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

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., 2008). In this study, we examined the role of overlapping tyrosine-based (YXXPhi) 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 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 these 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 membrane proximal 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 subtype C Vpu proteins, virus replication, and release.

Suppression of Tetherin-Restricting Activity upon Human Immunodeficiency Virus Type 1 Particle Release Correlates with Localization of Vpu in the trans -Golgi Network

“…These findings raise the possibility that subtype B Vpu TGN-to-endosome trafficking may not be actively regulated and may indeed occur by bulk flow. These results are in sharp contrast with recent observations made by Ruiz et al, which found that the mutation of the conserved Tyr residue in the hinge region of subtype C Vpu drastically decreased the viral replication of simian-HIV virus encoding subtype C Vpu (27). This same group also showed that the mutation of the second leucine (L39G) of the dileucine-based motif (EYRKLL) of subtype C Vpu leads to a protein that is transported to the cell surface less efficiently, with the majority retained within the Golgi complex (27).…”

“…These results are in sharp contrast with recent observations made by Ruiz et al, which found that the mutation of the conserved Tyr residue in the hinge region of subtype C Vpu drastically decreased the viral replication of simian-HIV virus encoding subtype C Vpu (27). This same group also showed that the mutation of the second leucine (L39G) of the dileucine-based motif (EYRKLL) of subtype C Vpu leads to a protein that is transported to the cell surface less efficiently, with the majority retained within the Golgi complex (27). It is therefore likely that the distinct requirement of subtype C and subtype B Vpu for tyrosine-and dileucine-based sorting signals is related to the activity of their respective sorting motifs as well as their different patterns of localization.…”

“…This notion is indeed consistent with results from our experiments showing that the retention of the Vpu wt or the Vpu R30A,K31A mutant in TGN-related structures following the disruption of clathrin-mediated TGN-to-endosome trafficking stimulates the Vpu-mediated enhancement of viral particle release. Interestingly, the mutation of the dileucine motif of subtype C Vpu, which resulted in an increased accumulation of the protein in the TGN, led to a similar stimulation of the Vpu-mediated enhancement of viral particle release (27). Thus, the TGN could conceivably represent one key intracellular site from where Vpu overcomes the restricting activity of tetherin on HIV-1 particle release.…”

“…Since the Vpu R30A,K31A mutant was found to be less efficiently targeted for degradation in the lysosomes than the Vpu wt, it seems therefore likely that these positively charged amino acids are affecting the efficiency and/or specificity of Vpu trafficking between the TGN and the endosomal system. Interestingly, Arg30 and Lys31 are part of sequences that were recently proposed to encode overlapping tyrosine-based (YXX⌽) and dileucine-based ([D/E]XXXL[L/I]) sorting motifs in subtype C Vpu (27) (Fig. 2A).…”

Eastern Chimpanzees, but Not Bonobos, Represent a Simian Immunodeficiency Virus Reservoir

“…Columns on the far right indicate the presence (ϩ) or absence (blank) of previously described transport and/or degradation motifs in the intracellular domain of Vpu. These include a Yxx⌽ motif scored as Yxx(L/M/V/I/F/W) (8,21,56), a ␤-TrCP ubiquitin-dependent degradation signal scored as D(S/D/E)Gxx(S/D/E) (28,39), and a putative trafficking signal scored as (D/E)xxxL(L/V/I/M) (33).…”