whether the HIV-2 Env EVR function was analogous to L domain activity. We observed that the production of all particles was stimulated by HIV-2 Env or Vpu, suggesting that the L domain and EVR activities play independent roles in the release of retroviruses. Interestingly, we found that the cytoplasmic tail of the murine leukemia virus (MLV) Env could functionally substitute for the HIV-2 Env tail, but it did so in a manner that did not require a Yxx motif or AP-2. The cellular distribution of the chimeric HIV-2/MLV Env was significantly less punctate than the wild-type Env, although confocal analysis revealed an overlap in the steady-state locations of the two proteins. Taken together, these data suggest that the essential GYxx motif in the HIV-2 Env tail recruits AP-2 in order to direct Env to a cellular pathway or location that is necessary for its ability to enhance virus release but that an alternate mechanism provided by the MLV Env tail can functionally substitute.
INTRODUCTION
The assembly and budding of C-type retroviruses are primarily driven by the Gag protein, and virus-like particles form efficiently in the absence of any other retroviral gene products or the RNA genome (20). Several distinct regions in Gag are necessary for virus-like particle formation, including a membrane-targeting domain at the N terminus of MA, a multimerization domain present in CA, and a late or L domain that is necessary for "pinching off" budding viral particles. L domains are located at various positions within Gag and have also been found in other enveloped RNA viruses, where they function as protein interaction motifs that recruit components of the cell's class E vacuolar protein synthesis (Vps) and endocytic trafficking pathways (for reviews, see references 12 and 29). In mammalian cells, the class E Vps pathway is involved in the export of cargo and the budding of exosomes into multivesicular bodies (MVBs). The process of exosome formation is similar to that of retrovirus budding since it involves the outward curvature of a membrane away from the cytosol and a membrane fusion event to allow release of the vesicle into the lumen of the MVB.
Although the retroviral Env protein is not absolutely required for the assembly and release of viral particles, it is now increasingly appreciated that Env plays an active role in these events. For example, the human immunodeficiency virus type 1 (HIV-1) Env is able to target virus assembly to the basolateral membrane of polarized epithelial cells (26, 37) and to sites of cell-cell contact in infected monocytes and lymphocytes (9, 15). In both cases, a conserved Y-x-x-hydrophobic (Yxx) motif in the cytoplasmic tail of Env is necessary for this redirection (15, 27). The clustering of HIV-1 assembly at cell-cell junctions has previously been documented (18, 40), and recent evidence suggests that the interaction of cell-associated HIV-1 Env with its cellular receptor(s) on target cells leads to the formation of a synapse that recruits the host cell proteins that are necessary for HIV entry as well as the viral Gag and Env proteins (24).
In HIV-2, the Env protein has an even more dramatic effect on virus assembly and release through its ability to boost production from certain human cell types (6, 44). We refer to this activity as the enhancement of virus release (EVR). In HIV-1, the Env protein does not generally possess EVR activity and this function is provided, instead, by the accessory protein Vpu (52, 53). Like HIV-2, most simian immunodeficiency virus (SIV) strains do not code for Vpu (notable exceptions being HIV-1-like monkey isolates, such as SIVcpz) and the Env proteins from SIVmac239 and SIVmnd have also been reported to have enhancing activity (7, 23). These observations lead us to suggest that Vpu evolved in HIV-1 to take over the EVR function from the Env protein.
In vitro, EVRs typically boost steady-state levels of virus production from cell lines by 4- to 10-fold and this activity has been observed in various different human cell types, including HeLa and HEp-2 cells, various T-cell lines, primary blood mononuclear cells, and macrophages (2, 8, 6, 7, 10, 14, 17, 21, 39, 44, 45, 46, 54). EVRs are able to stimulate the budding of heterologous viral particles from human cells (1), and it has been suggested that they counteract a natural human restriction factor that acts to inhibit retrovirus budding (21, 47). In contrast, simian cells do not appear to restrict HIV budding and do not support EVR activity (19, 47). Analysis of the heterokaryons formed between human and simian cells suggests that human cells contain a dominant factor that restricts virus production and which the EVRs counteract (1, 54).
Our previous studies of the HIV-2 Env defined two functional domains in the protein that are required for EVR activity, a conserved GYxx motif in the membrane-proximal part of the cytoplasmic tail (GYPRV) and an unidentified region in the ectodomain of the protein (1). Yxx motifs are found in the cytosolic domains of a variety of transmembrane proteins, where they play central roles in protein sorting (for a review, see reference 5). In mammalian cells, they act predominately to signal rapid internalization from the cell surface through interactions with components of the cellular trafficking machinery, most notably the adaptor protein (AP) complexes. Although the Yxx tetrapeptide is the minimal motif conferring sorting information, the presence of a conserved upstream glycine residue is characteristic of a lysosomal targeting motif.
The analogous membrane-proximal GYxx motif in the HIV-1 Env cytoplasmic tail, which we have previously shown to be functionally equivalent to the HIV-2 sequence (1), promotes endocytosis of HIV-1 Env from the cell surface and interacts specifically with the AP-2 complex (4). We therefore hypothesized that such an interaction could function to target the HIV-2 Env to an intracellular location that was important for its EVR activity. In the present study, we determined that AP-2 is indeed necessary for the EVR activity of the HIV-2 Env. Furthermore, by analyzing the cellular distribution of both a nonfunctional mutant of Env and a non-AP-2-requiring chimeric protein containing the cytoplasmic domain of the murine leukemia virus (MLV) Env, we have begun to examine whether targeting to a distinct cellular location is necessary for EVR activity.
