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Vol. 18, Issue 2, 414-425, February 2007
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*Cell Biology Unit, MRC-Laboratory for Molecular Cell Biology and Department of Biochemistry and Molecular Biology, University College London, London WC1E 6BT, United Kingdom; and Hematology-Oncology Division, Department of Medicine, University of Pennsylvania, Philadelphia, PA 19104
Submitted June 19, 2006;
Revised November 3, 2006;
Accepted November 6, 2006
Monitoring Editor: Jean Gruenberg
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In many cell types, HIV assembles at the plasma membrane and, in the course of Gag assembly, the particles derive their membrane from the plasma membrane. For these particles to be infectious, Env must be transported to the cell surface, but the level to which it is incorporated into budding virions is unclear. Recent data have suggested that fewer than 10 Env protein complexes (trimers) are incorporated per virion (Chertova et al., 2002
; Zhu et al., 2003), and early studies of HIV infected T-cells showed that much of the newly synthesized Env is transported to lysosomes and degraded (Willey et al., 1988). However, it has recently become evident that in macrophages the assembly of Env-containing infectious HIV occurs on intracellular membranes, that have some characteristics in common with late endosomes (Raposo et al., 2002; Pelchen-Matthews et al., 2003). The assembly of virus in distinct locations suggests that Env must contain the necessary trafficking information to ensure that its transport is coordinated with that of Gag. This information, and its appropriate interpretation in the infected cell, is likely to be essential for productive infection and pathogenesis. Indeed, deletion of an Env membrane proximal sorting/endocytosis motif in SIVmac239 enhances the viral cytopathic effect in vitro and abrogates pathogenesis in vivo (Sauter et al., 1996; Fultz et al., 2001).
HIV and SIV Envs are type I integral membrane proteins that are made as 160-kDa precursor proteins on the endoplasmic reticulum, where they undergo trimerization and extensive glycosylation before being exported to the secretory pathway (Braakman and van Anken, 2000). During transport through the secretory pathway, or possibly within an endosomal compartment (Franzusoff et al., 1995), the extracellular (luminal) domain of gp160 is proteolytically cleaved by furin or a furin-like protease to generate the surface unit (SU, gp120) and transmembrane (TM, gp41) proteins of the mature Env glycoprotein. The first 
650 amino acids (depending on the strain of virus) of gp160 form gp120 and the ectodomain of gp41, followed by a short transmembrane domain. The remaining amino acids, from approximately position 705 to the C-terminus, form the relatively long (150 amino acids) cytoplasmic domain. This domain is essential for viral replication and pathogenesis in vivo although, for SIV at least, much of the cytoplasmic domain is unnecessary for growth in culture (Kodama et al., 1989). The cytoplasmic domain is believed to play key roles in virus assembly and has been considered important for Env incorporation into virions and for Env interactions with the N-terminal matrix (MA) domain of HIV Gag (Cosson, 1996; Vincent et al., 1999).
Previously, we analyzed the trafficking properties of SIVmac239 Env using chimeras containing the ectodomain and transmembrane domains of human CD4 fused to the cytoplasmic domain of Env, as well as native Env expressed from an alpha virus expression vector (LaBranche et al., 1995; Sauter et al., 1996; Bowers et al., 2000). The CD4 chimeras offered the advantage that, while retaining similar trafficking properties to the native Env protein, they were more amenable to expression in stable cell lines, and as a consequence, to biochemical and morphological analysis (Sauter et al., 1996; Bowers et al., 2000). Using these approaches, we demonstrated that a conserved GYxxØ motif (where x is any amino acid and Ø is an amino acid with a large hydrophobic side chain) in the cytoplasmic domain of SIV Env can function as an endocytosis signal. The activity of this signal is dependent on a membrane proximal tyrosine (Y723 in SIVmac239) that is highly conserved in all HIV-1, HIV-2, and SIV isolates (LaBranche et al., 1995). The corresponding tyrosine in HIV-1 Env (Y712 in HIV-1HxB2) also appears to function as an endocytosis signal (Rowell et al., 1995; Ohno et al., 1997; Boge et al., 1998) and as a basolateral targeting motif in polarized cells (Lodge et al., 1997). In addition to the GYxxØ motif, SIV Env contains at least one more endocytosis signal that remains to be mapped in detail (Bowers et al., 2000). Previous studies had indicated that HIV and SIV Envs interacts with clathrin adaptor complexes and that the membrane proximal GYxxØ motif can bind both AP-1 and AP-2 adaptors (Bowers et al., 2000; Wyss et al., 2001). In addition, a conserved dileucine motif in the C-terminus of HIV-1HxB2 Env TM can also bind the clathrin adaptor complexes, though the role of this interaction has remained obscure (Ohno et al., 1997; Boge et al., 1998; Berlioz-Torrent et al., 1999; Wyss et al., 2001).
