The Role of Lipid Rafts in Vpu Function

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I型膜蛋白，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细胞中组装的特征是病毒颗粒在细胞表面的积累和拴系以及病毒成熟进入细胞内囊泡。这种增强的Vpu释放功能与Vpu的跨膜（TM）结构域有关。最近，Vpu已被证明可以拮抗骨髓基质细胞抗原2 (BST-2)（也称为CD317、HM1.24或tetherin）的抗病毒活性。BST-2是一种干扰素诱导的脂筏型II型整体膜蛋白，具有不同寻常的拓扑结构。它包含一个短的N端区域，随后是一个跨膜结构域，一个中央细胞外卷曲结构域，包含两个N样糖基化位点和一个c端糖基-磷脂酰肌醇（GPI）锚定位点。我们提出了Vpu蛋白可以在脂质或膜筏中检测到的初步数据。我们进一步提供的数据表明，Vpu可以在从感染细胞分离的脂筏中发现，并且胆固醇消耗药物如洛伐他汀/环糊精可以降低筏中Vpu的水平。在这项应用的研究中，我们假设Vpu定位到脂筏上是对抗BST-2和增强病毒释放的必要条件。在Specific Aim 1中，我们建议使用位点定向诱变技术来鉴定筏结合所必需的TM结构域中的氨基酸残基，并确定来自其他HIV-1亚型的Vpu蛋白是否也与脂筏相关。在Specific Aim 2中，我们建议研究是否需要Vpu的raft association来拮抗BST-2的抗病毒活性。我们将确定Vpu结合的时间动力学，如果BST-2破坏了这种结合，如果非筏Vpu仍然可以与BST-2相互作用，它将降低BST-2的细胞表面表达，仍然导致BST-2降解。最后，我们将确定表达非筏Vpu的病毒是否削弱了病毒释放。这些研究结果将为Vpu与膜筏和BST-2拮抗作用的关联提供新的信息，并为Vpu增强病毒释放功能的机制提供新的见解。

DESCRIPTION (provided by applicant): Human immunodeficiency virus type 1 (HIV-1) encodes for a small type I 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 accumulation of and tethering of virus particles at the cell surface and the maturation of viruses into intracellular vesicles. This enhanced release function of Vpu has been associated with the transmembrane (TM) domain of the Vpu. Recently, Vpu has been shown to antagonize the antiviral activities of bone marrow stromal cell antigen 2 (BST-2) also known as CD317, HM1.24, or tetherin. BST-2 is an interferon inducible, lipid raft , type II integral membrane protein with an unusual topology. It contains a short N- terminal region followed by a transmembrane domain, a central extracellular coiled-coiled domain containing two N-liked glycosylation sites and a C-terminal glycosyl-phosphatidylinositol (GPI) anchor. We present preliminary data that the Vpu protein can be detected in lipid or membrane rafts. We further present data that Vpu can be found in lipid rafts isolated from infected cells and that cholesterol depleting drugs as lovastatin/ cyclodextrin reduce the level of Vpu in raft fractions. In the studies proposed of this application, we hypothesize that Vpu localization to lipid rafts is necessary for antagonism of BST-2 and enhancement of virus release. In Specific Aim 1, we propose to use site-directed mutagenesis to identify amino acid residues within the TM domain that are necessary for raft association and determine if Vpu proteins from other HIV-1 subtypes also associate with lipid rafts. In the Specific Aim 2, we propose to examine if raft association of Vpu is required to antagonize the antiviral activities of BST-2. We will determine time kinetics of Vpu association and if BST-2 disrupts this association, if a non-raft Vpu can still interact with BST-2, it will decrease cell surface expression of BST-2 and still cause BST-2 degradation. Finally, we will determine if a virus expressing a non-raft Vpu has impaired virus release. The results of these studies will provide novel information on Vpu association with membrane rafts and BST-2 antagonism as well as provide mechanistic insight into enhanced virus release function of Vpu. PUBLIC HEALTH RELEVANCE: The Vpu protein of HIV-1 has two important functions in the virus replication cycle. One of this functions is to down-modulate the receptor for the virus, CD4. The other function of the Vpu protein is to enhance virus release from infected cells. Recently, bone marrow stromal cell antigen 2 (BST-2) was identified as a virus restriction factor that is targeted for Vpu. The exact mechanism by which Vpu antagonizes the antiviral activities of BST-2 is still unknown. These studies will provide novel information on whether the membrane raft properties of Vpu are required for the interaction with and antagonism of BST-2. The information gained from these studies may result in novel antiviral strategies against HIV-1.