MATERIALS AND METHODS
Cell lines and plasmid constructs. HeLa cells (obtained from the American Type Culture Collection) were maintained in Dulbecco's modified Eagle's medium (Cellgro, Herndon,VA) supplemented with 10% fetal bovine serum (U.S. Bio-Technologies, Pottstown, PA).
The HIV-1 Gag-Pol-Rev expression plasmid pHIV-1-pack, the vector genome plasmid pSMPU-MND-nlacZ, and the expression plasmids for the HIV-2ROD10 Env and HIV-1NL4-3 Vpu have been previously described (1). Chimeric proteins containing the ectodomain and membrane-spanning regions of the HIV-2 Env fused to the cytoplasmic domains of either CD8 or the MLV Env were generated by splice overlap PCR. The equine infectious anemia virus (EIAV) proviral clone, EIAVUK, and an L domain mutant containing the substitution YPDLSRSA were kindly provided by Ron Montelaro (25). An L domain mutant of pHIV-1-pack (PTAPLTAL) was generated by PCR mutagenesis and splice overlap PCR. This mutation was identical to a previously reported mutation that does not affect the function of the pol gene, which overlaps this region of gag in a different reading frame (13).
Generation of virus particles and Western blot analysis. HeLa cells grown to 80 to 90% confluence were transiently transfected with the appropriate plasmids using Lipofectamine 2000 (Invitrogen, Carlsbad, Calif.) as previously described (1). Cell lysates were harvested, and viral particles were collected from the supernatant by centrifugation through 2 ml of 20% (mass/vol) sucrose at 4°C for 2 h at 25,000 rpm using an SW40 rotor (Beckman Instruments, Inc., Palo Alto, Calif.). HIV-1 CA proteins were detected by using mouse anti-p24 monoclonal antibody 183-H12-5C (Bruce Chesebro and Kathy Wehrly, AIDS Research and Reference Reagent Program) at a 1:3,000 dilution. EIAV CA was detected by using a monoclonal antibody against p26 at a 1:1,000 dilution (11). The secondary antibody was horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G (1:10,000) (Pierce, Rockford, IL). Specific proteins were visualized using the enhanced chemiluminescence detection system (Amersham International, Arlington Heights, IL). Exposed and developed films were scanned with an HP Scanjet 4570c scanner, and bands were quantified by using the public domain NIH ImageJ software. Protein standards were run to ensure that the quantified gel bands remained within the linear range for analysis. The intensity of the CA-reacting bands on the Western blots was measured, and the ratio of the signal in virions to lysates was obtained. The enhancement of budding was calculated by normalizing all values to the Gag-Pol-only controls.
siRNA knockdowns. The 50-kDa μ2 subunit of the AP-2 complex was targeted by using an oligonucleotide with the sequence 5'-AAGUGGAUGCCUUUCGGGUCA-3' and a control nonfunctional oligonucleotide with the sequence 5'-AACACAGCAACCUCUACUUGG-3' (30). Both short interfering RNAs (siRNAs) were synthesized as option C by Dharmacon, Inc. (Lafayette, CO).
HeLa cells were seeded in 10-cm dishes at a density of 106 cells per dish 24 h before transfection. Forty microliters of Lipofectamine 2000 (Invitrogen) was added to 110 μl Opti-MEM I (Invitrogen) and incubated at room temperature for 10 min. This was added to a second solution of 800 μl Opti-MEM I plus 50 μl of a 20-μM solution of siRNA and incubated at room temperature for a further 20 min. Four milliliters of Opti-MEM I was added to the siRNA mixture for a final volume of 5 ml, and this was added to the cells, which were first rinsed with Opti-MEM I. The cells were incubated with the transfection mixture for 4 h, which was then replaced with Dulbecco's modified Eagle's medium plus 20% fetal bovine serum for overnight incubation. On the following day, the cells were trypsinized and reseeded into 10-cm dishes. Twenty-four hours later, a second transfection with siRNA was performed. Twenty-four hours after the second transfection, the cells were trypsinized and samples were taken from both the control and anti-AP-2 siRNA cells to assay the efficiency of AP-2 knockdown by using a mouse monoclonal antibody against the μ2 subunit of AP-2 (BD-Transduction Laboratories, San Jose, Calif.). The remaining cells were transfected 24 h later for a third time by using plasmids for virus release analysis as described above.
