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. Author manuscript; available in PMC: 2014 Jul 21.
Published in final edited form as: Virology. 2008 Jun 24;378(1):58–68. doi: 10.1016/j.virol.2008.05.022

Requirements of the Membrane Proximal Tyrosine and Dileucine Based Sorting Signals for Efficient Transport of the Subtype C Vpu Protein to the Plasma Membrane and in Virus Release

Autumn Ruiz 1, M Sarah Hill 1, Kimberly Schmitt 1, John Guatelli 2, Edward B Stephens 1,*
PMCID: PMC4104992  NIHMSID: NIHMS64678  PMID: 18579178

Abstract

Previously, we showed that the Vpu protein from HIV-1 subtype C is more efficiently transported to the cell surface than the well studied subtype B Vpu (Pacyniak et al., 2005) and that a SHIV expressing the subtype C Vpu exhibited a decreased rate of CD4+ T-cell loss following inoculation in macaques (Hill et al., 2007). In this study, we examined the role of overlapping tyrosine-based (YXXM) and dileucine-based ([D/E]XXXL[L/I]) motifs in the membrane proximal region of the subtype C Vpu (EYRKLL) in Vpu intracellular transport, CD4 surface expression and virus release from the cell surface. We constructed three site-directed mutants of the subtype C vpu and fused these genes to the gene for enhanced green fluorescent protein (EGFP). The first mutation made altered the tyrosine (EARKLL; VpuSCEGFPY35A), the second altered the dileucine motif (EYRKLG; VpuSCEGFPL39G), and the third contained both amino acid substitutions (EARKLG; VpuSCEGFPYL35,39AG) in this region of the Vpu protein. The VpuSCEGFPY35A protein was transported to the cell surface similar to the unmodified VpuSCEGFP1 while VpuSCEGFPL39G was expressed at the cell surface at significantly reduced levels. The VpuSCEGFPYL35,39AG was found to have an intermediate level of cell surface expression. All three mutant Vpu proteins were analyzed for the ability to prevent cell surface expression of CD4. We found that both single mutants did not significantly effect at CD4 surface expression while the double mutant (VpuSCEGFPYL35,39AG) was significantly less efficient at preventing cell surface CD4 expression. Chimeric simian-human immunodeficiency viruses were constructed with theses mutations in vpu (SHIVSCVpuY35A, SHIVSCVpuL39G and SHIVSCVpuYL35,39AG). Our results indicate that SHIVSCVpuL39G replicated much more efficiently and was much more cytopathic than SHIVSCVpu. In contrast, SHIVSCVpuY35A and SHIVSCVpuYL35,39AG replicated less efficiently when compared to the parental SHIVSCVpu. Taken together, these results show for the first time that the tyrosine-based sorting motif in the cytoplasmic domain of Vpu is essential for efficient virus release. These results also indicate that the dileucine-based sorting motif affects the intracellular trafficking of clade C Vpu proteins, virus replication, and release.

INTRODUCTION

Human immunodeficiency virus type 1 (HIV-1) and several strains of simian immunodeficiency viruses (SIV) encode for a small protein known as Vpu (Strebel et al., 1988; Cohen et al., 1988; Dazza et al., 2005; Huet et al., 1990; Barlow et al., 2003; Courgnaud et al., 2002; 2003). Vpu is an integral membrane protein with highly conserved and variable domains (Hout et al., 2004; Maldarelli et al., 1993; McCormick-Davis et al., 2000). The Vpu protein from a laboratory-adapted subtype B HIV-1 has been extensively studied with respect to its role in the virus life cycle and has identified two major functions in replication. Vpu interacts with the CD4 receptor in the rough endoplasmic reticulum (RER) and shunts it to the proteasome for degradation (Fujita et al., 1997; Schubert et al., 1998). Studies have shown that the highly conserved hinge region of the cytoplasmic domain of Vpu contains two casein kinase II sites that are required for the CD4 degradation (Paul and Jabbar, 1997; Schubert et al., 1994: Willey et al., 1994). Other studies have shown that the predicted two ∀-helical domains within the cytoplasmic domain and sequences within the transmembrane (TM) domain of the subtype B Vpu are also required for efficient degradation of CD4 (Tiganos et al., 1998; Hout et al., 2006). Additionally, Vpu also enhances virus release from infected cells, which has been associated with the transmembrane domain and its ion channel properties (Cordes et al., 2001; Ewart et al., 1996; 2002; Grice et al., 1997; Klimkait et al., 1990; Schubert et al., 1996a,b). Examination of cells infected with HIV-1 viruses containing large deletions within the vpu gene by electron microscopy revealed a different pattern of virus maturation with many particles tethered together at the cell surface and within intracellular vesicles (Klimkait et al., 1990).

Subtype C HIV-1 accounts for over 50% of the infections worldwide (Takebe et al., 2004; Hemelaar et al., 2006). While the reasons for the rapid spread of these viruses in the human population is currently unknown, several studies have suggested differences between the subtype C HIV-1 and viruses from the other subtypes of HIV-1 group M. In one study, both non-syncytia-inducing (NSI)/R5 and syncytia inducing (SI)/X4 subtype C HIV-1 isolates were found to be significantly less fit in peripheral blood mononuclear cells (PBMC) competition assays than all other group M isolates of the same phenotype (Ball et al., 2003). More recently, in a study that evaluated the replicative fitness of representative strains from subtypes A, B, C, D and CRF01_AE, the subtype C viruses were found to have less replicative fitness in PBMC compared to the other subtypes but were 100 fold more fit in these assays than HIV-2 or group O isolates (Arien et al., 2005). In addition to replication in in vitro assays, the Vpu proteins from subtype C HIV-1 isolates have biological properties and structural features that differ from the well studied subtype B Vpu protein. Previously, we showed that the subtype C Vpu protein was efficiently transported to the cell surface while the subtype B Vpu protein appears to be predominantly localized to intracellular compartments (Pacyniak et al., 2005). Recently, we also showed that the subtype C Vpu was less efficient at preventing surface expression of CD4 than the subtype B protein and that replacement of the subtype B vpu from a pathogenic molecular clone of SHIV (SHIVKU-1bMC33)with the vpu gene from a subtype C clinical isolate resulted in a decreased rate of CD4+ T cell loss following inoculation into macaques (Hill et al., 2008). In this study, we examined the role of the unique overlapping tyrosine (YXXM; with M being an amino acid with a large hydrophobic amino acid) and dileucine ([D/E]XXXL[L/I]) motifs on the intracellular trafficking of the subtype C Vpu, CD4 surface expression and viral release from infected cells. Our results show for the first time that a dileucine-based sorting signal can affect trafficking of the Vpu protein, the extent of cytopathology, the enhanced release function of Vpu and the efficiency of cell surface CD4 surface expression.

