Mechanistic investigations of HIV restriction by Serincs

Serincs 限制 HIV 的机制研究

• 批准号: 10360510
• 负责人: Amanda Elizabeth Ward
• 金额: $ 3.38万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-01 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10360510
• 关键词: Antiviral Agents; Biological; Biological Assay; Bulla; CCR5 gene; Cell membrane; Cells; Complement; Cryo-electron tomography; Cryoelectron Microscopy; Development; Family; Fetus; Fluorescence Microscopy; Freezing; HIV; HIV Budding; HIV Infections; Human; Image; Individual; Infant; Infection; Investigation; Kinetics; Lead; Maternal-Fetal Exchange; Membrane; Membrane Fusion; Membrane Lipids; Membrane Proteins; Modeling; Molecular Conformation; Mothers; Placenta; Plasma Cells; Process; Protein Isoforms; Proteins; Resolution; Speed; Structure; Techniques; Testing; Time; Tissues; Tomogram; Vertical Disease Transmission; Vesicle; Viral; Virus; Virus Replication; base; blocking factor; design; fetal blood; in utero; membrane model; novel; novel therapeutics; particle; pathogen; prevent; receptor; theories; transmission process

PROJECT SUMMARY: The placenta forms a physical barrier to HIV transmission by separating maternal and fetal blood and a functional barrier by expressing a plethora of restriction factors to inhibit viral replication. However, vertical transmission of HIV does occur in utero and shows that these defenses are imperfect. This is consistent with the recently described viral restriction factor, Serinc, which is highly expressed in the placenta and can incorporate into budding viral particles to inhibit their ability to infect. However, Serinc is an imperfect restriction factor as it is inactivated by the viral accessory protein, Nef, and some sequences of the HIV surface protein, Env. We plan to study the mechanism of how Serinc restricts HIV infection to gain a better understanding of host-pathogen interactions occurring in the placenta and with the hope of enabling the development of novel anti- virals that can exploit the same viral weakness as Serinc but are not susceptible to the same pitfalls. Serincs are a family of 5 human plasma membrane proteins expressed in select tissues. Isoforms Serinc3 and Serinc5 restrict HIV proliferation while Serinc2 incorporates into viral particles but does not restrict. The exact mechanism by which Serinc3 and 5 reduce infectivity is incompletely understood but is known to block infection at a step before cell entry. Two major theories have emerged: 1) Serinc inactivates Env by causing changes to the conformation and distribution of Env trimers in the viral particle or 2) Serinc slows or disrupts membrane fusion of the virus with the host cell plasma membrane. In this study, we will take advantage of the development of plasma membrane “blebs” as a model for viral membrane fusion to test the relative merits of two proposed mechanisms of Serinc. We will assess Serinc- containing or -lacking HIV pseudoviruses for changes in structure and distribution of Env by cryo- electron tomography and subtomogram averaging (aim 1). We will also assess whether Serincs block membrane fusion by observing HIV pseudoviruses containing or lacking Serincs as they fuse to bleb model membranes with high resolution cryo-electron tomography snapshots (aim 2.1) and higher throughput fluorescence microscopy single-particle viral fusion assays (aim 2.2). Preliminary results show Serinc3 and Serinc5 block membrane fusion by disrupting fusion pore opening but do not affect Env distribution or the speed at which viral particles undergo membrane fusion. With these two aims, we will be able to discriminate between competing hypotheses about the mechanism of Serinc restriction of HIV infection, gain a better understanding of how HIV transmits in the placenta, and potentially enable the development of new anti-virals.

项目摘要：PLAPETA形成了通过 通过表达大量 限制抑制病毒复制的因素。但是，艾滋病毒的垂直传播确实发生 在子宫内，表明这些防御是不完善的。这与最近一致 描述的病毒限制因子，序列，该因子在paceta中高度表达，可以 纳入萌芽的病毒颗粒以抑制其感染能力。但是，序列是 不完善的约束因子由于病毒辅助蛋白，NEF和某些人灭活 HIV表面蛋白的序列，Env。我们计划研究如何序列的机制 限制艾滋病毒感染以更好地了解宿主病原体相互作用 发生在放信中，并希望能够发展出新的反抗 可以利用与序列相同的病毒弱点但不易于相同的病毒病毒 陷阱。 序列是在某些组织中表达的5个人质膜蛋白的家族。同工型 Serinc3和Serinc5限制了HIV增殖，而Serinc2则掺入病毒颗粒，但不限制HIV 限制。 Serinc3和5减少感染的确切机制尚不完全理解，但是 已知会在细胞进入前的一步中阻止感染。出现了两个主要理论：1）序列 通过对病毒中的ENV三聚体的会议和分布进行更改而灭活Env 粒子或2）序列减慢或破坏病毒与宿主细胞质膜的膜融合。 在这项研究中，我们将利用质膜“ Blebs”的发展作为病毒的模型 膜融合以测试两种建议的序列机制的相对优点。我们将评估序列 - 含有或缩写的HIV假病毒，以通过冷冻的结构和ENV分布的变化 电子断层扫描和亚图平均图（AIM 1）。我们还将评估Serincs是否块 膜融合通过观察HIV伪病毒融合时含有或缺乏序列的膜融合 具有高分辨率的低分子电子层析成像快照（AIM 2.1）和更高的模型膜 吞吐量荧光显微镜单粒子病毒融合测定（AIM 2.2）。初步结果 通过破坏融合孔的打开，显示Serinc3和Serinc5嵌段膜融合，但不会影响 ENV分布或病毒颗粒经历膜融合的速度。以这两个目标，我们 将能够区分有关串行限制机制的竞争假设 艾滋病毒感染，更好地了解艾滋病毒在plapeta中的传播方式，并有可能启用 新反病毒的发展。