Using HIV-1HxB2 Env, and CD4-HxB2 Env cytoplasmic domain chimeras, we now show that the C-terminal dileucine motif also mediates endocytosis, that the activity of this motif is functionally equivalent to that of the membrane proximal GYxxØ motif, and that the two signals operate independently and their activities are not additive. In the presence of either motif Env accumulates in intracellular organelles and only remains on the cell surface when both motifs are functionally defective. RNAi knockdown experiments indicate that both motifs operate through interaction with AP-2 and the clathrin-mediated endocytic pathway. This functional redundancy in endocytosis signals suggests that internalization of cell surface Env is important for viral pathogenesis. Moreover, as a functional GYxxØ motif is required for SIV pathogenesis (Fultz et al., 2001), the membrane proximal motif may have roles other than endocytosis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) was obtained from Dr. Q. Sattentau through the Centralised Facility for AIDS Reagents at the National Institute for Biological Standards and Control (Potters Bar, United Kingdom). The recombinant human mAb 2G12 (IgG1) against HIV gp120 (Buchacher et al., 1994) was obtained from Dr. H. Katinger through the same program. MAbs against clathrin heavy chain (# 23, IgG1) and the µ2 subunit of AP-2 (AP50, IgG1) were purchased from BD Transduction Laboratories (Mannheim, Germany), and the mAb against 
-adaptin (100/3, IgG2b) was from Sigma-Aldrich. Human transferrin coupled to Alexa Fluor 594 (594Tf) was purchased from Molecular Probes Invitrogen (Paisley, United Kingdom). Alexa Fluor 488– and 594–conjugated goat anti-mouse IgG and isotype specific anti-IgG1 and IgG2b antibodies were from Molecular Probes Invitrogen. Goat anti-human IgG-FITC was purchased from Perbio Science United Kingdom (Cheshire, United Kingdom).
Q4120 was radioiodinated using 125I-labeled 3-(p-hydroxyphenyl)-propionic acid N-hydroxy-succinimide ester (Bolton and Hunter reagent) essentially as described (Pelchen-Matthews et al., 1998). Specific activity was 500 Ci/mmol. The radioiodinated protein was stored in 100-µl aliquots at –20°C and was stable for several months.
HeLa cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) containing 10% fetal calf serum (FCS; Biowest, Nuaille, France), 100 U/ml penicillin, and 0.1 mg/ml streptomycin. For stable cell lines, expressing CD4-HIV Env constructs, the medium was supplemented with 750 µg/ml G418.
