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）：人类免疫缺陷病毒1型（HIV-1）编码一种称为Vpu的小型I型膜蛋白，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。这种增强的Vpu释放功能与Vpu的跨膜（TM）结构域有关。最近，已显示Vpu拮抗骨髓基质细胞抗原2（BST-2）的抗病毒活性，也称为CD 317、HM 1.24或系链蛋白。BST-2是一种干扰素诱导的脂筏II型整合膜蛋白，具有不寻常的拓扑结构。它含有一个短的N-末端区域，随后是一个跨膜结构域，一个含有两个N-样糖基化位点的中央胞外卷曲螺旋结构域和一个C-末端糖基-磷脂酰肌醇（GPI）锚。我们目前的初步数据表明，Vpu蛋白可以检测到脂筏或膜筏。我们进一步提出的数据表明，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/环糊精降低脂筏组分中Vpu的水平。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鉴定TM结构域内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，我们建议检查是否需要Vpu的筏缔合来拮抗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的病毒是否会损害病毒释放。这些研究的结果将提供新的信息与膜筏和BST-2拮抗作用的Vpu协会，以及提供机制洞察增强病毒释放功能的Vpu。 公共卫生相关性：HIV-1的Vpu蛋白在病毒复制周期中有两个重要功能。这种功能之一是下调病毒的受体CD 4。Vpu蛋白的另一个功能是增强病毒从感染细胞中释放。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尚不清楚。这些研究将提供新的信息是否Vpu的膜筏性能所需的相互作用和拮抗BST-2。从这些研究中获得的信息可能会导致新的抗HIV-1的抗病毒策略。