Immunofluorescence and confocal microscopy. HeLa cells were transfected with pHIV-1-pack, pSMPU-MND-nlacZ, and the appropriate HIV-2 Env expression plasmid in 10-cm dishes and then, 12 h later, were seeded on coverslips that were coated with poly-L-lysine (Sigma). The cells were incubated for an additional 48 h and then processed for antibody staining. Cells were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde for 20 min at room temperature, washed three times in PBS, permeabilized for 10 min in 0.1% Triton X-100 at room temperature, washed three times in PBS, and then blocked for 1 hour in 1% bovine serum albumin. HIV-2 Env proteins were stained by using a rabbit polyclonal serum against the HIV-2ST SU protein (Raymond Sweet, AIDS Research and Reference Reagent Program) at a 1:5,000 dilution. Various cellular organelles were costained as follows: Golgi, by using anti-human Golgin97 mouse monoclonal antibody (Molecular Probes, Eugene, OR) at 1:100 dilution; trans-Golgi network, by using sheep anti-TGN46 (Serotec, Oxford, United Kingdom) at 1:1,000 dilution; early endosomes, by using anti-EEA1 mouse monoclonal antibody (Abcam, Cambridge, MA) at 1:200 dilution; lysosomal associated protein-3/CD63, by using anti-LAMP-3 mouse monoclonal antibody (Santa Cruz Biotechnologies, Santa Cruz, CA) at a 1:500 dilution; and lysosomal-associated membrane protein-2/CD107b, by using mouse monoclonal antibody (BD Pharmingen, San Diego, CA) at 1:100 dilution. The secondary antibodies used were either donkey anti-rabbit AlexaFluor 488-conjugated, donkey anti-mouse AlexaFluor 594-conjugated, or donkey anti-sheep AlexaFluor 594-conjugated secondary antibodies (Molecular Probes). Nuclei were stained with DAPI (4',6'-diamidino-2-phenylindole) and To-Pro-3 (Molecular Probes). Images were acquired with a Leica TCS-SP1 spectral confocal microscope (Leica Microsystems, Bannockburn, IL) and processed with LCS Lite software.
RESULTS
AP-2 is needed for the HIV-2 Env-mediated enhancement of virus release. Our previous studies identified a membrane-proximal GYxx motif, centered at tyrosine-707 in the HIV-2 Env cytoplasmic tail, that was absolutely essential for the protein's EVR activity (1). The analogous motif in the HIV-1 Env has been shown to promote the internalization of Env through an interaction with AP-2 (4). We therefore examined whether the recruitment of AP-2 was necessary for the HIV-2 Env EVR activity by using RNA interference targeting the μ2 subunit of the AP-2 complex as described previously (30). Two hundred nanomolar siRNA was transfected into HeLa cells on days 1 and 3, and the cells were transfected a third time on day 5 with an HIV-1-based vector system comprising a plasmid expressing the HIV-1 Gag-Pol and Rev proteins and a plasmid transcribing a packageable vector genome. In addition, we cotransfected expression vectors for either the HIV-2ROD10 Env or the HIV-1NL4-3 Vpu protein (Fig. 1A). An inactive siRNA was included as a control, and AP-2 depletion was confirmed by Western blotting (Fig. 1B).
The levels of p24 (CA)-reacting bands in the cell lysates and viral particles that were harvested from culture supernatants were measured by Western blot analysis, and the enhancement of virus release resulting from the HIV-2 Env or Vpu proteins was determined (Fig. 1C). This revealed that, in the HeLa cells that received the control siRNA, the cotransfection of either HIV-2 Env or Vpu resulted in an approximately fivefold increase in virus production. However, the depletion of AP-2 completely abrogated the EVR activity of the HIV-2 Env, while having no effect on the Vpu activity. Interestingly, we also observed that the loss of AP-2 increased the baseline level of HIV-1 release in the absence of any EVR proteins by an average of 1.6-fold (n = 3), which is in agreement with a recent report (3). However, this increase was considerably lower than the stimulation achieved by Vpu or HIV-2 Env, suggesting that the inability of the HIV-2 Env to enhance release in the absence of AP-2 is a direct effect on the HIV-2 Env rather than resulting from abrogation of the budding restriction.
Position dependence of the GYRPV motif in the HIV-2 Env tail. We next asked whether the GYxx motif was the only contribution of the HIV-2 Env tail to EVR activity and, therefore, whether the motif could function in the context of a heterologous cytoplasmic domain. We have previously described a nonfunctional chimeric protein comprising the ectodomain and a membrane-spanning domain of the HIV-2 Env fused to the cytoplasmic domain of CD8 (protein E2M2T8) (1). The insertion of the HIV-2 GYRPV motif into this protein at the same location as that in the native Env protein (5 to 9 amino acids from the membrane-spanning domain) resulted in a fully functional protein (Fig. 2).
It has been previously noted that the distance of a Yxx motif from the membrane can influence both the cellular partner and function of the motif. For example, purely endocytic signals are typically situated 10 to 40 residues from the membrane, while lysosomal targeting motifs are present 6 to 9 residues from the membrane (5). We therefore examined whether the HIV-2 Env motif was position dependent by inserting the GYRPV motif at various sites within the CD8 tail and assessing the ability of the chimeric proteins to enhance virus release (Fig. 2). Interestingly, we obtained only a fully functional protein when the motif was placed in the analogous position to its native location in the HIV-2 Env, i.e., at 5 to 9 amino acids downstream from the membrane-spanning region. The insertion of the motif at more distal sites did not result in any EVR activity. These data are in agreement with a model whereby the GYRPV motif acts to recruit AP-2 and is a sorting signal for both endocytosis and subsequent trafficking towards lysosomes. Furthermore, they indicate that that the only contribution of the HIV-2 Env cytoplasmic tail to EVR activity is to provide a correctly positioned GYRPV sequence.
