The Role of Vpu in HIV-1 Pathogenesis

描述(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，在受感染的细胞中有两个主要功能。众所周知，VPU与粗面内质网(RER)中的CD4分子相互作用，并将其分流到蛋白酶体进行降解。此外，已知VPU可促进受感染细胞释放病毒。HIV-1病毒在缺乏VPU基因的CD4+T细胞上组装的特征是病毒成熟为细胞内小泡并在细胞表面积聚病毒颗粒。VPU的这种增强的释放功能与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(也称为病毒菌素)。利用被称为SHIVKu-ibMC33的猴人类免疫缺陷病毒(SHIV)的致病分子克隆，我们证明了VPU蛋白的跨膜区和细胞质结构域在猕猴体内参与了该病毒的致病作用。此外，我们已经证明，用来自C亚型HIV-1分离株的VPU替换来自SHIVKu-ibMC33的B亚型VPU基因可以降低猕猴的CD4+T细胞损失率。在前两个具体目标中，我们建议继续我们对VPU蛋白在病毒粒子释放中的TM/离子通道的研究。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，但不能抑制表达带有扰乱TM结构域的VPU的SIV(SHIVrw)-在特定目标1中，我们建议研究BIT225抑制SHIVKu-ibMcss复制和病毒释放的部位/机制。在具体目标2中，我们建议检查这些化合物在猕猴巨噬细胞培养中减少突变和亲本SHIV复制的能力。在第三和第四个目标中，我们建议继续对C亚型VPU的生物学特性进行研究。在特定的目标3中，我们建议研究高度保守的二亮氨酸基序与适配器复合体(AP-1、AP-2和AP-3)的作用，并确定该结构域是否影响感染细胞的病毒释放。在特定的目标4中，我们建议产生一系列表达B/C型嵌合亚型VPU蛋白的SHIV，以确定哪个结构域是导致猕猴CD4+T细胞损失率降低的原因。这些研究的结果将提供C亚型HIV-1的VPU蛋白的新信息，该亚型是全世界感染HIV-1最多的病毒。

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.