Plasmids and Transfection
Chimeric proteins containing the CD4 ectodomain and membrane-spanning domains linked to the cytoplasmic domain of HxB2 Env (see Figure 2) were made using a unique HindIII restriction site engineered into the pT4b molecular clone of CD4 (provided by D. Littman, New York University, New York) at nt 1351 such that four residues from the CD4 cytoplasmic domain (–RCRH) were included in the constructs, as previously described (Sauter et al., 1996; Bowers et al., 2000). These residues act as a spacer to place the HIV Env membrane proximal endocytosis motif at a requisite distance from the membrane for functional activity (Jing et al., 1990). Cloning of the CD4 fragment into pSP65 has been described (Sauter et al., 1996). Restriction sites for HindIII and EcoRI were introduced into the HIV-1HxB2 Env cytoplasmic domain by PCR with the forward primer 5'-ATAGTGAATAAGCTTAGGCAGGGATATTCACCATTA-3' and the reverse primer 5'-GCCGAATTCTTATAGCAAAATCCTTTCCAAGCCCTGTCTTATTC-3'. The fragment was digested with HindIII and EcoRI and cloned into pSP64 (Promega Biotec, Southampton, United Kingdom). The CD4-containing sp65 and HIV Env cytoplasmic domain–containing sp64 plasmids were then digested with PvuI and HindIII and ligated to create an EcoRI fragment encoding CD4/HIV Env chimeric proteins. Site-specific mutagenesis using the Quickchange system (Stratagene, Amsterdam, The Netherlands) was used to convert the HindIII site to the original sequence using the forward primer 5'AGGTGCCGGCACCGAAGAGTTAGGCAGGGATATTCACC-3' and the reverse primer 5'- GGTGAATATCCCTGCCTAACTCTTCGGTGCCGGCACCT-3' according to the manufacturer's instructions. The constructs were cloned into the expression vector pCR3.1 (Invitrogen) using a KpnI restriction site, introduced by PCR, upstream of the CD4 gene and the EcoRI site 3' of the HIV Env sequence. Mutations of potential trafficking motifs (see Figure 2) were introduced using the Quickchange system. Short tail constructs were generated by mutating the Gly codon at the position equivalent to 726 in HxB2 to a stop codon using the same method. All constructs were verified by sequencing and proteins with an appropriate molecular mass were identified by Western blotting for the CD4 domain.
Plasmids containing the genes encoding the CD4/HIV Env chimeras were transfected into HeLa cells using FuGENE (Roche, Lewes, United Kingdom) according to the manufacturer's instructions. Stable transfectants were selected using G418 (750 µg/ml). Selected colonies were expanded and screened for expression by immunofluorescence using anti-CD4 antibodies.
A pSVIII plasmid encoding HIV-1HxB2 gp160 was provided by Dr. Robin Weiss (Wohl Virion Center, Windeyer Institute, UCL, London) and has been described (Helseth et al., 1990). Mutations of potential trafficking motifs (see Figure 2) were introduced using Quickchange mutagenesis. Env expression plasmids were transfected into HeLa cells using FuGENE according to the manufacturer's instructions and expressed for 48 h before analysis. Sixteen hours before analysis the transfected cells were treated with 5 mM sodium butyrate to enhance Env expression.
Endocytosis Assays
Quantitative endocytosis assays were performed essentially as described (Pelchen-Matthews et al., 1991). Briefly, 1.2–1.8 x 105 CD4/Env expressing cells were seeded in 16-mm-diameter wells in 24-well plates and grown to confluency over 2 d. The cells were cooled on ice, washed with BM (RPMI-1640 without bicarbonate, containing 0.2% bovine serum albumin [BSA] and 10 mM HEPES, pH 7.4), and incubated with 250 µl BM containing 0.5 nM 125I-Q4120 for 2 h on ice. Free antibody was then washed away, and samples set aside to determine the cell surface levels of CD4/Env. The remaining cells were then warmed to 37°C by immersion of the plates into 37°C BM. At the indicated times the plates were transferred to 4°C BM. For each time point at least four wells were used. For half of the wells, the cells were washed in 4°C PBS and harvested directly in 400 µl 0.2 M NaOH and transferred to tubes for -counting to determine the total cell-associated radioactivity. To determine the intracellular activity, the remaining cells were rinsed twice with 0.5 ml 4°C BM adjusted to pH 2.3 and then incubated twice for 3 min with the same medium to remove cell surface–bound antibody. These cells were then harvested in NaOH as above. The proportion of the internalized activity for each time point was determined by dividing the acid-resistant activity by the total cell-associated activity, and the endocytosis rates were calculated by analysis of data from the first 5 min of warm-up as described (Bowers et al., 2000).
