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蛋白质组由所需的结构和必需的调节蛋白以及 增加病毒适应性的辅助蛋白。这些辅助蛋白，Vif，Vpr，Vpu和Nef，参与 对抗宿主免疫应答并拮抗所谓的限制因子，从而增加病毒感染。 感染性，即病毒粒子感染靶细胞并在靶细胞中复制的能力。 Nef，即负性因子，具有多种活性，其中大部分涉及信号调节 转导或膜蛋白分选。Nef增加病毒颗粒（病毒体）的感染性， 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）。 丝氨酸蛋白酶（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。SERINC 5（SERC 5）是最活跃的;它 可能通过改变病毒体-靶细胞的动力学， 融合，Env蛋白的功能。尽管流行的模型是SERC 5整合到病毒体中， 抑制Env功能，它似乎不与Env共免疫沉淀，这表明需要 辅因子或其他间接相互作用。此外，不同的Env蛋白对SERC 5的敏感性不同， 但SERC 5敏感性的结构和分子决定因素尚不清楚。抑制SERC 5抗病毒 活性，Nef与内体机制协调，将SERC 5从质膜重新定位到晚期， 核内体然而，该SERC 5抑制模型仅来源于其中外源性抑制的实验。 SERC 5被表达。内源性SERC 5是否被类似地调节是未知的。 在这里，研究提出，以确定背后的机制SERC 5介导的限制病毒粒子 感染性和Nef介导的这种作用的抵消。进化枝B Envs的初步分析导致了 假设gp 41膜近端外部区域（MPER）内的区域负责 SERC 5-灵敏度。该区域和其他推定的决定簇将通过定点诱变进行探测。到 确定SERC 5与Env的直接相互作用是否可以解释抗病毒活性，或者相反， SERC 5-辅因子是负责的，使用APEX 2的基于邻近的标记方法，与质量 基于光谱的蛋白质组学，将用于探测SERC 5和Env的相互作用。最后 Nef下调来自细胞表面的内源性SERC 5并将其捕获在晚期内体中的假说 通过网格蛋白介导的内吞作用将使用差速离心的细胞分级分离进行测试， 靶向蛋白质组学追踪天然的、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SERC 5对HIV-1病毒粒子感染性的影响。这 理解可能提供新的方法来限制传播，这是NIAID使命的支柱。