RESULTS

Transport to the cell surface is reduced in subtype C Vpu fusion protein with mutations in the dileucine motif

The sequences of the Vpu mutants analyzed in this study are shown in Figure 1. We first analyzed the ability of the Vpu mutants to be transported to the cell surface using two different assays. 293 cells were transfected with vectors expressing either VpuSCEGFP1, VpuSCEGFPY35A, VpuSCEGFPL39G, or VpuSCEGFPYL35,39AG. At 36 hours post-transfection, cells were analyzed by laser scanning confocal microscopy. As shown in Figure 2A–D, the VpuSCEGFP1 and VpuSCEGFPY35A localized at the cell plasma membrane. In contrast, the VpuSCEGFPL39G and VpuSCEGFPYL35,39AG clearly had reduced expression at the cell surface (Figure 2E–H). Of these two constructs, the VpuSCEGFPL39A consistently had the lowest surface expression. As the results in Figure 2 suggested that VpuSCEGFPL39G and VpuSCEGFPYL35,39AG were expressed at reduced levels, we analyzed which compartment these proteins were predominantly localized. 293 cells were transfected with vectors expressing VpuSCEGFP1, VpuSCEGFPY35A, VpuSCEGFPL39G, or VpuSCEGFPYL35,39AG and DsRed2-Golgi. As shown in Figure 3, VpuSCEGFPY35A (Figure E–H) was predominately expressed at the cell surface while the majority of the VpuSCEGFPL39G (Figure 3I–L) and VpuSCEGFPYL35,39AG (Figure 3M–P) colocalized with the DsRed2-Golgi marker. We next analyzed the ratio of the fluorescent signal at the cell surface to that within the Golgi complex as described in the Materials and Methods section. As shown in Figure 4, we found that the ratio of surface to Golgi fluorescent signal was 0.29 for VpuSCEGFP1 and 0.32 for VpuSCEGFPY35A. In contrast, VpuSCEGFPL39G and VpuSCEGFPYL35,39AG had ratios of 0.06 and 0.18, respectively, which were consistent with what was observed in Figure 2. To confirm these findings, transfected cells were surface biotinylated followed by immunoprecipitation with an anti-EGFP antibody. As shown in Figure 5, biotinylated VpuSCEGFP1 (100%) and VpuSCEGFPY35A (110–120%) were easily detected at the cell surface. In contrast, decreased levels of VpuSCEGFPL39G (30%) and VpuSCEGFPYL35,39AG (40%) were found at the cell surface. Together, these results indicate that VpuSCEGFPY35A had slightly higher (but not statistically significant) levels at the cell surface, VpuSCEGFPYL35,39AG had an intermediate level of expression and VpuSCEGFPL39G had the lowest level of surface expression.

Figure 1.

Figure 1

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Sequence of the proteins analyzed in this study. The bolded residues represent the tyrosine and dileucine motifs. The bolded residues in italics represent the amino acid substitutions.

Figure 2.

Figure 2

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Expression of EGFP, VpuSCEGFP1, VpuSCEGFPY35A, VpuSCEGFPL39G, or VpuSCEGFPYL35, 39AG in 293 cells. 293 cells were co-transfected with vectors expressing VpuSCEGFP1, VpuSCEGFPY35A, VpuSCEGFPL39G, VpuSCEGFPYL35, 39AG or EGFP. At 48 hours, cells expressing EGFP were identified and images collected using laser scanning confocal microscopy as described in the Materials and Methods section.

Panel A. Phase image merged with fluorescence micrograph of a cell expressing VpuSCEGFP1. Panel B. Fluorescence micrograph of a cell expressing VpuSCEGFP1. Panel C. Phase image merged with fluorescence micrograph of a cell expressing VpuSCEGFPY35A. Panel D. Fluorescence micrograph of a cell expressing VpuSCEGFPY35A. Panel E. Phase image merged with fluorescence micrograph of a cell expressing VpuSCEGFPL39G. Panel F. Fluorescence micrograph of a cell expressing VpuSCEGFPL39G. Panel G. Phase image merged with fluorescence micrograph of a cell expressing VpuSCEGFPYL35,39AG. Panel H. Fluorescence micrograph of a cell expressing VpuSCEGFPYL35,39A.

Figure 3.

Figure 3

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Expression of EGFP, VpuSCEGFP1, VpuSCEGFPY35A, VpuSCEGFPL39G or VpuSCEGFPYL35, 39AG and DsRed2-Golgi in 293 cells. 293 cells were co-transfected with vectors expressing VpuSCEGFP1, VpuSCEGFPY35A, VpuSCEGFPL39G, VpuSCEGFPYL35,39AG or EGFP and DsRed2-Golgi. At 48 hours, cells expressing EGFP were identified and images collected using laser scanning confocal microscopy as described in the Materials and Methods section.

Panels A–D. 293 cells co-transfected with VpuSCEGFP1 and DsRed2-Golgi. Panel A. Phase contrast image of a cell expressing VpuSCEGFP1 and DsRed2-Golgi. Panel B. Fluorescence micrograph of a cell expressing VpuSCEGFP1. Panel C. Fluorescence micrograph of a cell expressing DsRed2-Golgi. Panel D. Merge of panels B and C. Panels E–H. 293 cells co-transfected with VpuSCEGFPY35A and DsRed2-Golgi. Panel E. Phase contrast image of a cell expressing VpuSCEGFPY35A and DsRed2-Golgi. Panel F. Fluorescence micrograph of a cell expressing VpuSCEGFPY35A. Panel G. Fluorescence micrograph of a cell expressing DsRed2-Golgi. Panel H. Merge of panels F and G. Panels I–L. 293 cells co-transfected with VpuSCEGFPL39G and DsRed2-Golgi. Panel I. Phase contrast image of a cell expressing VpuSCEGFPL39G and DsRed2-Golgi. Panel J. Fluorescence micrograph of a cell expressing VpuSCEGFPL39G. Panel K. Fluorescence micrograph of a cell expressing DsRed2-Golgi. Panel L. Merge of panels J and K.