RNAi Knockdown
Small interfering RNAs (siRNA) against the µ1 subunit of AP-1 (Hirst et al., 2003) and the µ2 subunit of AP-2 (Fraile-Ramos et al., 2003) have been described and were purchased from Xeragon (Zurich, Switzerland). Cells were transfected with siRNA by nucleofection (Amaxa, Koeln, Germany) using the nucleofection program A-28 for HeLa cells. Typically a 70–80% confluent 10-cm dish of cells was used for up to four nucleofection reactions. For µ1 knockdown, the cells were transfected with 300 pmol of siRNA, and the entire reaction was plated onto one 10-cm dish and incubated for 3 d at 37°C. The cells were then nucleofected again with 150 pmol siRNA and incubated for a further 3 d before analysis (Lui-Roberts et al., 2005). For µ2 knockdown, cells were nucleofected once with 150 pmol siRNA, plated directly onto coverslips, and incubated for 56 h before analysis by antibody- and transferrin-uptake experiments.
Antibody and Transferrin Uptake
For antibody feeding, HeLa cells expressing CD4/HIV Env constructs or HxB2 Env were grown on coverslips for 2 d and then washed twice in BM and incubated for 15min to 2h, as indicated, in 37°C BM containing 4.5 µg/ml anti-CD4 (Q4120) or 20 µg/ml anti-gp120 (2G12). The cells were then cooled on ice, washed twice in 4°C BM to remove unbound antibody, fixed in 2.5% formaldehyde, and processed for immunofluorescence microscopy.
For transferrin (Tf) feeding experiments, µ2 siRNA-transfected HeLa cells were washed with BM and incubated for 30 min at 37°C to remove endogenous Tf. The cells were then incubated in BM containing 200 nM Tf coupled to Alexa594 (Molecular Probes, Invitrogen) at 37°C. After 10 min the coverslips were placed on ice and washed with cold PBS. The cells were then fixed and processed for immunofluorescence microscopy. Alternatively, 594Tf was added for the last 10 min of antibody feeding.
Immunofluorescence
Cells on glass coverslips were fixed for 20 min with 2.5% formaldehyde in serum-free medium supplemented with 20 mM HEPES (pH 7.0) at room temperature. The cells were then washed twice with serum-free medium containing 20 mM HEPES, followed by incubation in phosphate-buffered saline containing 1 mM Mg2+ and 0.5 mM Ca2+ (PBS2+) at 4°C for 10 min. Subsequently, the cells were permeabilized and blocked in PBS2+ containing 20% fetal calf serum, 15 mM glycine, 20 mM HEPES, and 0.05% saponin for 15 min at room temperature. For surface staining, the same buffer without saponin was used. All subsequent incubations and washing steps were carried out in the permeabilization/blocking buffer. The cells were incubated for 45 min at room temperature with antibodies against CD4 or gp120. After extensive washing, the cells were incubated at room temperature in the dark for 30 min with Alexa-labeled secondary antibodies. The coverslips were then washed five times in permeabilization buffer, twice in PBS2+, and quickly in water before mounting on glass slides with Mowiol. The cells were examined at ambient temperature through a 60x oil immersion lens (NA 1.4) on a Nikon Optiphot 2 microscope (Kingston upon Thames, United Kingdom) fitted with a MRC 1024 confocal laser scanner (Bio-Rad Laboratories, Hemel Hempstead, Herts, United Kingdom). Images were acquired using the Bio-Rad Lasersharp software and imported into Adobe Photoshop CS2 and Illustrator CS2 (San Jose, CA) to generate figures.
