Allostery and Hijacking of Host Membrane Traffic by HIV-1 Accessory Proteins

HIV-1 辅助蛋白对宿主膜运输的变构和劫持

基本信息

批准号：
10460353
负责人：
James H Hurley
金额：
$ 64.68万
依托单位：
UNIVERSITY OF CALIFORNIA BERKELEY
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-06-18 至 2025-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10460353
关键词：
Acquired Immunodeficiency Syndrome Adaptor Protein Complex 2 Adaptor Signaling Protein Affect Alleles Antiviral Agents Automobile Driving Basic Science Binding Sites Biochemistry Biophysics CD28 gene CD3 Antigens CD8B1 gene CXCR4 gene Cell Surface Proteins Cell membrane Cell surface Cells Clathrin Clathrin-Coated Vesicles Complement Complex Cryoelectron Microscopy Cytosol Detergents Down-Regulation Family Funding Goals HIV HIV-1 Host Defense Human Image Immune system Immunity Infection Integral Membrane Protein Lipids Location Lysosomes Mediating Membrane Membrane Lipids Membrane Protein Traffic Monomeric GTP-Binding Proteins Mutation Pathway interactions Pattern Phenotype Process Production Proteins Reporting Research Proposals Role SIV Seminal Signal Transduction Site Sorting - Cell Movement Structure Tail Viral Viral Antigens Virion Virus Virus Latency base design follow-up insight member prevent protein complex reactivation from latency receptor reconstitution trans-Golgi Network viral fitness

项目摘要

PROJECT SUMMARY Nef is an HIV-1 accessory protein whose function is to undermine host defenses. Long term infection with HIV strains bearing defective nef alleles leads to AIDS only over many years, suggesting Nef could be targeted as part of functional cure strategies. This basic research proposal seeks will elucidate the mechanisms of action of Nef. Nef targets include CD3, CD4, CD8, CD28, CXCR4, MHC-I, SERINC3/5, and for HIV-1 group O and SIV, BST2/tetherin. Nef substrates are downmodulated by via the clathrin-coated vesicle (CCV) pathway. Nef does not interact directly with clathrin, but rather with various members of a family of heterotetrameric adaptors known as the adaptor protein (AP) complexes, AP-1 and AP-2. The ability of the human immune system to detect and kill virally infected cells relies on proper presentation of viral antigens on the cell surface by MHC-I complexes. Nef subverts this process by promoting MHC-I complex downregulation by hijacking AP-1 and its associated small GTPase Arf1 at the TGN. MHC-I contains an incomplete version of the normal Tyr-based sorting motif. Nef complements this defective motif and converts MHC-I into a substrate for AP-1 mediated sorting to the lysosome for degradation. Structures of Nef assembled with AP-1 and the MHC-I cytosolic tail in solution suggested that Nef promotes the assembly of hexagonal lattices whose symmetry matches that of clathrin. Now, the previous solution studies will be followed up by reconstitution and structure determination of Nef, MHC-I tail, AP-1 and Arf1 in their functional setting on lipid membranes. Downmodulation of CD28 by Nefs is conserved across SIV and HIV and is mediated by AP-2. CD28 downmodulation phenotypes of Nef mutations follow a distinct pattern from other receptors, and the structural basis for this mode of CD28 is unknown. The structure of the CD28:HIV-1 Nef:AP-2 complex will be determined and leveraged to design mutations that uniquely perturb the CD28 binding site. Of the many host substrates of Nef, the most significant for viral infectivity are the multipass integral membrane proteins SERINC3 and 5. The SERINC binding site appears, on the basis of Nef phenotypes, to overlap with the site used by SIVsmm Nef to downmodulate simian tetherin, but is otherwise distinct from the known locations of CD3, CD4, and MHC-I sites. SERINCs do not share any obvious motifs with other substrates. SERINCs have been purified in monodisperse form suitable for structure determination. The cryo-EM structure of lipid- or detergent embedded SERINC3 or 5 in complex with AP-2 and HIV-1 Nef will be determined, completing a major goal in the field.

项目概要 Nef 是一种 HIV-1 辅助蛋白，其功能是破坏宿主防御。长期感染艾滋病毒携带有缺陷的 nef 等位基因的菌株仅在多年后才会导致艾滋病，这表明 Nef 可以作为目标功能性治疗策略的一部分。这项基础研究提案旨在阐明作用机制内夫. Nef 靶标包括 CD3、CD4、CD8、CD28、CXCR4、MHC-I、SERINC3/5 以及 HIV-1 O 组和 SIV、BST2/系链蛋白。 Nef 底物通过网格蛋白包被的囊泡 (CCV) 途径下调。内夫不直接与网格蛋白相互作用，而是与异四聚体适配器家族的各个成员相互作用称为衔接蛋白 (AP) 复合物、AP-1 和 AP-2。人体免疫系统的能力检测并杀死病毒感染的细胞依赖于 MHC-I 在细胞表面正确呈递病毒抗原复合物。 Nef 通过劫持 AP-1 及其促进 MHC-I 复合物下调来颠覆这一过程 TGN 处相关的小 GTPase Arf1。 MHC-I 包含正常 Tyr 基的不完整版本排序主题。 Nef 补充了这个有缺陷的基序，并将 MHC-I 转化为 AP-1 介导的底物分选至溶酶体进行降解。 Nef 与 AP-1 和 MHC-I 胞质尾部组装的结构解决方案表明 Nef 促进了六方晶格的组装，其对称性与网格蛋白。现在，之前的解决方案研究将继续进行重构和结构确定 Nef、MHC-I 尾部、AP-1 和 Arf1 在脂膜上的功能设置。 Nefs 对 CD28 的下调在 SIV 和 HIV 中保守，并由 AP-2 介导。 Nef 的 CD28 下调表型突变遵循与其他受体不同的模式，CD28 这种模式的结构基础是未知。 CD28:HIV-1 Nef:AP-2 复合物的结构将被确定并用于设计独特地干扰 CD28 结合位点的突变。在 Nef 的众多宿主底物中，最重要的是用于病毒感染性的是多通道整合膜蛋白 SERINC3 和 5。SERINC 结合位点根据 Nef 表型，似乎与 SIVsmm Nef 用于下调的位点重叠猿系绳蛋白，但在其他方面与 CD3、CD4 和 MHC-I 位点的已知位置不同。 SERINC 可以不与其他基材共享任何明显的图案。 SERINC 已以单分散形式纯化适用于结构测定。嵌入脂质或去污剂的 SERINC3 或 5 的冷冻电镜结构 AP-2 和 HIV-1 Nef 的复合物将被确定，从而完成该领域的一个主要目标。