The Role of Lipid Rafts in Vpu Function

脂筏在 Vpu 功能中的作用

• 批准号: 8017001
• 负责人: Edward Brice Stephens
• 金额: $ 22.5万
• 依托单位: UNIVERSITY OF KANSAS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-15 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8017001
• 关键词: Affect; Alanine; Amino Acids; Anticholesteremic Agents; Antigens; Antiviral Agents; Binding Proteins; Bone Marrow; C-terminal; CD4 Antigens; CD4 Positive T Lymphocytes; Cell membrane; Cell surface; Cells; Cholesterol; Coiled-Coil Domain; Complex; Cullin 1; Cyclodextrins; Data; Degradation Pathway; Glycosylphosphatidylinositols; HIV-1; Human; Integral Membrane Protein; Interferons; Kinetics; Libraries; Lipids; Lovastatin; Mediating; Membrane; Membrane Microdomains; Membrane Proteins; Messenger RNA; Molecular; N-terminal; Nature; Peripheral Blood Lymphocyte; Pharmaceutical Preparations; Production; Property; Proteins; Role; Rough endoplasmic reticulum; Series; Shunt Device; Site; Site-Directed Mutagenesis; Sphingolipids; Stromal Cells; Time; Transmembrane Domain; Vesicle; Viral Proteins; Virion; Virus; Virus Assembly; Virus Receptors; Virus Replication; cell type; env Gene Products; extracellular; glycosylation; insight; multicatalytic endopeptidase complex; mutant; novel; prevent; public health relevance; research study; ubiquitin-protein ligase; vpu Genes

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.

描述（由申请人提供）：1 型人类免疫缺陷病毒 (HIV-1) 编码一种称为 Vpu 的小型 I 型膜蛋白，在受感染的细胞中具有两个主要功能。已知 Vpu 与 CD4 分子相互作用并将 CD4 分子从粗面内质网 (RER) 分流至蛋白酶体进行降解。此外，已知 Vpu 可以增强受感染细胞的病毒释放。 HIV-1病毒在缺乏vpu基因的CD4+T细胞中组装的特点是病毒颗粒在细胞表面的积累和束缚以及病毒成熟为细胞内囊泡。 Vpu 的这种增强的释放功能与 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 是一种干扰素诱导型脂筏 II 型整合膜蛋白，具有不寻常的拓扑结构。它包含一个短的 N 端区域，后跟一个跨膜结构域、一个包含两个 N 样糖基化位点的中央细胞外卷曲螺旋结构域和一个 C 端糖基磷脂酰肌醇 (GPI) 锚定点。我们提供了可以在脂质或膜筏中检测到 Vpu 蛋白的初步数据。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，并且洛伐他汀/环糊精等胆固醇消耗药物可降低脂筏部分中的 Vpu 水平。在本申请提出的研究中，我们假设 Vpu 定位于脂筏对于拮抗 BST-2 和增强病毒释放是必要的。在具体目标 1 中，我们建议使用定点诱变来识别 TM 结构域内对于筏关联所必需的氨基酸残基，并确定来自其他 HIV-1 亚型的 Vpu 蛋白是否也与脂筏关联。在具体目标 2 中，我们建议检查 Vpu 的筏关联是否需要拮抗 BST-2 的抗病毒活性。我们将确定 Vpu 关联的时间动力学，如果 BST-2 破坏这种关联，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，并仍然导致 BST-2 降解。最后，我们将确定表达非筏 Vpu 的病毒是否损害了病毒释放。这些研究的结果将提供有关 Vpu 与膜筏和 BST-2 拮抗作用的关联的新信息，并为 Vpu 增强病毒释放功能提供机制见解。 公共卫生相关性：HIV-1 的 Vpu 蛋白在病毒复制周期中具有两个重要功能。该功能之一是下调病毒受体 CD4。 Vpu 蛋白的另一个功能是增强病毒从受感染细胞中的释放。最近，骨髓基质细胞抗原2（BST-2）被鉴定为针对Vpu的病毒限制因子。 Vpu 拮抗 BST-2 抗病毒活性的确切机制仍不清楚。这些研究将提供关于 Vpu 的膜筏特性是否是与 BST-2 相互作用和拮抗所必需的新信息。从这些研究中获得的信息可能会产生针对 HIV-1 的新型抗病毒策略。