Western Blotting
Mock and µ2 siRNA transfected cells were cultured in six-well plates, and the cells were scraped into 50 µl lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 5 mM EDTA, 5% glycerol, 1% Triton X-100, complete protease inhibitor cocktail [Roche, Lewes, United Kingdom]) and lysed for 30 min on ice. Cellular debris were removed by centrifugation at 13,200 rpm for 15 min in a bench top microfuge, and the supernatants were diluted 3:1 in 4x nonreducing SDS-PAGE sample buffer. The samples were separated on 10% SDS-PAGE gels, transferred to nitrocellulose, and analyzed essentially as described (Fraile-Ramos et al., 2001).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Ohno et al., 1997; Boge et al., 1998; Wyss et al., 2001). However, mutation of Y712 did not abrogate internalization, indicating that additional motifs C terminal to Y712 must be also capable of mediating endocytosis (Rowell et al., 1995). Moreover, in in vitro assays, the GY712xxØ motif as well as two dileucine motifs at positions 814/815 and 855/856 in the cytoplasmic domain can bind the µ1 subunit of the clathrin adaptor AP-1 (Ohno et al., 1997; Boge et al., 1998; Berlioz-Torrent et al., 1999; Wyss et al., 2001).
To characterize the signals mediating Env trafficking and endocytosis, we analyzed the distribution of HxB2 Env (referred to as construct "Y wt") in HeLa cells by immunofluorescence 48 h after transfection. We stained permeabilized cells with a human mAb that recognizes a carbohydrate epitope on gp120 (2G12) and reacts specifically with post-ER forms of the protein (Buchacher et al., 1994). We found that little Env was expressed at the plasma membrane and that the bulk of the protein localized to the perinuclear area of the cell in a vesicular pattern (Figure 1A, top left panel). To further analyze Env distribution, we performed antibody uptake assays. HeLa cells transiently expressing Env constructs were incubated with 2G12 for 2 h at 37°C and then fixed and labeled, intact or after permeabilization, with a secondary FITC-coupled anti-human antibody. This protocol allowed us to examine exclusively the Env population that had reached the plasma membrane and either remained at this site or undergone endocytosis. Little labeling was seen on intact cells (not shown), but on permeabilized cells a labeling pattern similar to that observed above was seen (top left panel in Figure 1B). These observations indicated that the steady state distribution of Env was achieved, at least in part, by transport of the protein to the plasma membrane and subsequent endocytosis.
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The C-terminal Dileucine Acts as an Endocytosis Signal
To compare the functional activities of the GYxxØ and LL855/856 motifs as endocytosis signals, we determined the internalization rates of proteins carrying either one or both of the putative signals. Because gp160 constructs are not expressed efficiently in stable cell lines, reproducible biochemical assays with native Env are not currently feasible. We therefore chose to use chimeric proteins, where the cytoplasmic domain of HxB2 gp41 was fused to the luminal and membrane spanning domains of CD4, as previously described for SIV Env (Sauter et al., 1996; Bowers et al., 2000). The set of constructs used is shown in Figure 2. We also included two short tail variants, where a stop codon was introduced to replace the G726 codon. Thus, construct "CD4-Y short" contained only the membrane proximal GYxxØ signal, and "CD4-I short" was not expected to contain any endocytosis information, similar to SIVCP-mac Env that contains a Tyr to Cys mutation on the background of a truncated cytoplasmic tail (LaBranche et al., 1994). These constructs were used to generate stable HeLa cell lines.
To confirm that the CD4-Env chimeras are a reliable model for native Env, the subcellular localization of the chimeras was analyzed by immunofluorescence (Figure 3). As expected, staining for a chimera containing the native HxB2 Env tail (CD4-Y wt) showed almost no surface labeling and localized to the perinuclear area of the transfected cells. This was also the case for CD4-Y712I, CD4-Y + LL814/815AA, and CD4-Y + LL855/56AA (Figure 3A). CD4-Y short localized predominantly to internal vesicles, but these were more dispersed than for CD4-Ywt, suggesting that the cytoplasmic domain may contain information crucial for correct intracellular trafficking. Mutation of LL814/815AA in combination with Y712I (CD4-I + LL814/815AA) also generated a protein that was predominantly intracellular, though the distribution of this chimera appeared to be more disperse and present at the plasma membrane to a greater extent than the corresponding Env variant. The CD4-I short and CD4-I + LL855/856AA constructs were localized predominantly at the plasma membrane as expected, because these mutants should lack both of the putative endocytosis motifs (Figure 3A).