Panels M–P. 293 cells co-transfected with VpuSCEGFPYL35,39AG and DsRed2-Golgi. Panel M. Phase contrast image of a cell expressing VpuSCEGFPYL35,39AG and DsRed2-Golgi. Panel N. Fluorescence micrograph of a cell expressing VpuSCEGFPYL35,39AG. Panel O. Fluorescence micrograph of a cell expressing DsRed2-Golgi. Panel P. Merge of panels N and O.

Figure 4.

Figure 4

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The membrane to Golgi ratios for VpuSCEGFP1, VpuSCEGFPY35A, VpuSCEGFPL39G, and VpuSCEGFPYL35,39AG. Z-stacks were complied for 42 cells expressing each of the above proteins at 36 hours post-transfection. Using the Zeiss software package, the fluorescence intensity of the cell plasma membrane and Golgi complex were analyzed and the ratio computed. Shown are the mean ratio of membrane to Golgi fluorescence for each of the four constructs. The control for the studies was VpuEGFP, which is primarily localized in the Golgi complex with little on the cell surface.

Figure 5.

Figure 5

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Detection of subtype C Vpu mutants by surface biotinylation. 293 cells were transfected with vectors expressing VpuSCEGFP1, VpuSCEGFPY35A, VpuSCEGFPL39G, VpuSCEGFPYL35,39AG, or EGFP. At 48 hours, cells were radiolabeled and surface biotinylated as described in the Materials and Methods section. EGFP containing proteins were immunoprecipitated using an anti-EGFP serum and run on SDS-PAGE. The proteins were transferred to membranes and reacted with a substrate to visualize the biotinylated proteins. Lane 1. Cells transfected with EGFP. Lane 2. Cells transfected with VpuSCEGFP1. Lane 3. Cells transfected with VpuSCEGFPY35A. Lane 4. Cells transfected with VpuSCEGFPL39G. Lane 5. Cells transfected with VpuSCEGFPYL35,39AG. Lane 6. Untransfected cells, control.

The reduced level of expression of VpuSCEGFPL39G and VpuSCEGFPYL39AG was not due to enhanced turnover of the protein

One possible explanation for the reduced expression of VpuSCEGFPL39G and VpuSCEGFPYL35,39AG at the cell surface could be due to the stability of the viral proteins. Pulse-chase experiments were performed to examine the rate of turnover of VpuSCEGFP1 and the three mutant Vpu proteins. As shown in Figure 6A–D, VpuSCEGFP1 and the three mutant Vpu proteins had 62–69% of the protein remaining at the 6 hour chase period. These results indicate that protein stability was not the reason for the observed results in Figures 2–5.

Figure 6.

Figure 6

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Pulse-chase analyses of the Vpu fusion proteins. To determine if the amino acid substitutions resulted in altered turnover of the Vpu fusion proteins, 293 cells were transfected with vectors expressing either VpuSCEGFP1, VpuSCEGFPY35A, VpuSCEGFPL39G, or VpuSCEGFPYL35,39AG. At 48 hours post-transfection, the medium was removed and cells were incubated in methionine/cysteine-free medium for 2 hours. The cells were then radiolabeled for 30 minutes with 1 mCi of 35S-Translabel (methionine and cysteine, ICN Biomedical, Costa Mesa, CA) and the radiolabel chased for various periods of time (0–6 hours) in DMEM containing 100X unlabeled methionine/cysteine. Vpu fusion proteins were immunoprecipitated using an anti-EGFP serum as described in the Material and Methods. Non-transfected 293 cells, radiolabeled and chased for 6 hours served as a negative control (lane C). All immune precipitates were collected on protein A Sepharose, the beads washed three times with RIPA buffer, and the samples resuspended in sample reducing buffer. Samples were boiled and the SHIV specific proteins analyzed by SDS-PAGE (10% gel). Proteins were then visualized by standard autoradiographic techniques. Panel A. Results of pulse-chase analysis of viral proteins immunoprecipitated from VpuSCEGFP1 transfected cells. Panel B. Results of pulse-chase analysis of viral proteins immunoprecipitated from VpuSCEGFPY35A transfected cells. Panel C. Results of pulse-chase analysis of viral proteins immunoprecipitated from VpuSCEGFPL39G transfected cells. Panel D. Results of pulse-chase analysis of viral proteins immunoprecipitated from VpuSCEGFPYL35,39AG transfected cells.

Cell surface expression of CD4 by the tyrosine and dileucine mutants

We examined the ability of VpuSCEGFP1 and the three Vpu mutants to down-regulate cell surface CD4 expression. HeLa CD4+ cells were transfected with vectors expressing VpuSCEGFP1 and the three Vpu mutants. At 48 hours, cells were removed from the plates, stained for surface CD4 and analyzed by flow cytometry. The results in Figure 7 show that CD4 surface expression in cells expressing either VpuSCEGFPL39G or VpuSCEGFPY35A were not statistically significant when compared to VpuSCEGFP1. However, cells transfected with VpuSCEGFPYL35,39AG had a statistically significant reduction in CD4 cell surface expression when compared to VpuSCEGFP1 (p<0.05). These results indicate that removal of both the tyrosine and dileucine signals effected CD4 down-regulation.

Figure 7.

