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：胎盘通过以下方式形成了艾滋病毒传播的物理屏障： 通过表达大量的蛋白来分离母体和胎儿的血液以及功能屏障 抑制病毒复制的限制因素。然而，艾滋病毒的垂直传播确实存在 在子宫内并表明这些防御是不完善的。这与近期的情况相符 描述了病毒限制因子 Serinc，它在胎盘中高度表达，并且可以 掺入出芽的病毒颗粒中以抑制其感染能力。然而，Serinc 是一个 不完美的限制因子，因为它被病毒辅助蛋白 Nef 和一些病毒失活 HIV 表面蛋白 Env 的序列。我们计划研究 Serinc 的机制 限制艾滋病毒感染，以更好地了解宿主与病原体的相互作用 发生在胎盘中，希望能够开发出新型抗 可以利用与 Serinc 相同的病毒弱点，但不易受到相同病毒的影响 陷阱。 Serinc 是在特定组织中表达的 5 种人类质膜蛋白家族。同工型 Serinc3和Serinc5限制HIV增殖，而Serinc2融入病毒颗粒但不 限制。 Serinc3 和 5 降低传染性的确切机制尚不完全清楚，但已被证实 已知可在进入细胞之前的步骤阻止感染。出现了两种主要理论：1）Serinc 通过改变病毒中 Env 三聚体的构象和分布来灭活 Env 颗粒或2)丝氨酸减缓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“泡”的发展作为病毒模型 膜融合来测试 Serinc 的两种提议机制的相对优点。我们将评估 Serinc- 含有或缺乏 HIV 假病毒，通过冷冻改变 Env 的结构和分布 电子断层扫描和次断层扫描平均（目标 1）。我们还将评估 Serincs 是否会阻止 通过观察含有或缺乏丝氨酸的 HIV 假病毒与泡融合时的膜融合 具有高分辨率冷冻电子断层扫描快照（目标 2.1）及更高版本的模型膜 高通量荧光显微镜单颗粒病毒融合测定（目标 2.2）。初步结果 显示 Serinc3 和 Serinc5 通过破坏融合孔开放来阻止膜融合，但不影响 包膜分布或病毒颗粒进行膜融合的速度。带着这两个目标，我们 将能够区分有关 Serinc 限制机制的相互竞争的假设 HIV 感染，更好地了解 HIV 如何在胎盘中传播，并有可能使 新型抗病毒药物的开发。