HIV-2 Env and Vpu EVR activities are independent from L domain activity. It is increasingly clear that retrovirus assembly and budding use the cell's protein trafficking and sorting machinery. L domains interact with various members of the class E Vps pathway, and both AP-3 and AP-2 have been implicated in HIV-1 Gag trafficking and release (3, 16). Several different classes of L domains have been identified, including Yxx motifs in EIAV and influenza virus. Interestingly, the EIAV L domain has been reported to interact with both AP-2 (41) and AIP1/ALIX (28, 51) and EIAV is unusual among the lentiviruses in that its cytoplasmic tail does not contain a membrane-proximal Yxx motif. We therefore considered the possibility that the membrane-proximal GYxx motif of the HIV-2 Env and the Yxx motif in the EIAV L domain could both be functioning to enhance virus release through similar mechanisms involving the recruitment of AP-2 and that the HIV-2 Env EVR activity was therefore analogous to L domain activity.
To test this hypothesis, we examined whether the HIV-2 Env or Vpu could compensate for the loss of L domain activity in either HIV-1 or EIAV particles and, conversely, whether L domain mutants of these two viruses were still responsive to HIV-2 Env or Vpu. HeLa cells were transfected with either wild-type or L domain mutants of HIV-1 and EIAV and cotransfected with expression plasmids for either Vpu or HIV-2 Env. For the HIV-1 particles, we used a previously reported specific mutation of the PTAP L domain sequence (13), while for EIAV, we used proviral clones of either the wild-type virus or a YPDLSRSA L domain mutant (25). For both HIV-1 and EIAV, as was expected, the loss of the L domain significantly reduced overall virus production, giving only 14 and 12%, respectively, of the amount of particles released when the L domain sequences were present. Despite the lower levels of release, both mutant viruses remained fully responsive to the enhancing effects of Vpu and HIV-2 Env (Fig. 3). However, even when stimulated by the presence of the EVRs, the level of release of the mutant constructs was increased to only 37 to 49% of the level produced by the wild-type constructs in the absence of EVRs, indicating that the loss of an L domain cannot be fully compensated for by EVR activity. In addition, the characteristic processing defect in the HIV-1 L domain mutant that leads to an increase in the level of p25 was not corrected by coexpression of an EVR (Fig. 3A). Taken together, these data suggest that L domain and EVR activities are independent.
Mutation of Y707 or depletion of AP-2 alters the cellular distribution of the HIV-2 Env. We speculated that the interaction between the GYPRV motif and AP-2 contributed to HIV-2 Env EVR function by targeting the protein to a specific cellular pathway or location necessary for its activity. Our previously reported finding of a requirement for the upstream glycine residue (1), combined with the position dependence we observed in the present study, suggests that this motif acts to target Env towards lysosomal compartments, possibly including the late endosomes/MVBs that can serve as sites of retrovirus budding (31, 33, 35, 49).
As a first analysis, we examined the overall pattern of Env distribution in HeLa cells and compared the wild-type protein to a defective tyrosine mutant, HIV-2Y707A (1). For each Env protein, we saw both cells showing a predominantly punctate pattern of Env distribution throughout the cell and others with a mostly diffuse distribution. However, the relative amounts of each pattern differed considerably; the wild-type Env was observed in a punctate distribution in 87% of the cells examined, while only 41% of the cells expressing the Y707A mutant showed this pattern (Fig. 4A). In addition, we noted that the punctate spots for the Y707A mutant were considerably larger than those observed for the wild-type Env (Fig. 4C).
We also analyzed the pattern of staining for the wild-type HIV-2 Env protein when AP-2 was depleted by RNA interference. Similar to the pattern resulting from the Y707A mutation, we observed that the loss of AP-2 caused a significant decrease in the percentage of cells showing a punctate pattern (Fig. 4D and E).
The MLV Env tail can substitute for the HIV-2 tail, despite not using its Yxx motif or requiring AP-2. We next asked whether the presumed trafficking function of the HIV-2 Env cytoplasmic tail could be substituted by the cytoplasmic domains of another retroviral Env protein. Accordingly, we constructed a chimeric protein containing the MLV Env tail [protein E2M2T(MLV)] and observed that this protein also supported wild-type levels of EVR activity (Fig. 5).
The MLV Env cytoplasmic tail contains a Yxx motif, located in the C-terminal half of the tail, which is referred to as the R peptide. This peptide is proteolytically cleaved from the Env protein during virion maturation, an event that increases the fusogenic potential of the Env protein (42, 43). Although the MLV motif is positioned 22 to 25 residues from the membrane and therefore does not fit the criteria for functionality that we had established with the HIV-2 Env/CD8 chimeras, we examined whether it played a role in the EVR activity of the chimeric protein. However, the mutation of the tyrosine had no effect on EVR activity (Fig. 5). This finding suggested that the MLV tail may not have an AP-2 interacting motif and therefore that AP-2 might not be required for the activity of the chimeric protein. In order to address this possibility, we examined whether the E2M2T(MLV) Env was affected by the loss of AP-2 by RNA interference and found that the chimeric Env was insensitive to the loss of AP-2 (Fig. 5).
Finally, we examined the overall pattern of cellular distribution of E2M2T(MLV) Env and found that it differed from the wild-type Env, as only 33% of the cells exhibited a punctate staining pattern, with spots that were often smaller and less distinct than those we observed with the wild-type Env (see the supplemental material). Together, these results indicate that, while the HIV-2 Env tail provides an essential function that utilizes AP-2, the same activity can be provided by the MLV Env tail through a different mechanism that results in a different gross cellular distribution.