Figure 7

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Substitution of tyrosine and leucine residues at positions 35 and 39 results in a protein that is less efficient at down-regulating cell surface CD4 expression. HeLa CD4+ cells were transfected with plasmids expressing EGFP, VpuSCEGFP1, VpuSCEGFPY35A, VpuSCEGFPL39G, or VpuSCEGFPYL35,39AG. At 48 hours, live cells were immunostained for CD4. Cells expressing EGFP or EGFP fusion proteins were assessed for CD4 surface expression using flow cytometry. CD4 expression in cells expressing the various Vpu proteins was normalized to CD4 expression in EGFP expressing cells. P-values above bars are those Vpu proteins that were less efficient at CD4 down-regulation and reached significance.

SHIVs expressing Vpu proteins with mutations in the dileucine motifs replicate faster than those with mutations in tyrosine motif

We constructed simian human immunodeficiency viruses that expressed a Vpu protein with either the Y35A (SHIVSCVpuY35A), the L39G (SHIVSCVpuL39G) or the YL35,39AG (SHIVSCVpuYL35,39AG) amino acid substitutions. The replication of these viruses was assessed using p27 growth curves and compared to parental SHIVSCVpu. The results of these assays are shown in Figure 8 and indicate that SHIVSCVpuL39G released p27 into the culture medium faster and was 5.6-fold higher than parental SHIVSCVpu at day 7 post-inoculation. Additionally, we observed that cultures inoculated with SHIVSCVpuL39G developed syncytial cytopathology at a faster rate (and larger) compared to the other three viruses (data not shown). In contrast, the SHIVSCVpuY35A and SHIVSCVpuYL35,39AG released p27 into the culture medium at a slower rate and at day 7 were 10.3 and 7.4 -fold less, respectively, when compared to the parental SHIVSCVpu. It should be noted that in four attempts to prepare stocks of SHIVSCVpuY35A, three of the stocks selected for a highly cytopathic variant. Sequence analysis of the vpu gene amplified from cells isolated from infected cultures revealed a premature stop codon in vpu at amino acid position 28 (data not shown). Since we have never encountered this problem in construction of other SHIVs, it suggests that the lack of a tyrosine residue at this position is being compensated for by truncation of the protein. Taken together, these results indicate that the tyrosine residue appeared to be required for efficient virus particle release and that removal of the dileucine motif resulted in a virus that replicated much more efficiently.

Figure 8.

Figure 8

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Growth curves of SHIVSCVpu, SHIVSCVpuY35A, SHIVSCVpuL39G and SHIVSCVpuYL35,39AG in C8166 cells. Cultures of C8166 cells were inoculated with either SHIVSCVpu (), SHIVSCVpuY35A (), SHIVSCVpuL39G () and SHIVSCVpuYL35,39AG () as described in the text. Aliquots of the culture medium was assayed for the presence of p27 antigen. The growth curves were performed in triplicate and the mean of the three experiments plotted.

SHIVSCVpuL39G processes Gag and Env precursors faster than SHIVSCVpuY35A and SHIVSCVpuYL35,39AG

Pulse-chase experiments were used to analyze the processing of viral Gag and Env proteins. C8166 cells were inoculated with either SHIVSCVpu, SHIVSCVpuY35A SHIVSCVpuL39G, or SHIVSCVpuYL35,39AG for 5 days at which time pulse-chase analyses were performed. The results of the pulse-chase analysis for SHIVSCVpu is shown in Figure 9A (Hill et al., 2008). The results of the pulse-chase analyses for SHIVSCVpuL39G (Figure 9B) indicate that Env and Gag protein precursors were processed similar to SHIVSCVpu but faster than either SHIVSCVpuY35A (Figure 9 C) but not SHIVSCVpuYL35,39AG (Figure 9D). This was also reflected in the release of the viral proteins into the culture medium (data not shown). Together, these data correlated well with the p27 growth curves.

Figure 9.

Figure 9

Figure 9

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Pulse-chase analyses revealed that SHIVSCVpuY35A, SHIVSCVpuL39G and SHIVSCVpuYL35,39AG have altered processing of viral proteins. To determine if viral structural proteins were released with reduced efficiency in SHIVSCVpu-inoculated cultures, C8166 cells were inoculated with 103 TCID50 of either SHIVSCVpu, SHIVSCVpuY35A, SHIVSCVpuL39G or SHIVSCVpuYL35,39AG. At 7 days post-infection, the medium was removed and infected cells were incubated in methionine/cysteine-free medium for 2 hours. The cells were then radiolabeled for 30 minutes with 1 mCi of 35S-Translabel (methionine and cysteine, ICN Biomedical, Costa Mesa, CA) and the radiolabel chased for various periods of time (0–6 hours) in DMEM containing 100X unlabeled methionine/cysteine. SHIV proteins were immunoprecipitated from cell lysates using plasma pooled from several pig-tailed monkeys infected previously with SHIV as described in the Material and Methods. Uninfected C8166 cells, radiolabeled and chased for 6 hours served as a negative control (lane C). All immune precipitates were collected on protein A Sepharose, the beads washed three times with RIPA buffer, and the samples resuspended in sample reducing buffer. Samples were boiled and the SHIV specific proteins analyzed by SDS-PAGE (10% gel). Proteins were then visualized by standard autoradiographic techniques. Panel A. Results of pulse-chase analysis of viral proteins immunoprecipitated from SHIVSCVpu infected cell lysates. Panel B. Results of pulse-chase analysis of viral proteins immunoprecipitated from SHIVSCVpuL39G infected cell lysates. Panel C. Results of pulse-chase analysis of viral proteins immunoprecipitated from SHIVSCVpuY35A infected cell lysates. Panel D. Results of pulse-chase analysis of viral proteins immunoprecipitated from SHIVSCVpuYL35,39AG infected cell lysates.