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To measure the endocytosis activity of the different constructs, we used 125I-Q4120 in endocytosis assays, as previously described (Pelchen-Matthews et al., 1991). Representative endocytosis curves for all constructs are shown in Figure 4B, and the results of several experiments are summarized in Table 1. Chimeras containing the complete HIV Env cytoplasmic domain (CD4-Ywt) were internalized rapidly (6%/min in the first 5 min after warming to 37°C) and to a high extent (80% of the total after 60 min; Figure 4B and Table 1). A similar endocytosis rate was also seen for CD4-Y short, confirming that the GYxxØ signal can mediate efficient internalization as previously shown (Boge et al., 1998; Wyss et al., 2001). However, mutation of Y712 to I did not affect the rates of uptake of constructs with a full-length cytoplasmic domain, indicating the presence of a second signal capable of mediating efficient internalization. By contrast, the CD4-I short construct was internalized slowly (approximately 0.8%/min; Table 1 and Figure 4B) and to low levels (10% of the total after 60 min) comparable to the bulk flow endocytosis of CD4 molecules lacking a cytoplasmic domain (Pelchen-Matthews et al., 1992; Pitcher et al., 1999). These results were consistent with the high surface expression of this construct (Figures 3 and 4A). For LL814/815AA mutants, with either Y712 or Y712I, the rates and extents of internalization were reduced only moderately (i.e., 20%) compared with CD4-Y wt, indicating that the CD4-I + LL814/815AA construct retained a functional endocytosis motif. By contrast, the CD4-I + LL855/856AA construct showed a reduced internalization rate similar to that found for CD4-I short, indicating that the functional endocytosis motifs had been removed.
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Endocytosis of CD4/HIV Env Chimeras Is Dependent on the Clathrin Adaptor AP-2
It was previously shown that HIV and SIV Env cytoplasmic domains can bind the clathrin AP-1 and AP-2 adaptor complexes through the conserved GYxxØ motif (Ohno et al., 1997; Boge et al., 1998; Berlioz-Torrent et al., 1999; Bowers et al., 2000; Wyss et al., 2001). Pulldown assays have also suggested that LL814/815 and LL855/856 bind AP-1 (Wyss et al., 2001), but no previous evidence has suggested that either of these motifs bind AP-2 or that the C terminal LL motif can mediate endocytosis of HIV Env. To examine the functional activities of these signals in more detail, we used siRNA against the µ2 subunit of AP-2, which have been demonstrated to inhibit AP-2–mediated endocytosis (Fraile-Ramos et al., 2003; Motley et al., 2003). HeLa cells expressing CD4-Env constructs were treated with the siRNA, and the levels of µ2 expression were analyzed 56 h after nucleofection by Western blot. To check protein loading, blots were also probed with an anti-clathrin heavy-chain antibody. Typically siRNA reduced the expression of µ2 by 80% (Figure 5A). To test whether loss of µ2 affected endocytosis, siRNA and mock-treated cells were incubated with Alexa 594–coupled human diferric transferrin (594Tf) for 10 min at 37°C, and the distribution of 594Tf was assessed by fluorescence microscopy. In mock-treated cells, Tf was seen within endocytic vesicles, with little labeling at the cell surface (Figure 5B). By contrast, the majority of µ2 siRNA-treated cells showed Tf accumulation at the plasma membrane, suggesting that endocytosis was inhibited. A minor fraction of siRNA treated cells did internalize Tf, suggesting that these cells had not taken up the siRNA or that AP-2 knockdown was incomplete (Figures 5, B and C, and 6A).
