Nef Interaction Networks at the Membrane

NEF是使HIV-1能够破坏宿主防御的四种辅助蛋白之一。NEF不是任何 目前的抗病毒药物，但是一个令人兴奋的潜在目标，因为它对病毒粒子的传染性至关重要。长 长期感染携带有缺陷的nef等位基因的HIV毒株，即使进展到艾滋病，进展也非常缓慢。许多 Nef的功能已被归因于，包括通过蛋白酪氨酸激酶和 下调CD4、MHC-I、BST2/tetherin(针对O-群Nef)和其他细胞表面受体。在此基础上 在2015年的突破性发现中，Nef赋予HIV-1传染性的机制被证明是 下调完整的膜蛋白SERINC5。SERINC5含有10个可能的跨膜蛋白 跨越螺旋，没有暗示Nef结合位点的基序。关于生物化学的文献很少 SERINC5，目前尚不清楚SERINC是直接结合Nef，还是通过一个或多个额外因素结合Nef。 在Nef下调SERINC5的其他因素中，四聚体网状蛋白接头AP-2是 已知对SERINC5下调和直接与Nef结合是必不可少的。AP-2参与网状蛋白介导的 内吞作用，但不直接参与降解性分选。另一个因素Alix与 ESCRT分选溶酶体的机制，并与Nef相互作用。NEF在以下环境中与Alix交互 Gag-Pol融合。所有这些蛋白质都在细胞膜的背景下发挥作用。该项目的目标1将 重组SERINC5、AP-2、Alix和Gag-Pol的相互作用网络。NEF通过以下途径下调MHC-I 笼状蛋白接头AP-1，这是一个严重依赖于小G蛋白Arf1的过程。Arf1不会推广 然而，AP-2依赖内吞作用。在目标2中，我们将分离AP-2形成的笼蛋白包裹的囊泡。 NEF的诱导作用，并与无Nef时AP-2/clathrin囊泡的成分进行比较。这将使我们能够 鉴定与Nef依赖的AP-2囊泡形成相关的Arf1类似物。小说的组成部分(S)将 然后被纳入到目标1的重建实验中，使项目循环完整。在目标3中，我们 将优化包括3，5在内的几种SERINC的表达条件，寻求纯均一和稳定的 用于结构分析的产品和复杂构型。

Nef is one of four accessory proteins that enable HIV-1 to undermine host defenses. Nef is not targeted by any current antivirals, but is an exciting potential target because it is essential for the infectivity of virions. Long term infection with HIV strains bearing defective nef alleles progresses to AIDS very slowly, if at all. Many functions have been imputed to Nef, including modulating signaling by protein tyrosine kinases and downregulating CD4, MHC-I, BST2/tetherin (for O-group Nefs) and other cell surface receptors. On the basis of breakthrough findings in 2015, the mechanism whereby Nef confers infectivity on HIV-1 was shown to be the downregulation of the integral membrane protein, SERINC5. SERINC5 contains ten putative transmembrane spanning helices and no motifs suggestive of a binding site for Nef. Little biochemical literature exists on SERINC5 and it is currently unclear whether SERINC binds Nef directly or via one or more additional factors. Of the additional factors involved in Nef downregulation of SERINC5, the tetrameric clathrin adaptor AP-2 is known to be essential for SERINC5 downregulation and to directly bind to Nef. AP-2 is involved in clathrin-mediated endocytosis, but is not directly involved in degradative sorting. Another factor, Alix, is associated with the ESCRT machinery of lysosomal sorting, and interacts with Nef. Nef interacts with Alix in the context of the Gag-Pol fusion. All of these proteins function in the context of cell membranes. Aim 1 of this project will reconstitute the interaction network of SERINC5, AP-2, Alix, and Gag-Pol. Nef downregulates MHC-I via the clathrin adaptor AP-1, a process that is critically dependent on the small G-protein Arf1. Arf1 does not promote AP-2 dependent endocytosis, however. In Aim 2, we will isolate clathrin-coated vesicles formed by AP-2 upon Nef induction and compare their components to AP-2/clathrin vesicles in the absence of Nef. This will allow us to identify the putative Arf1 counterpart for Nef-dependent AP-2 vesicle formation. The novel component(s) will then be incorporated into the reconstitution experiments in Aim 1, bringing the project full circle. In Aim 3, we will refine conditions for expression of several SERINCs including 3,5 seeking a pure homogeneous and stable product and complex formation for structure analysis.