Do EVR-active proteins [HIV-2 Env and E2M2T(MLV)] share common cellular locations Despite the fact that the wild-type Env and the functional E2M2T(MLV) chimera gave distinct staining patterns when expressed in HeLa cells, it remained possible that they were present in similar subcellular locations that are important for EVR activity. To address this question, we examined the extent of colocalization of each of the Env proteins used in this study with subcellular markers. Cells exhibiting both punctate and diffuse staining patterns were examined separately (Table 1; see the supplemental material). This analysis revealed that a significant portion of the wild-type HIV-2 Env was found to colocalize with markers for the Golgi apparatus and the trans-Golgi network (TGN), especially in the punctate-staining cells. We also saw some costaining with the early endosome marker, EEA1, and with LAMP-2 (found in early endosomes, the plasma membrane, late endosomes/MVBs, and lysosomes). HIV-1 budding has been reported to occur at late endosomes/MVBs and tetraspanin-enriched microdomains at the cell surface (34), both of which contain CD63, and the HIV-1 Env has been shown to colocalize with surface CD63 (34). However, we observed no significant overlap with CD63, although it is possible that the sensitivity of our assay is not sufficient to detect such an interaction.
The Y707A mutant of the HIV-2 Env displayed a markedly different distribution than the wild-type Env. In addition to having a more diffuse overall staining pattern, we noticed that, in those cells that did display a punctate staining pattern, the spots appeared larger than those we observed with either the wild-type Env or the E2M2T(MLV) chimera. The Y707A protein exhibited reduced colocalization with the Golgi and the TGN and gave no overlap with the marker EEA1, CD63, or LAMP-2. The marked decrease in punctate staining for the Y707A mutant, together with the loss of EEA1 costaining, is in agreement with the GYxx motif of the HIV-2 Env functioning as an endocytosis signal. It also suggests that endocytosis from the cell surface is a prerequisite for entry of the HIV-2 Env into internal compartments containing EEA1, CD63, or LAMP-2.
As noted above, although the substitution of the MLV tail in chimera E2M2T(MLV) resulted in a fully functional protein, this chimera did not exhibit the same overall staining pattern as the wild-type Env. Only 33% of cells expressing the chimera displayed a punctate distribution, and the spots we observed were often smaller and less distinct than those observed for the wild-type Env. However, despite these gross differences in distribution, the costaining studies with subcellular markers revealed a distribution that resembled that of wild-type Env, with colocalization with markers for the Golgi apparatus, TGN, early endosomes, and LAMP-2 but not with CD63. It is therefore possible that both the HIV-2 and MLV Env cytoplasmic tails contain signals that direct the HIV-2 Env to a common compartment that is necessary for its EVR activity but that, while the HIV-2 tail uses AP-2-mediated endocytosis, the MLV tail uses a different route. Our ongoing studies are aimed at elucidating the pathway used by the MLV cytoplasmic domain.
DISCUSSION
The HIV-2 Env and HIV-1 Vpu proteins provide analogous functions in the HIV life cycle that we refer to as the enhancement of virus release. They act to increase the production of viral particles from various human cell types, including primary lymphocytes and macrophages, and can also stimulate the release of heterologous retroviral particles. The importance of this activity is suggested by the fact that intact Vpu open reading frames are maintained in AIDS patients, and studies of chimeric SIV/HIV infections in macaques have demonstrated that Vpu is essential for pathogenicity (22, 50).
A current model of EVR activity is that human cells contain a restriction factor that suppresses retroviral budding and whose activity is counteracted by the EVRs. This is supported by analyses of the heterokaryons formed between restrictive (human) and nonrestrictive (simian) cell types, where the human cell phenotype of restricted budding/EVR responsiveness is dominant (1, 54). It is something of a philosophical question as to whether EVR activity is viewed as counteracting a host defense mechanism that has evolved to restrict retrovirus budding or as simply reflecting a viral mechanism to override normal cellular checkpoints. Either way, the cellular proteins or pathways that the EVRs target are presently unknown. As part of our investigations into these activities, we are mapping functional domains within the EVR proteins and elucidating their contribution to the activity. In the present study, we show that the cytoplasmic tail of the HIV-2 Env contributes a membrane-proximal GYRPV motif that interacts with AP-2 and which, by its sequence and position dependence characteristics, shares properties with lysosomal targeting motifs.
We speculated that two possible mechanisms could account for the requirement for the GYRPV motif and AP-2 in the HIV-2 Env EVR activity. First, this sequence could be recruiting AP-2 as part of an activity that is similar to L domain function. This hypothesis was based on the fact that the L domain in the EIAV p9 protein contains a Yxx sequence that is capable of interacting with both AIPI/ALIX and AP-2 (41, 51), with both interactions being critical for L domain activity (32). Intriguingly, the EIAV L domain has characteristics that are distinct from other L domains (36, 38, 48) and the cytoplasmic tail of the EIAV Env is unusual among the lentiviruses in having no membrane-proximal Yxx motif. However, our analysis of the response of both wild-type and L domain mutants of EIAV and HIV-1 revealed that all virions were stimulated by both HIV-2 Env and Vpu. Furthermore, although virus production was significantly decreased for both viruses when the L domains were mutated, this could not be fully restored by the addition of an EVR. Taken together, these findings indicate that L domain activity is a distinct process from that of the EVR activity of either HIV-2 Env or Vpu.
