VPU mediated enhancement of human immunodeficiency virus pathogenesis: The role of conserved and unique domains in protein function.

摘要

The work in this dissertation examined the biological characteristics of different HIV-1 Vpu subtypes, with an emphasis on subtypes B and C, and the potential impact of these proteins on SHIV pathogenesis. Different Vpu subtypes exhibited distinct biological properties that potentially could affect HIV-1 pathogenesis and/or transmission efficiency, including intracellular localization, efficiency in down-modulating CD4 surface expression, and the ability to enhance virion release in HeLa cells. We show for the first time that the subtype B and C Vpu proteins partitioned into detergent resistant membranes (DRMs), a property characteristic of lipid raft association. We also identified two mutants, IVV19-21AAA and W22A, that prevented this association. Additionally, we found a correlation between the ability of Vpu to stably associate with DRMs and the ability to enhance virion release in HeLa cells. Analysis of different Vpu proteins from clinical isolates identified a membrane proximal tyrosine motif (YXXphi) that is highly conserved among all Vpu subtypes and an overlapping dileucine motif ([D/E]xxxL[L/I]) that is conserved among subtype C Vpu proteins. Substitution of the tyrosine residue in the YXXphi motif with an alanine (Y35A) significantly inhibited SHIV replication while substitution of the primary leucine residue with a glycine (L39G) in the overlapping [D/E]xxxL[L/I] motif significantly increased the amount of virus released from C8166 cells and the mean number of viral particles per cell compared to cells inoculated with the parental SHIVSCVpu. Recently, the enhanced virion release function of Vpu has been attributed to the antagonism of bone marrow stromal antigen 2 (BST-2) protein. This has been shown to involve the transmembrane domains (TMD) of both proteins. Our results indicate that the length of the BST-2 TMD is more important than the primary sequence both for the function and sensitivity of the protein. Additionally, we showed that the BST-2 protein expressed in pig-tailed macaques is not antagonized by HIV-1 Vpu, but rather by SIV Nef, thus indicating a species-specific basis for this antagonism. Based on these results we continued our analyses by examining the biological characteristics of SHIV expressing either a subtype B (SHIV KU-1bMC33) or C (SHIVSCVpu) Vpu protein. SHIVSCVpu caused a more gradual rate of CD4+ T cell loss and lower peak viral loads in infected pig-tailed macaques compared to macaques inoculated with SHIVKU-1bMC33. The identification of the TMD and the putative sorting signals proximal to the membrane as key determinants in Vpu-mediated enhanced virion release prompted the hypothesis that these two regions may dictate these differences in pathogenesis. Therefore, we constructed chimeric Vpu proteins in which the N-terminus/TMD regions of the subtype B and C Vpu proteins were exchanged (VpuBC and VpuCB). Inoculation of pig-tailed macaques with SHIVVpuCB resulted in a more gradual loss of circulating CD4+ T cells compared to SHIVVpuBC-inoculated macaques, but more rapid than resulted in macaques inoculated with parental SHIVSCVpu. Since both of these proteins down-modulated CD4 surface expression similar to the unmodified VpuSCEGFP1 protein, our results indicate that the differences observed in CD4 surface down-modulation in vitro are most likely not physiologically relevant. Finally, as pig-tailed macaques express a BST-2 protein that is not affected by the HIV-1 Vpu protein, our results suggest that the enhanced virion release function of different Vpu subtypes as observed during an intravenous inoculation may be dependent upon a different factor(s). The work presented here demonstrates a clear potential for differential signaling and functional efficiency among all HIV-1 Vpu subtypes with the ability to modify pathogenesis. Additionally, our analyses identified the TMD and membrane proximal regions as crucial components to Vpu enhancement of pathogenesis providing novel information essential for anti-retroviral therapeutic development.