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Analysis of the steady state distribution could not determine whether the increased plasma membrane expression was indeed due to a reduction in endocytosis or resulted from an increase in transport to the plasma membrane. We therefore performed antibody uptake experiments, incubating mock- and µ2 siRNA-transfected CD4-Env–expressing HeLa cells with anti-CD4 for 15 min and adding 594Tf for the last 10 min of incubation. In mock-treated cells the majority of the antibody labeled CD4-Env was internalized within the 15-min incubation (Figure 6A) and gave a distribution similar to that seen for the steady state staining (Figure 5B). Efficient internalization was also observed for the construct with the wt cytoplasmic domain as well as for the two mutants lacking one of the two internalization motifs. µ2 siRNA treatment led to accumulation of CD4-Env at the plasma membrane, indicating that AP-2 is required for efficient endocytosis (Figure 6A). This effect was observed for all three constructs, providing evidence for a role of AP-2 in Env internalization mediated by either the GYxxØ or the C-terminal dileucine motif. However, when antibody feeding was extended to 30 min or longer, internal pools of CD4-Env were also seen in the siRNA-treated cells (Figure 6B). Whether this uptake was due to some remaining AP-2 activity or reflects either AP-2–independent clathrin-mediated endocytosis (Motley et al., 2003) or a clathrin-independent mechanism for Env internalization is unclear.
To analyze whether the previously described interaction between HIV Env and the AP-1 clathrin adaptor and the observed recruitment of AP-1 by HIV Env to the TGN (Wyss et al., 2001) are directly relevant to HIV Env distribution and to examine a role for AP-1 in Env endocytosis, we used siRNA against the µ1 subunit of AP-1 (Hirst et al., 2003). Because the incomplete AP-1 complexes in µ1-deficient cells cannot be recruited onto membranes (Meyer et al., 2000) and the remaining subunits are destabilized, the -subunit of AP-1 became cytosolic in µ1 RNAi treated cells (Figures 7A and 7B; Lui-Roberts et al., 2005), compared with its normal Golgi-associated, perinuclear/vesicular distribution in mock-treated cells. The impact of µ1 depletion on CD4 Env distribution was again assessed by steady state staining (Figure 7A) and antibody feeding (Figure 7B). We found that in both cases CD4 Y wt distribution was similar in mock- and in RNAi-treated cells, with the CD4-Env protein found predominantly in perinuclear vesicles. Thus, in µ1-depleted cells, the CD4-Env protein traffics to the plasma membrane and is then internalized, suggesting that AP-1 is not required for CD4-Env endocytosis and is not essential for its transport to the plasma membrane.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). As an enveloped virus HIV assembles at a membrane interface where oligomerization of Gag complexes is coordinated with the incorporation of Env into budding particles. Thus, it is essential that Env is targeted to virus assembly and budding sites in the cell. However, the precise site of assembly varies in different HIV target cells. In T-cells assembly occurs predominantly at the plasma membrane, whereas in macrophages HIV assembles on intracellular membrane-bound multivesicular organelles (Raposo et al., 2002; Pelchen-Matthews et al., 2003). Thus Env and Gag must contain appropriate trafficking signals to allow infectious particle assembly in distinct locations. Although some knowledge of intracellular Gag trafficking has been gained (Perlman and Resh, 2006; Ryzhova et al., 2006), little is known about Env targeting in infected cells.
Previously, several labs have identified trafficking signals in HIV and SIV Env that mediate endocytosis (LaBranche et al., 1995; Rowell et al., 1995; Bowers et al., 2000; Wyss et al., 2001), polarized sorting (Lodge et al., 1997), and recycling to the TGN (Blot et al., 2003), through interactions with diverse components of the endocytic trafficking machinery including the clathrin-adaptors AP-1 and AP-2 and the putative late endosome-Golgi recycling protein TIP47 (Blot et al., 2003). Of these motifs, the highly conserved membrane proximal GYxxØ motif is the most extensively characterized. This motif binds both AP-1 (Boge et al., 1998; Bowers et al., 2000) and AP-2 adaptors (Ohno et al., 1997; Boge et al., 1998; Berlioz-Torrent et al., 1999; Wyss et al., 2001) and mediates endocytosis from the cell surface (LaBranche et al., 1995; Rowell et al., 1995; Bowers et al., 2000; Wyss et al., 2001). Quantitative experiments with CD4-SIV Env constructs suggest that any Env delivered to the plasma membrane resides there for <5 min before removal by endocytosis (Bowers et al., 2000). Moreover, for SIVmac239 at least, the GYxxØ motif is essential for pathogenesis (Fultz et al., 2001).