SHIVs with the L39G amino acid substitution exhibit more particles on the cell surface

We examined the maturation of SHIVSCVpu, SHIVSCVpuY35A, SHIVSCVpuL39G and SHIVSCVpuYL35,39AG by electron microscopy to determine if these amino acid substitutions altered the pattern of virus maturation. As shown in Figure 10A, the SHIVSCVpu was found to mature at the cell surface as we recently reported (Hill et al., 2008). SHIVSCVpuY35A, SHIVSCVpuL39G, and SHIVSCVpuYL35,39AG were also found to mature predominantly at the cell surface (Figure 10B–D) although viral particles were occasionally observed within vesicles of SHIVSCVpuL39G infected cells (Figure 10E). The salient feature of the electron microscopy studies was the number of viral particles associated per infected cell. We determined the mean number of particles per 50 cells at five days post-inoculation. As shown in Figure 11A, the mean number of virus particles from 50 SHIVSCVpu-infected C8166 cells was approximately 16. The number of particles per cell for SHIVSCVpuY35A and SHIVSCVpuYL35,39AG-infected cells was approximately 3 and 8, respectively (Figure 11B–C). Contrasting with these results, SHIVSCVpuL39G had a mean of approximately 75 particles per cell or approximately 5 times as many particles as parental SHIVSCVpu (Figure 11D). The difference in the mean number of particles per cell between SHIVSCVpu and SHIVSCVpuL39G, SHIVSCVpu and SHIVSCVpuYL35,39AG, and SHIVSCVpu and SHIVSCVpuY35A were found to be very significant (p<0.001). Analysis of the distribution of the number of particles per cell also showed a clear difference between SHIVSCVpuL39G and the other three viruses. For SHIVSCVpu, the number of particles per cell was generally less than 30 (Figure 11A) while for SHIVSCVpuYL35,39AG and SHIVSCVpuY35A the number of particles per cell was less than 20 and 10, respectively (Figure 11B–C). These results were in contrast with those for SHIVSCVpuL39G, which showed a more even distribution in the number of particles per cell (Figure 11D). Combined with the p27 growth curves, while there were increased numbers of virus particles on the surface, the virus replicated with increased kinetics and released more p27 into the culture medium.

Figure 10.

Figure 10

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Electron microscopic examination of C8166 cells inoculated with SHIVSCVpu, SHIVSCVpuY35A, SHIVSCVpuL39G, and SHIVSCVpuYL35,39AG. C8166 cells were inoculated with each virus for 7 days. Cells were washed three times with PBS and processed for electron microscopy as described in the Materials and Methods section. Panel A. C8166 cells inoculated with parental SHIVSCVpu. Panel B. C8166 cells inoculated with SHIVSCVpuY35A. Panel C and D. C8166 cells inoculated with SHIVSCVpuYL35,39AG. Panel E and F. C8166 cells inoculated with SHIVSCVpuL39G.

Figure 11.

Figure 11

Figure 11

Figure 11

Figure 11

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Enumeration of the number of viral particles associated with the infected C8166 cells following inoculation with SHIVSCVpu (Panel A), SHIVSCVpuY35A (Panel B), SHIVSCVpuYL35,39AG (Panel C) and SHIVSCVpuL39G (Panel D). Cells were inoculated with equivalent doses of infectious virus and at 5 days processed for electron microscopy as described in the Material and methods section. The number of viral particles associated with 50 cells per virus and mean values calculated.

DISCUSSION

The subtype C Vpu proteins have canonical tyrosine (YXXM) and dileucine ([D/EXXXL[L/I]) motifs that could be involved in the targeting of this protein to different intracellular compartments. The targeting of proteins containing these motifs is generally mediated through interactions with various adaptor protein complexes (AP-1, AP-2, AP-3, AP-4). The YXXM signals have been shown to be involved in endocytosis of membrane proteins from the cell plasma membrane as well as targeting of membrane proteins to lysosomes and lysosome-related vesicles. The YXXM motifs that are involved in endocytic functions generally have a glycine residue at the Y+1 position and are located 10–40 amino acids from the transmembrane of the protein. Those YXXM motifs that are involved in lysosomal targeting generally have an acidic residue at the Y+1 position and are generally located 6–9 residues from the transmembrane domain (Bonificano and Traub, 2003). Tyrosine signals generally interact with the : subunit of adaptor protein complexes. The best-studied interactions with the AP complexes have been predominantly with :2, although the :1, :3, and :4 subunits can interact with these motifs. The :3A and :3B subunits have a preference for binding to tyrosine signals with an acidic amino acid before and/or after the tyrosine residue. The most characteristic feature of the :4 subunit binding is the presence of aromatic amino acids near the tyrosine residue. With dileucine motifs, the first leucine is generally invariant and replacement of the first leucine with an isoleucine reduces the potency of the signal. However, the second leucine can be replaced with an isoleucine and in some cases a methionine residue with an acidic amino acid at the +4 and/or +5 position and retain signal function. Dileucine motifs also interact with adaptor protein complexes AP-1, AP-2, and AP-3 (Bonifacino and Traub, 2003), but they appear to interact with a hemi-complex formed by the small subunit and the large specific subunit (Janvier et al., 2003). Similar to the tyrosine motifs, the proximity to the transmembrane domain appears to affect whether it is involved in endocytosis or lysosomal targeting. As the overlapping tyrosine and dileucine motifs of Vpu are membrane proximal, it suggests that they may be involved in lysosomal targeting.

In this study we analyzed the role of overlapping tyrosine and dileucine signals within the cytoplasmic domain of the subtype C Vpu in intracellular targeting. The two other membrane proteins of HIV-1, Env and Nef, use tyrosine and dileucine motifs for either trafficking within the cell and/or removal of cellular proteins from the cell surface (Boge et al., 1998; Bresnahan et al., 1998; Byland et al., 2007; Chaudhuri et al., 2007; Coleman et al., 2005; Craig et al., 1998; Day et al.,2006; Greenberg et al.., 1998; Lodge et al., 1997; Novello et al. 2007; Roeth et al., 2004; Schwartz et al., 1996). Our findings indicate that the tyrosine motif within Vpu may have a role in enhanced virus release. The finding that the tyrosine based motif was important to virus release is not entirely surprising as this motif is conserved in virtually all of the Vpu sequences from the different HIV-1 subtypes in the Los Alamos National Laboratory (LANL)-HIV-1 group M database. Recently, BST-2/CD317 or “tetherin” was identified as a target for Vpu (Neil et al., 2008; Van Damme et al., 2008). This protein was shown to prevent BST-2/CD317 expression at the cell surface and that expression of this protein correlated with the requirement of Vpu for enhanced virion release. However, a direct interaction of Vpu and BST-2/CD317 has yet to be demonstrated. As changing the tyrosine residue at position 35 to an alanine in the subtype C Vpu protein resulted in a virus, SHIVSCVpuY35A, that replicated very poorly in C8166 cells, it raises the question, “Is the membrane proximal tyrosine motif important in the Vpu interactions with BST-2?” If Vpu interacts directly with BST-2/CD317, it may be possible that Vpu could either prevent the transport of this protein to the cell surface and/or sequester this protein to the intracellular compartment(s) such that it will not effect virus release.