The second hypothesis that we explored was that the GYRPV/AP-2 interaction was critical for HIV-2 Env EVR activity by virtue of targeting the protein to a specific cellular location or pathway that was necessary for its activity. In particular, the lysosome-targeting characteristics of the GYPRV sequence are consistent with a model whereby Env is directed to an endosomal/lysosomal compartment, where it may interact with other components involved in virus assembly that are also targeted towards the late endosome/MVB that is proposed to be a site of virus assembly (31, 33, 35, 49). We explored the possibility that a distinct subcellular location is correlated with HIV-2 Env EVR activity by comparing the distributions of both EVR-active proteins [wild-type HIV-2 Env and the E2M2T(MLV) chimera] and the nonfunctional HIV-2Y707A mutant. We observed that the mutation Y707A in the HIV-2 tail as well the loss of AP-2 by RNA interference altered the distribution of Env from a predominantly punctate to a more diffuse staining pattern. Furthermore, the Y707A mutant had significantly less colocalization with markers of the endocytic pathway such as EEA1, which is in keeping with a role for the GYPRV sequence in promoting endocytosis through an AP-2 interaction. However, the finding that the MLV tail could substitute for this activity in a manner that did not use AP-2, that did not depend on a Yxx motif, and that resulted in a cellular distribution that was more diffuse than punctate, suggests that AP-2-mediated endocytosis is not the only route whereby the HIV-2 Env can be directed to such an active cellular compartment. Identification of the cellular partner(s) used by the MLV tail may shed light on the mechanism involved. Finally, it remains a possibility that only a fraction of the Env needs to be localized to a specific site for activity and sufficient protein is correctly targeted by either Env tail. Such an active cellular location may be obscured by the background distribution that we observe for different constructs, especially when we consider that the EVR proteins have other functions in the HIV life cycle. Our ongoing analyses are aimed at elucidating both the functional subcellular compartment for EVR activities and the cellular partners that are involved.
ACKNOWLEDGMENTS
We acknowledge the NIH AIDS Research and Reference Reagent Program for several reagents that were vital to this project. We thank Chaoping Chen and Ronald Montelaro for the EIAV constructs, Markus Thali for helpful discussions, and George McNamara at the CHLA Imaging Core for technical assistance with the confocal microscopy.
This work was supported by Public Health Service grant CA-59318 and the Universitywide AIDS Research Program, ID03-CHLA-036.
Supplemental material for this article may be found at http://jvi.asm.org/.
REFERENCES
Abada, P., B. Noble, and P. M. Cannon. 2005. Functional domains within the human immunodeficiency virus type 2 envelope protein required to enhance virus production. J. Virol. 79:3627-3638.
Balliet, J. W., D. L. Kolson, G. Eiger, F. M. Kim, K. A. McGann, A. Srinivasan, and R. Collman. 1994. Distinct effects in primary macrophages and lymphocytes of the human immunodeficiency virus type 1 accessory genes vpr, vpu, and nef: mutational analysis of a primary HIV-1 isolate. Virology 200:623-631.
Batonick, M., M. Favre, M. Boge, P. Spearman, S. Honing, and M. Thali. 2005. Interaction of HIV-1 Gag with the clathrin-associated adaptor AP-2. Virology 342:190-200.
Boge, M., S. Wyss, J. S. Bonifacino, and M. Thali. 1998. A membrane-proximal tyrosine-based signal mediates internalization of the HIV-1 envelope glycoprotein via interaction with the AP-2 clathrin adaptor. J. Biol. Chem. 273:15773-15778.
Bonifacino, J. S., and L. M. Traub. 2003. Signals for sorting of transmembrane proteins to endosomes and lysosomes. Annu. Rev. Biochem. 72:395-447.
Bour, S., U. Schubert, K. Peden, and K. Strebel. 1996. The envelope glycoprotein of human immunodeficiency virus type 2 enhances viral particle release: a Vpu-like factor J. Virol. 70:820-829.
Bour, S., and K. Strebel. 1996. The human immunodeficiency virus (HIV) type 2 envelope protein is a functional complement to HIV type 1 Vpu that enhances particle release of heterologous retroviruses. J. Virol. 70:8285-8300.
Bour, S. P., C. Aberham, C. Perrin, and K. Strebel. 1999. Lack of effect of cytoplasmic tail truncations on human immunodeficiency virus type 2 ROD Env particle release activity. J. Virol. 73:778-782.
Carr, J. M., H. Hocking, P. Li, and C. J. Burrell. 1999. Rapid and efficient cell-to-cell transmission of human immunodeficiency virus infection from monocyte-derived macrophages to peripheral blood lymphocytes. Virology 265:319-329.
Cervantes-Acosta, G., R. Lodge, G. Lemay, and E. A. Cohen. 2001. Influence of human immunodeficiency virus type 1 envelope glycoprotein YXXL endocytosis/polarization signal on viral accessory protein functions. J. Hum. Virol. 4:249-259.
Chong, Y. H., S. L. Payne, C. J. Issel, R. C. Montelaro, and K. E. Rushlow. 1991. Characterization of the antigenic domains of the major core protein (p26) of equine infectious anemia virus. J. Virol. 65:1007-1012.
