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，在感染细胞中具有两种主要功能。已知Vpu与CD 4分子相互作用并将其从粗面内质网（RER）分流至蛋白酶体进行降解。此外，已知Vpu可增强病毒从受感染细胞的释放。HIV-1病毒在缺乏vpu基因的CD 4 + T细胞中的组装的特征在于病毒成熟为细胞内囊泡和病毒颗粒在细胞表面的积累。Vpu的这种增强的释放功能与Vpu分子的跨膜结构域相关，并且研究人员已经表明VpuTM具有离子通道特性（也称为病毒孔蛋白）。使用猴人类免疫缺陷病毒（SHIV）的致病性分子克隆SHIVKu-ibMC 33，我们已经证明Vpu蛋白的跨膜（TM）和胞质结构域都有助于这种病毒在猕猴中的发病机制。此外，我们已经表明，用来自C亚型HIV-1分离株的vpu取代来自SHIVKu-ibMC 33的B亚型vpu基因降低了猕猴中CD 4 + T细胞的损失率。在前两个具体目标中，我们建议继续研究Vpu蛋白在病毒体释放中的TM/离子通道。我们最近获得了一种新的化合物，BIT 225（来自Biotron LTD.），其在我们的初步研究中抑制病毒颗粒从接种SHIVKu-ibwc 33但不抑制表达具有乱序TM结构域（SHIVrw）的Vpu的SHIV的培养物中的释放。在具体目标1中，我们提出检查BIT 225抑制SHIVKu-ibMcss复制和病毒释放的位点/机制。在具体目标2中，我们建议检查这些化合物降低猕猴巨噬细胞培养物中突变体和亲本SHIV复制的能力。在第三和第四个目标中，我们建议继续研究C亚型Vpu的生物学特性。在特定目标3中，我们建议检查高度保守的双亮氨酸基序与接头复合物（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释放。在具体目标4中，我们提出产生一系列表达嵌合亚型B/C Vpu蛋白的SHIV，以确定哪个结构域负责猕猴中CD 4 + T细胞损失率降低。这些研究的结果将提供来自C亚型HIV-1的Vpu蛋白的新信息，C亚型HIV-1占全球最多的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.