Dileucine motifs are found in a high percentage of subtype C Vpu sequences in the LANL-HIV-1 database, with approximately 80% of the sequences having a dileucine, leucine-isoleucine or leucine-valine motif at the membrane proximal location. In contrast, of the 271 subtype B Vpu sequences in the LANL-HIV database, the majority (~95%) of the sequences have an isoleucine in place of the primary leucine (EYRKIL) with only 5.2% of the sequences having dileucine, leucine-isoleucine or leucine-valine motif. Similar to YXXM signals, [D/E]XXXL[L/I] signals in mammalian membrane proteins can mediate both rapid internalization as well as targeting to endosomal and lysosomal compartments. This indicates that they can be recognized at the plasma membrane and the intracellular compartments. In addition, [D/E]XXXL[L/I] signals have been implicated in trafficking of membrane proteins to basolateral membranes in polarized epithelial cells (Hunziler et al., 1994; Matter et al., 1994; Miranda et al., 2001). Our results indicate that the dileucine sorting signal within the subtype C Vpu protein was important for efficient transport and expression at the cell surface. Substitution of the second leucine with a glycine resulted in a protein (VpuSCEGFPL39G) that was transported to the cell surface less efficiently with the majority being retained within the Golgi complex. The virus constructed with this mutation, SHIVSCVpuL39G, was found to replicate much better than the SHIV with the unmodified subtype C Vpu protein (SHIVSCVpu). We also find that SHIVSCVpuL39G replicates as well as the SHIV expressing the subtype B Vpu protein (SHIVKU-1bMC33). While speculative at this juncture, one explanation for these observations is that the presence of the [D/E]XXXL[L/I] motif may interfere with the function of the tyrosine-based signal. Possibly, the subtype C Vpu protein does not interact with AP complexes for sorting of the protein to intracellular compartments resulting in the transport of the protein to the cell surface. By removing the dileucine sequence, the protein may be allowed to interact with cellular components that target the protein to an intracellular compartment.

Our results raise the important question, “Why would the subtype C viruses select for a dileucine signal that would hinder virus replication?” It has been hypothesized that subtype C HIV-1 may be evolving to a less virulent form (Ariens et al., 2007). Data to support this hypothesis include that the subtype C viruses were found to be less fit in PBMC competition assays (Ball et al., 2003; Arien et al., 2005). Additional support comes from our recent study using SHIVSCVpu where we exchanged the subtype B vpu with one from a clinical subtype C isolate (Hill et al., 2008). In this study, we showed that inoculation of pig-tailed macaques with this virus resulted in a slower rate of CD4+ T cell loss compared to our highly pathogenic subtype B SHIVKU-1bMC33. It will be of interest to determine if inoculation of macaques with the SHIVSCVpuL39G causes a more rapid loss of CD4+ T cells.

MATERIALS AND METHODS

Cells, viruses and vectors

The 293 cell line was maintained in Dulbecco’s minimal essential medium supplemented with 10% fetal bovine serum, gentamicin (5 ug per ml) and penicillin/streptomycin (100U per ml and 100:g per ml). A C8166 cell line was used as the indicator cells to measure infectivity and cytopathicity of the viruses used in this study. C8166 cells were maintained in RPMI-1640, supplemented with 10mM Hepes buffer pH 7.3, 2 mM glutamine, 5 :g per ml gentamicin and 10% fetal bovine serum (R10FBS). HeLa CD4+ cells were obtained from the NIH AIDS Research and Reference Reagents Program and were maintained in Dulbecco’s minimal essential medium supplemented with 10% fetal bovine serum, 5 :g per ml gentamicin, and 1mg per ml G-418. The derivation and pathogenicity of SHIVKU-1bMC33 has been described (McCormick-Davis et al., 2000b; Stephens et al., 2002; Singh et al., 2003). A derivative of this virus, known as SHIVSCVpu, is identical to SHIVKU-1bMC33 except that this virus expresses the subtype C Vpu protein (Hill et al., 2008). Vectors expressing the subtype B (pcvpuegfp) and C Vpu (pcvpuscegfp1) proteins fused to enhanced green fluorescent protein (eGFP) have been previously described (Singh et al., 2003; Gomez et al., 2005; Pacyniak et al., 2005). The vectors expressing the DsRED2-Golgi was obtained from Clontech.

Construction of vectors expressing the subtype C mutants

For the construction of the subtype C Vpu mutants, site-directed mutagenesis was performed on the parental plasmid, pcvpuSCegfp. Site directed mutagenesis was performed using this plasmid in the Quick Change Mutagenesis kit (Stratagene) and oligonucleotides (only sense strand shown) 5’- GCATATATAGAAGCTAGAAAATTGTTAAGACAG-3' (to change the tyrosine residue at position 35 to an alanine), 5'-GAATATAGAAAATTGGGAAGACAGAAA-AAGATAGAC-3' (to change the leucine at position 39 to a glycine), and 5’-GAAGCTAGAAAATTGGGAAGACAGAAAAAGATAGAC-3’ (to change the tyrosine and leucine at positions 35 and 39) according to the manufacturers instructions. With all mutants, the entire insert sequenced (both vpu and egfp genes) to ensure the validity of the mutations introduced and that no additional changes were introduced during the mutagenesis process.