Demirov, D. G., and E. O. Freed. 2004. Retrovirus budding. Virus Res. 106:87-102.
Demirov, D. G., J. M. Orenstein, and E. O. Freed. 2002. The late domain of human immunodeficiency virus type 1 p6 promotes virus release in a cell type-dependent manner. J. Virol. 76:105-117.
Deora, A., and L. Ratner. 2001. Viral protein U (Vpu)-mediated enhancement of human immunodeficiency virus type 1 particle release depends on the rate of cellular proliferation. J. Virol. 75:6714-6718.
Deschambeault, J., J. P. Lalonde, G. Cervantes-Acosta, R. Lodge, E. A. Cohen, and G. Lemay. 1999. Polarized human immunodeficiency virus budding in lymphocytes involves a tyrosine-based signal and favors cell-to-cell viral transmission. J. Virol. 73:5010-5017.
Dong, X., H. Li, A. Derdowski, L. Ding, A. Burnett, X. Chen, T. R. Peters, T. S. Dermody, E. Woodruff, J. J. Wang, and P. Spearman. 2005. AP-3 directs the intracellular trafficking of HIV-1 Gag and plays a key role in particle assembly. Cell 120:663-674.
Ewart, G. D., K. Mills, G. B. Cox, and P. W. Gage. 2002. Amiloride derivatives block ion channel activity and enhancement of virus-like particle budding caused by HIV-1 protein Vpu. Eur. Biophys. J. 31:26-35.
Fais, S., M. R. Capobianchi, I. Abbate, C. Castilletti, M. Gentile, P. Cordiali Fei, F. Ameglio, and F. Dianzani. 1995. Unidirectional budding of HIV-1 at the site of cell-to-cell contact is associated with co-polarization of intercellular adhesion molecules and HIV-1 viral matrix protein. AIDS 9:329-335.
Geraghty, R. J., K. J. Talbot, M. Callahan, W. Harper, and A. T. Panganiban. 1994. Cell type-dependence for Vpu function. J. Med. Primatol. 23:146-150.
Gheysen, D., E. Jacobs, F. de Foresta, C. Thiriart, M. Francotte, D. Thines, and M. De Wilde. 1989. Assembly and release of HIV-1 precursor Pr55gag virus-like particles from recombinant baculovirus-infected insect cells. Cell 59:103-112.
Gottlinger, H. G., T. Dorfman, E. A. Cohen, and W. A. Haseltine. 1993. Vpu protein of human immunodeficiency virus type 1 enhances the release of capsids produced by gag gene constructs of widely divergent retroviruses. Proc. Natl. Acad. Sci. USA 90:7381-7385.
Hout, D. R., E. R. Mulcahy, E. Pacyniak, L. M. Gomez, M. L. Gomez, and E. B. Stephens. 2004. Vpu: a multifunctional protein that enhances the pathogenesis of human immunodeficiency virus type 1. Curr. HIV Res. 2:255-270.
Iida, S., T. Fukumori, Y. Oshima, H. Akari, A. H. Koyama, and A. Adachi. 1999. Compatibility of Vpu-like activity in the four groups of primate immunodeficiency viruses. Virus Gene 18:183-187.
Jolly, C., K. Kashefi, M. Hollinshead, and Q. J. Sattentau. 2004. HIV-1 cell to cell transfer across an Env-induced, actin-dependent synapse. J. Exp. Med. 199:283-293.
Li, F., C. Chen, B. A. Puffer, and R. C. Montelaro. 2002. Functional replacement and positional dependence of homologous and heterologous L domains in equine infectious anemia virus replication. J. Virol. 76:1569-1577.
Lodge, R., H. Gottlinger, D. Gabuzda, E. A. Cohen, and G. Lemay. 1994. The intracytoplasmic domain of gp41 mediates polarized budding of human immunodeficiency virus type 1 in MDCK cells. J. Virol. 68:4857-4861.
Lodge, R., J. P. Lalonde, G. Lemay, and E. A. Cohen. 1997. The membrane-proximal intracytoplasmic tyrosine residue of HIV-1 envelope glycoprotein is critical for basolateral targeting of viral budding in MDCK cells. EMBO J. 16:695-705.
Martin-Serrano, J., A. Yarovoy, D. Perez-Caballero, and P. D. Bieniasz. 2003. Divergent retroviral late-budding domains recruit vacuolar protein sorting factors by using alternative adaptor proteins. Proc. Natl. Acad. Sci. USA 100:12414-12419.
Morita, E., and W. I. Sundquist. 2004. Retrovirus budding. Annu. Rev. Cell Dev. Biol. 20:395-425.
Motley, A., N. A. Bright, M. N. Seaman, and M. S. Robinson. 2003. Clathrin-mediated endocytosis in AP-2-depleted cells. J. Cell Biol. 162:909-918.
Nguyen, D. G., A. Booth, S. J. Gould, and J. E. Hildreth. 2003. Evidence that HIV budding in primary macrophages occurs through the exosome release pathway. J. Biol. Chem. 278:52347-52354.
Noble, B., and P. M. Cannon. Unpublished observations.
Nydegger, S., M. Foti, A. Derdowski, P. Spearman, and M. Thali. 2003. HIV-1 egress is gated through late endosomal membranes. Traffic 4:902-910.
