Investigating the Env-dependent antiviral activity and Nef-mediated antagonism of SERINC5 in HIV-1 infection

研究 HIV-1 感染中 SERINC5 的 Env 依赖性抗病毒活性和 Nef 介导的拮抗作用

• 批准号: 10237249
• 负责人: Aaron Louis Oom
• 金额: $ 1.82万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2021-10-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10237249
• 关键词: Affect; Antiviral Agents; Biochemical; Biological Assay; Biotinylation; Cell Fractionation; Cell fusion; Cell membrane; Cell physiology; Cell surface; Cells; Centrifugation; Chimeric Proteins; Clathrin; Complement; Coupled; Data; Dynamin; Dynamin 2; Endocytosis; Fluorescence; Future; Gene Expression; Genes; Glycoproteins; HIV-1; Immune response; Infection; Kinetics; Label; Link; Mass Spectrum Analysis; Mediating; Medical; Membrane; Membrane Proteins; Mission; Modeling; Molecular; Movement; National Institute of Allergy and Infectious Disease; Nature; Ontology; Pathway interactions; Peptides; Peroxidases; Prevention strategy; Property; Protein Family; Protein Sortings; Proteins; Proteome; Proteomics; Regimen; Reporting; Resistance; Serine; Signal Transduction; Site-Directed Mutagenesis; Small Interfering RNA; Specificity; Structure; Surface; Testing; Therapeutic; Time; Viral; Viral Genes; Viral Genome; Viral Proteins; Virion; Virus Activation; Virus Replication; Western Blotting; ascorbate; base; cofactor; computerized tools; enhancer-binding protein AP-2; experimental study; gene complementation; genetic regulatory protein; insight; knock-down; late endosome; member; novel strategies; particle; recruit; trafficking; transmission process; viral fitness; viral transmission

The HIV-1 proteome consists of required structural and essential regulatory proteins as well as accessory proteins that increase viral fitness. These accessory proteins, Vif, Vpr, Vpu, and Nef, are involved in countering host immune responses and antagonizing so-called restriction factors, thereby increasing viral infectivity, the ability of a virion to infect and replicate in target cells. Nef, or negative factor, has a variety of activities, most of which involve modulation of signal transduction or membrane protein sorting. Nef increases the infectivity of viral particles (virions), with a mechanism that depends on cellular endosomal proteins as well as on sequences within the viral Envelope glycoprotein (Env). Two members of the serine incorporator (SERINC) protein family were identified recently as host proteins that inhibit viral infectivity and are antagonized by Nef. SERINC5 (SERC5) is the most active; it incorporates into virions and inhibits virion infectivity, potentially by altering the kinetics of virion-target cell fusion, the function of the Env protein. Despite the prevailing model that SERC5 incorporates into virions to inhibit Env function, it does not seem to co-immunoprecipitate with Env, suggesting the requirement of cofactors or other indirect interactions. Moreover, different Env proteins are differentially sensitive to SERC5, but the structural and molecular determinants of SERC5-sensitivity are unclear. To inhibit SERC5 antiviral activity, Nef coordinates with endosomal machinery to relocalize SERC5 from the plasma membrane to late endosomes. However, this model of SERC5-inhibition is derived solely from experiments in which exogenous SERC5 is expressed. Whether endogenous SERC5 is similarly modulated is unknown. Here studies are proposed to determine the mechanisms behind SERC5-mediated restriction of virion infectivity and Nef-mediated counteraction of this effect. Preliminary analysis of clade B Envs leads to the hypothesis that a region within the gp41 membrane proximal external region (MPER) is responsible for SERC5-sensitivity. This region and other putative determinants will be probed by site-directed mutagenesis. To determine whether a direct interaction of SERC5 with Env can explain antiviral activity, or instead whether SERC5-cofactors are responsible, a proximity-based labeling approach using APEX2, coupled with mass spectrometry-based proteomics, will be used to probe the interactions of SERC5 and Env. Lastly, the hypothesis that Nef downregulates endogenous SERC5 from the cell surface and traps it in late endosomes via clathrin-mediated endocytosis will be tested using cell fractionation by differential centrifugation and targeted proteomics to track the native, endogenous, unlabeled protein. When these experiments are completed, the field will have a better explanation of the effect of SERC5 on HIV-1 virion infectivity. This understanding might provide new approaches to restricting transmission, a pillar of the NIAID mission.

HIV-1蛋白质组由必需的结构和必需的调节蛋白以及 增强病毒适应性的辅助蛋白。这些辅助蛋白Vif、Vpr、VPu和Nef参与了 对抗宿主免疫反应和对抗所谓的限制因素，从而增加病毒 感染性，病毒粒子在目标细胞中感染和复制的能力。 NEF，或负性因子，有多种活动，其中大部分涉及信号的调制 转导或膜蛋白分选。NEF增加了病毒颗粒(病毒粒子)的传染性，具有 依赖于细胞内体蛋白以及病毒包膜内序列的机制 糖蛋白(Env)。 丝氨酸结合蛋白(SERINC)家族的两个成员最近被鉴定为宿主 抑制病毒感染性并被Nef拮抗的蛋白质。SERINC5(SERC5)最活跃；它 结合到病毒粒子中并抑制病毒粒子的感染性，可能是通过改变病毒粒子靶细胞的动力学 融合，包膜蛋白的功能。尽管SERC5整合到病毒粒子中的流行模型 抑制Env的功能，它似乎不与Env共沉淀，提示需要 辅因或其他间接相互作用。此外，不同的Env蛋白对SERC5具有不同的敏感性， 但SERC5敏感性的结构和分子决定因素尚不清楚。抑制SERC5抗病毒药物 活性，Nef与内体机械协调，将SERC5从质膜重新定位到晚期 内噬菌体。然而，这种SERC5抑制模型完全来自于外源激素的实验 表达了SERC5。内源性SERC5是否也受到类似的调节尚不清楚。 本研究旨在确定SERC5介导的病毒粒子限制机制。 感染性和Nef介导的这一效应的抵消。B分支环境的初步分析导致了 假设gp41膜近端外部区域(MPER)内的一个区域负责 SERC5-敏感性。这个区域和其他假定的决定因素将通过定点突变进行探测。至 确定SERC5与Env的直接相互作用是否可以解释抗病毒活性，或者相反 SERC5-辅因子起作用，一种使用APEX2的基于邻近的标记方法，结合质量 基于光谱的蛋白质组学，将被用来探索SERC5和Env的相互作用。最后， Nef下调细胞表面内源性SERC5并将其捕获到晚期内吞体内的假说 通过网状蛋白介导的内吞作用将通过差速离心法进行细胞分级和 靶向蛋白质组学跟踪天然的、内源性的、未标记的蛋白质。当这些实验 完成后，该领域将更好地解释SERC5对HIV-1病毒粒子传染性的影响。这 了解可能会为限制传播提供新的方法，这是NIAID任务的一个支柱。