Transfections and laser scanning confocal fluorescence microscopy analysis

VpuEGFP and other plasmids were transfected in human 293 cells to assess their subcellular localization using a cationic polymer (polyethylenimine) transfection reagent (ExGen™ 500, MBI Fermentas) using the manufacturer’s protocol. Briefly, 1–3 × 105 cells were seeded onto coverslips in each well of a 6 well tissue culture plate 24 hours prior to transfection. Transfection was carried out on cultures that were 50–60% confluent using 4.75 :g of plasmid DNA and 15.5 :l of ExGen™500 corresponding to 6 equivalents. Each plasmid DNA sample was diluted in 300 :l of 150 mM sodium chloride solution. Samples were vortexed gently and immediately centrifuged at low revolution for a few seconds. Polyethylenimine was then added to the plasmid DNA solution, mixed with a vortex and allowed to stand at room temperature for 10 minutes. The 293 cells were washed with serum free media twice and 3.0 ml of serum free DMEM was added. Polyethylenimine/DNA mixture was added to the cells and the plate swirled by slow hand rotation for a couple of seconds. Culture plates were centrifuged at 280 × g for 5 minutes and incubated at 37EC for 30 minutes. The medium from transfected cultures was replaced with fresh complete growth media and cells were incubated at 37≅C in 5% CO2 atmosphere.

At 48 hours post-transfection, cells were rinsed briefly in phosphate buffered saline (PBS, pH 7.2) at room temperature. The cells were fixed in freshly prepared, ice cold 1% paraformaldehyde in 0.13M sodium phosphate pH 7.2 for 2 minutes. The fixative was removed and the cells briefly rinsed in phosphate buffered saline. The saline was removed, and one drop of mounting media (Slowfade Antifade, Molecular Pprobes) was placed on a slide and the coverslip mounted. The cells were imaged with a Zeiss LSM 510 confocal microscope in the upright configuration. The objective used was a 63X 1.4 n.a. Plan Apochromat. Images were captured at 12 bit resolution with a pixel array of 2048 × 2048 and a zoom of 2.0 X. The signal was averaged 4 times per line. The EGFP was excited with light at 488 nm (laser intensity 75% for all images), and the emitted light was collected after passing through a 505 nm long pass filter. The amplifier offset and gain were identical for all images. The pinhole was set to 96 :m which at this wavelength represents one airey unit. The optical section had a width of 0.7 :m. Simultaneous to the acquisition of the confocal signal from the EGFP, a non-confocal transmitted light image was obtained to provide a reference point for the EGFP signal. Individual confocal slices were overlaid onto the greyscale transmitted light image.

To confirm the presence of the Vpu fusion protein at different subcellular compartments, a series of co-transfection studies were performed using vectors expressing a Golgi marker fused to the fluorescent protein DsRed2 (DsRed2-Golgi). 293 cells were co-transfected with vectors expressing the various Vpu fusion proteins and a vector expressing one of the subcellular markers. At 48 hours post-transfection, cells were processed for confocal microscopy as described above, and cells identified expressing both proteins. Fluorescent digital images were obtained using a Zeiss LSM510 confocal microscope equipped with Argon and HeNe2 lasers (25 mW) for the excitation (488 nm, 50% laser power) and detection (band pass 505–530 nm filter; BP505–530) of EGFP and for excitation (558 nm, 100% laser power) and detection (band pass 583 nm filter; LP560) of DsRed2. Images were acquired in Multitrack channel mode (sequential excitation/emission) with LSM510 (v 3.2) software and a Plan-Apochromat 63/1.4 Oil DIC objective with frame size of 2048 × 2048 pixels. Detector gain was set initially to cover the full range of all the samples and background corrected by setting the amplifier gain, and all images were then collected under the same photomultiplier detector conditions and pinhole diameter.

In order to compare the level of expression of the Vpu fusion proteins on the cell surface, the ratio of the expression on the cell surface versus the Golgi complex was determined. For this, 293 cells were plated, transfected with various fusion proteins, fixed and mounted as described above. Using the Z function within LSM510, the depth of the top and bottom of the individually fluorescent cells were found. A 0.5:m slice picture was taken at a depth equidistant from the top and bottom of the cell. All pictures were taken with the same pinhole and detector gain for uniformity. A profile line was drawn from outside the cell through the Golgi, and reference points chosen in the cell surface and Golgi. The fluorescence intensity from those two reference points was recorded and a ratio of membrane to Golgi intensity was calculated. Ratios were taken from at least 42 separate cells, averaged and the standard deviation calculated. Student's t-test was used to determine significance, with p<0.05 considered significant.

Construction of viruses with the tyrosine and dileucine mutations

For the construction of a subtype C SHIV containing tyrosine and dileucine mutations, we used plasmid pUCvpuSC, which contains the tat and rev of HIV-1 (HXB2), the subtype C vpu and the 5' end of the env gene. To introduce the tyrosine and dileucine mutations, we used this plasmid and the same oligonucleotides and procedures used for site-directed mutagenesis as described above for the construction of the Vpu/EGFP fusion proteins. Simian human immunodeficiency viruses expressing subtype C Vpu proteins with the Y35A (SHIVSCVpuY35A), the L39G (SHIVSCVpuL39G), and YL35,39AG (SHIVSCVpuYL35,39AG) amino acid substitutions were constructed as previously described (McCormick-Davis et al., 2000b; Stephens et al., 2002; Singh et al., 2003; Hout et al., 2005; 2006; Hill et al., 2008). Stocks were prepared, titrated in C8166 cells and frozen at −86C until used.

Pulse-chase analysis of viral proteins

To analyze the viral proteins synthesized and released from cells, C8166 cells were inoculated with 104 TCID50 of either SHIVSCVpuY35A, SHIVSCVpuL39G, SHIVSCVpuYL35,39AG or SHIVSCVpu. At 7 days post-inoculation, the medium was removed and infected cells were incubated in methionine/cysteine-free Dulbecco's modified Eagle's medium (DMEM) for 2 hours. The cells were then radiolabeled for 30 minutes with 1 mCi per ml of 35S-Translabel (methionine and cysteine, ICN Biomedical, Costa Mesa, CA) and the radiolabel chased for various periods of time in DMEM containing 100X unlabeled methionine/cysteine. SHIV proteins were immunoprecipitated from the cell culture medium and infected cell lysates using plasma pooled from several rhesus monkeys infected previously with non-pathogenic SHIV-4. Briefly, the cell culture medium was clarified (16,000 × g) for 2 minutes. The supernatant was transferred and made 1X with respect to cell lysis buffer (50 mM Tris-HCl, pH 7.5; 50 mM NaCl; 0.5% deoxycholate; 0.2% SDS; 10 mM EDTA) and SHIV proteins were immunoprecipitated with 10 :l of a pooled serum.For immunoprecipitation of cell associated SHIV proteins, cell lysates were prepared as previously described (Stephens et al., 1995; McCormick-Davis et al., 2000b; Hout et al., 2005) prior to incubation with antiserum. Lysates were centrifuged in a microfuge to remove nuclei prior to the addition of antibody. Cell lysates and culture medium were incubated with antibody for 16 hours at 4°C. All immunoprecipitates were collected on protein A Sepharose, the beads washed three times with RIPA buffer, and the samples resuspended in sample reducing buffer. Samples were boiled and the SHIV specific proteins analyzed by SDS-PAGE. Proteins were then visualized by standard autoradiographic techniques.