Nydegger, S., S. Khurana, M. Foti, and M. Thali. High resolution mapping of tetraspanin-enriched microdomains (TEMs) that function as gateways for HIV-1. Submitted for publication.
Ono, A., and E. O. Freed. 2004. Cell-type-dependent targeting of human immunodeficiency virus type 1 assembly to the plasma membrane and the multivesicular body. J. Virol. 78:1552-1563.
Ott, D. E., L. V. Coren, R. C. Sowder, II, J. Adams, K. Nagashima, and U. Schubert. 2002. Equine infectious anemia virus and the ubiquitin-proteasome system. J. Virol. 76:3038-3044.
Owens, R. J., J. W. Dubay, E. Hunter, and R. W. Compans. 1991. Human immunodeficiency virus envelope protein determines the site of virus release in polarized epithelial cells. Proc. Natl. Acad. Sci. USA 88:3987-3991.
Patnaik, A., V. Chau, F. Li, R. C. Montelaro, and J. W. Wills. 2002. Budding of equine infectious anemia virus is insensitive to proteasome inhibitors. J. Virol. 76:2641-2647.
Paul, M., S. Mazumder, N. Raja, and M. A. Jabbar. 1998. Mutational analysis of the human immunodeficiency virus type 1 Vpu transmembrane domain that promotes the enhanced release of virus-like particles from the plasma membrane of mammalian cells. J. Virol. 72:1270-1279.
Phillips, D. M. 1994. The role of cell-to-cell transmission in HIV infection. AIDS 8:719-731.
Puffer, B. A., S. C. Watkins, and R. C. Montelaro. 1998. Equine infectious anemia virus Gag polyprotein late domain specifically recruits cellular AP-2 adapter protein complexes during virion assembly. J. Virol. 72:10218-10221.
Ragheb, J. A., and W. F. Anderson. 1994. pH-independent murine leukemia virus ecotropic envelope-mediated cell fusion: implications for the role of the R peptide and p12E TM in viral entry. J. Virol. 68:3220-3231.
Rein, A., J. Mirro, J. G. Haynes, S. M. Ernst, and K. Nagashima. 1994. Function of the cytoplasmic domain of a retroviral transmembrane protein: p15E-p2E cleavage activates the membrane fusion capability of the murine leukemia virus Env protein. J. Virol. 68:1773-1781.
Ritter, G. D., Jr., G. Yamshchikov, S. J. Cohen, and M. J. Mulligan. 1996. Human immunodeficiency virus type 2 glycoprotein enhancement of particle budding: role of the cytoplasmic domain. J. Virol. 70:2669-2673.
Sakai, H., K. Tokunaga, M. Kawamura, and A. Adachi. 1995. Function of human immunodeficiency virus type 1 Vpu protein in various cell types. J. Gen. Virol. 76:2717-2722.
Schubert, U., K. A. Clouse, and K. Strebel. 1995. Augmentation of virus secretion by the human immunodeficiency virus type 1 Vpu protein is cell type independent and occurs in cultured human primary macrophages and lymphocytes. J. Virol. 69:7699-7711.
Schwartz, M. D., R. J. Geraghty, and A. T. Panganiban. 1996. HIV-1 particle release mediated by Vpu is distinct from that mediated by p6. Virology 224:302-309.
Shehu-Xhilaga, M., S. Ablan, D. G. Demirov, C. Chen, R. C. Montelaro, and E. O. Freed. 2004. Late domain-dependent inhibition of equine infectious anemia virus budding. J. Virol. 78:724-732.
Sherer, N. M., M. J. Lehmann, L. F. Jimenez-Soto, A. Ingmundson, S. M. Horner, G. Cicchetti, P. G. Allen, M. Pypaert, J. M. Cunningham, and W. Mothes. 2003. Visualization of retroviral replication in living cells reveals budding into multivesicular bodies. Traffic 4:785-801.
Stephens, E. B., C. McCormick, E. Pacyniak, D. Griffin, D. M. Pinson, F. Sun, W. Nothnick, S. W. Wong, R. Gunderson, N. E. Berman, and D. K. Singh. 2002. Deletion of the vpu sequences prior to the env in a simian-human immunodeficiency virus results in enhanced Env precursor synthesis but is less pathogenic for pig-tailed macaques. Virology 293:252-261.
Strack, B., A. Calistri, S. Craig, W. Popova, and H. G. Gottlinger. 2003. AIP1/ALIX is a binding partner for HIV-1 p6 and EIAV p9 functioning in virus budding. Cell 114:689-699.
Strebel, K., T. Klimkait, F. Maldarelli, and M. A. Martin. 1989. Molecular and biochemical analyses of human immunodeficiency virus type 1 vpu protein. J. Virol. 63:3784-3791.
Terwilliger, E. F., E. A. Cohen, Y. C. Lu, J. G. Sodroski, and W. A. Haseltine. 1989. Functional role of human immunodeficiency virus type 1 vpu. Proc. Natl. Acad. Sci. USA 86:5163-5167.
Varthakavi, V., R. M. Smith, S. P. Bour, K. Strebel, and P. Spearman. 2003. Viral protein U counteracts a human host cell restriction that inhibits HIV-1 particle production. Proc. Natl. Acad. Sci. USA 100:15154-15159.(Beth Noble, Paolo Abada, )