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

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 (YXXΦ) 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; Vpu_SCEGFPY35A), the second altered the dileucine motif (EYRKLG; Vpu_SCEGFPL39G), and the third contained both amino acid substitutions (EARKLG; Vpu_SCEGFPYL35,39AG) in this region of the Vpu protein. The Vpu_SCEGFPY35A protein was transported to the cell surface similar to the unmodified Vpu_SCEGFP1 while Vpu_SCEGFPL39G was expressed at the cell surface at significantly reduced levels. The Vpu_SCEGFPYL35,39AG 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 (Vpu_SCEGFPYL35,39AG) was significantly less efficient at preventing cell surface CD4 expression. Chimeric simian human immunodeficiency viruses were constructed with these mutations in vpu (SHIV_SCVpuY35A, SHIV_SCVpuL39G and SHIV_{SCVpuYL35,39AG}). Our results indicate that SHIV_SCVpuL39G replicated much more efficiently and was much more cytopathic than SHIV_SCVpu. In contrast, SHIV_SCVpuY35A and SHIV_{SCVpuYL35,39AG} replicated less efficiently when compared to the parental SHIV_SCVpu. 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.