The Role of Vpu in HIV-1 Pathogenesis

描述（由申请人提供）：人类免疫缺陷病毒1型（HIV-1）编码一种称为Vpu的小膜蛋白，并在感染细胞中具有两种主要功能。已知Vpu与CD4分子相互作用并将其从粗内质网（RER）转移到蛋白酶体进行降解。此外，已知Vpu可以增强病毒从感染细胞中的释放。HIV-1病毒在缺乏vpu基因的CD4+ T细胞中组装的特点是病毒成熟进入细胞内囊泡，并在细胞表面积聚病毒颗粒。这种增强的Vpu释放功能与Vpu分子的跨膜结构域有关，研究人员已经证明Vpu TM具有离子通道特性（也称为病毒孔蛋白）。利用猴人免疫缺陷病毒（SHIV）的致病分子克隆SHIVKu-ibMC33，我们发现Vpu蛋白的跨膜结构域和细胞质结构域都参与了该病毒在猕猴中的发病机制。此外，我们已经证明，用来自C亚型HIV-1分离物的vpu取代SHIVKu-ibMC33的B亚型vpu基因可以降低猕猴CD4+ T细胞的失失率。在前两个Specific Aims中，我们建议继续研究Vpu蛋白的TM/离子通道在病毒粒子释放中的作用。我们最近获得了一种新的化合物BIT225（来自Biotron LTD.），在我们的初步研究中，它可以抑制接种SHIVKu-ibwc33的培养物中病毒颗粒的释放，而不是表达带有混乱TM结构域的Vpu （SHIVrw）的SHIV。在Specific Aim 1中，我们建议研究BIT225抑制SHIVKu-ibwc33复制和病毒释放的位点/机制。在Specific Aim 2中，我们建议研究这些化合物在猕猴巨噬细胞培养中减少突变体和亲代shiv复制的能力。在第三和第四个目标中，我们建议继续对C - Vpu亚型的生物学特性进行研究。在特异性目标3中，我们建议研究高度保守的二苯环基序与接头复合物（AP-1、AP-2和AP-3）的作用，并确定该结构域是否影响病毒从感染细胞中的释放。在Specific Aim 4中，我们提出产生一系列表达嵌合亚型B/C Vpu蛋白的shiv，以确定是哪个结构域导致猕猴CD4+ T细胞损失率降低。这些研究的结果将提供来自C型HIV-1的Vpu蛋白的新信息，C型HIV-1在世界范围内占大多数HIV-1感染。

DESCRIPTION (provided by applicant): Human immunodeficiency virus type 1 (HIV-1) encodes for a small membrane protein known as Vpu and has two major functions in the infected cell. Vpu is known to interact with and shunt the CD4 molecule from the rough endoplasmic reticulum (RER) to the proteasome for degradation. In addition, Vpu is known to enhance virus release from infected cells. The assembly of HIV-1 viruses in CD4+ T cells lacking a vpu gene is characterized by the maturation of viruses into intracellular vesicles and the accumulation of virus particles at the cell surface. This enhanced release function of Vpu has been associated with the transmembrane domain of the Vpu molecule and investigators have shown that Vpu TM has ion channel properties (also known as a viroporin). Using pathogenic molecular clones of simian human immunodeficiency viruses (SHIV) known as SHIVKu-ibMC33, we have shown that both the transmembrane (TM) and cytoplasmic domains of Vpu protein contribute to the pathogenesis of this virus in macaques. Further, we have shown that substitution of the subtype B vpu gene from SHIVKu-ibMC33 with vpu from a subtype C HIV-1 isolate reduces the rate of CD4+ T cell loss in macaques. In the first two Specific Aims, we propose to continue our studies on the TM/ion channel of the Vpu protein in virion release. We have recently obtained a novel compound, BIT225 (from Biotron LTD.), which in our preliminary studies inhibits the release of viral particles from cultures inoculated with SHIVKu-ibwc33 but not a SHIV expressing a Vpu with a scrambled TM domain (SHIVrw)- In Specific Aim 1, we propose to examine the site/mechanism by which BIT225 inhibits SHIVKu-ibMcss replication and virus release. In the Specific Aim 2, we propose to examine the ability of these compounds to decrease replication of mutant and parental SHIVs in macaque macrophage cultures. In the third and fourth Aims, we propose to continue our studies on the biological properties of the subtype C Vpu. In Specific Aim 3, we propose to examine the role of the highly conserved dileucine motif with adaptor complexes (AP-1, AP-2, and AP-3) and to determine if this domain influences virus release from infected cells. In Specific Aim 4, we propose to generate a series of SHIVs expressing chimeric subtype B/C Vpu proteins to determine what domain is responsible for the decreased rate of CD4+ T cell loss in macaques. The results of these studies will provide novel information of the Vpu protein from the subtype C HIV-1, which accounts for the most HIV-1 infections worldwide.