Our previous experiments with SIVmac239 Env suggested that, in addition to the conserved GYxxØ signal, more endocytosis information is present in the full-length Env protein. Thus the internalization rate of a CD4-SIV Env chimera was reduced only 50% when the GYxxØ signal was eliminated (Bowers et al., 2000). Here we show that the same is true for HIVHxB2 Env and identify the C-terminal dileucine as the relevant second signal. Dileucine motifs have previously been identified as endocytosis and trafficking signals in studies with cellular proteins, including MHC-II, CD4, and CI-M6PR, and are believed to interact with clathrin adaptors (Marks et al., 1996), though the mode of binding is different from that of GYxxØ motifs (Bonifacino et al., 1996). Indeed the dileucine motifs at positions 814/815 and 855/856 in HxB2 Env have previously been shown to bind AP-1 complexes. However, the roles of these motifs in HIV Env trafficking have remained obscure (Wyss et al., 2001). Although our previous experiments suggested the C-terminal dileucine motif in SIVmac239 Env does not contribute to endocytic trafficking (Bowers et al., 2000), we demonstrate here that the C-terminal dileucine in HxB2 Env clearly acts as an endocytosis signal operating through clathrin (data not shown) and the clathrin adaptor AP-2. This signal is independent of the GYxxØ motif, but is equally efficient, and both the C-terminal dileucine, and the GYxxØ motifs must be removed to allow efficient cell surface expression of Env. Such double mutants show only basal levels of endocytosis, indicating that the two motifs account for all the endocytosis information. In some cases, dileucine-based sorting signals require an acidic amino acid at position –4 and/or –5 relative to the first leucine (Letourneur and Klausner, 1992; Pond et al., 1995). The C-terminal dileucine of HIV-1 has a glutamic acid at position –3 (aa 852 in HxB2). However, mutation of this residue did not change the distribution of Env in cells, nor did it reduce the endocytosis of the CD4-Y712I mutant for which internalization is entirely governed by the dileucine motif (data not shown). By contrast, LL814/815 does not apparently contribute to endocytosis and its role in Env trafficking remains unclear. Potential involvement of LL814/815 in Env targeting to the viral assembly site is currently under investigation. Significantly, our results here suggest that there are differences in the trafficking signals in SIV and HIV Env, which may explain why short tail Env variants emerge during SIV propagation in cultured human T-cell lines, but similar HIV variants do not (Kodama et al., 1989; Luciw et al., 1998; Shacklett et al., 2000).
How trafficking signals in Env are coordinated with Gag in relevant host cells remains to be established. In the main, HIV is thought to assemble at the plasma membrane of T-cells. The finding that endocytosis signals function to maintain low levels of Env on the cell surface may explain why HIV particles collected from cultured T-cell lines have relatively low Env levels (average 7–10 spikes/virion; Chertova et al., 2002; Zhu et al., 2003). In addition, the levels of Env expression on the cell surface and on virions could be a determinant for host humoral immune responses and viral evasion (Yuste et al., 2004, 2005). In contrast, endocytosis signals may play a different role in infected macrophages where HIV assembles on intracellular membranes. Interestingly, the fact that SIVmac239-carrying mutations in the GYxxØ motif is nonpathogenic, even though these proteins continue to undergo endocytosis through the activity of the second yet to be identified signal (Bowers et al., 2000), suggests the GYxxØ signal has additional roles and that its genetic conservation throughout the primate lentiviruses reflects a key functional importance. Further work analyzing the activities of the viral trafficking motifs in cellular systems and animal models will indicate their role in viral assembly and in pathogenesis.
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ACKNOWLEDGMENTS |
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Footnotes |
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Address correspondence to: Mark Marsh (m.marsh{at}ucl.ac.uk)
Abbreviations used: Env, envelope glycoprotein; gp, glycoprotein; HIV, human immunodeficiency virus; SIV, simian immunodeficiency virus; SU, surface subunit of the viral envelope glycoprotein; TM, transmembrane subunit of the viral envelope glycoprotein.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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