Assays for assessing the cell surface Vpu expression

To determine if Vpu proteins were expressed on the plasma membrane, 293 cells were transfected with 4.75 :g of plasmid DNA expressing EGFP, VpuSCEGFP,1 VpuSCEGFPY35A, VpuSCEGFPL39G, or VpuSCEGFPYL35,39AG. At 48 hours post-transfection, cells were incubated in methionine/cysteine-free media for 2 hours and then labeled with 200 :Ci of 35S for 1 hour. Cells were washed three times in ice-cold 1X PBS and the surface of cells labeled with EZ-Link Sulfo-NHS-LC-Biotin (Pierce) at a concentration of 10mg/ml for 1 hour on ice. Cells were then washed three times in 1X PBS containing 100mM glycine. The cells were lysed in 1ml of 1X RIPA buffer and nuclei were removed by centrifugation at 14,000 rpm for 15 minutes. Cell lysates were incubated overnight at 4° C with a rabbit polyclonal anti-EGFP antibody and protein A Sepharose beads. Lysate immunoprecipitates were washed three times in 1X RIPA, resuspended in 100 :l of 2X sample reducing buffer, and boiled for 5 minutes. Proteins were separated on a 10% SDS-PAGE gel and densitometry was used to quantify the total amount of VpuEGFP fusion expressed in each sample. Equal amounts of fusion proteins were then separated on a 10% SDS-PAGE gel, the proteins were transferred to nitrocellulose and a Western blot was used to detect biotin-labeled proteins. Biotin-labeled proteins were detected using a Vectastain-ABC-AmP chemiluminescent detection kit (Vector Laboratories). A BioRad chemiluminescent imager was used to quantify the total amount of biotin-labeled VpuEGFP in each sample. The experiments were conducted in triplicate and a Student’s t-test was used to determine statistical significance with p<0.05 considered significant.

Assays for assessing cell surface expression of CD4

For analysis for cell surface CD4 expression in the presence of various fluorescent proteins, HeLa CD4+ cells were seeded into six well plates one day in advance, such that hours later they would be 70–80% confluent. Cells were transfected with plasmids expressing EGFP or various Vpu proteins fused to EGFP. Cultures were monitored for 48 hours, cells removed from the six well plate using Ca++ /Mg++-free PBS containing 1mM EDTA and stained with PE-Cy5 conjugated anti-CD4 (BD Bioscience). Cells were analyzed using an LSR II flow 19 cytometer, determining mean fluorescence intensity (MFI) of PE-Cy5 for transfected (EGFP positive) and untransfected (EGFP negative) cells within the same well. An MFI ratio was calculated for each sample with EGFP controlnormalized to 1.0. Normalized ratios from at least five separate experiments were averaged and the standard deviation calculated. All groups were compared using one way ANOVA (performed using SAS software, alpha = 0.5) to determine variance. Post hoc analysis was perfomed using Tukey’s HSD test.

p27 growth curve assays

Standard p27 assays (Zeotometrix Incorporated, SIV core antigen kit) were used to assess release of viral particles from cells infected with SHIVSCVpu, SHIVSCVpuY35A, SHIVSCVpuL39G or SHIVSCVpuYL35,39AG. Cultures of 106 C8166 cells were inoculated with 10 ng of p27 (determine by commercial p27 antigen kits) for 4 hours. At the end of 4 hours, the cells were centrifuged at 400 × g for 10 minutes and the pellet washed with 10 ml of medium. This was repeated two additional times. The cells were resuspended in RPMI-1640 supplemented with 10% FBS and antibiotics and this was considered the 0 time point of the assay. Cultures were incubated at 37C and aliquots of the culture were removed at 0, 1, 3, 5, 7, and 9 days with fresh media added to cultures at days 3 and 6. The culture medium was separated from the cells by centrifugation and assayed for p27 according to the manufacturer’s instructions.

Electron microscopy

To determine the site(s) of intracellular maturation, infected cells were examined by transmission electron microscopy. Cultures of 106 C8166 cells were inoculated with 10 ng of SHIVSCVpu, SHIVSCVpuY35A, SHIVSCVpuL39G or SHIVSCVpuYL35,39AG. Cells were incubated for 5 days at which time cells were pelleted at 400 × g for 10 minutes. Cells were washed three times with 10 ml of phosphate buffered saline (pH 7.4) and fixed in 2% glutaraldehyde overnight at 4C. Cells were post-fixed in 2% osmium tetroxide (OsO4) for 1 hour. The cells were washed twice with water and dehydrated through a series of alcohols (30–100%) followed by embedding in Embed 812 resin. Thin sections were cut at 80 A, stained with uranyl acetate and lead citrate and examined under a JEOL 100CXII transmission electron microscope. The number of virus particles associated with 50 infected cells (either at the surface or within the cell) were enumerated and data analyzed by planned comparisons using unpaired t-test.

ACKNOWLEDGMENTS

The work reported here is supported by grant NIH grant AI51981 to E.B.S. The anti-Vpu serum and the Hela CD4+ cell line was kindly provided by the NIH AIDS Research and Reference Reagent Program. We thank members of the KUMC Biotechnology Support Facility for their assistance with the sequence analysis and oligonucleotide synthesis.

Footnotes

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