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：胎盘通过以下途径形成防止艾滋病毒传播的物理屏障 通过过量表达母血和胎儿血液和功能障碍 抑制病毒复制的限制因素。然而，艾滋病毒的垂直传播确实发生了 并表明这些防御系统是不完美的。这与最近的 被描述的病毒限制因子Serinc.它在胎盘中高表达，可以 加入萌芽中的病毒颗粒中，以抑制其感染能力。然而，Serinc是一种 不完善的限制因子，因为它被病毒辅助蛋白Nef和一些 人类免疫缺陷病毒表面蛋白Env.我们计划研究丝氨酸的作用机制 限制HIV感染以更好地了解宿主和病原体的相互作用 发生在胎盘中，并希望能够开发出新的抗 病毒可以利用与Serinc相同的病毒弱点，但不会对相同的病毒敏感 陷阱。 Serincs是一个由5个人的质膜蛋白组成的家族，在特定的组织中表达。异构体 Serinc3和Serinc5抑制HIV的增殖，而Serinc2结合到病毒颗粒中，但不 限制。Serinc3和Serinc5降低传染性的确切机制尚不完全清楚，但 已知在进入细胞之前的一步就能阻止感染。出现了两种主要的理论：1)Serinc. 通过改变病毒中环境三聚体的构象和分布来灭活环境病毒 颗粒或2)丝氨酸锌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”的发育作为病毒的模型。 膜融合，以检验两种提出的丝氨酸锌作用机制的相对优劣。我们会评估赛琳娜- 含或不含HIV伪病毒对冷藏病毒包膜结构和分布的影响 电子断层扫描和亚断层图像平均(目标1)。我们还将评估Serincs是否会阻止 通过观察HIV假病毒与水泡融合时含有或缺失丝蛋白的膜融合 具有高分辨率冷冻电子断层扫描快照的模型膜(目标2.1)和更高版本 通过荧光显微镜单颗粒病毒融合分析(AIM 2.2)。初步结果 显示Serinc3和Serinc5通过破坏融合孔开放来阻断膜融合，但不影响 Env分布或病毒粒子经历膜融合的速度。有了这两个目标，我们 将能够区分关于丝氨酸限制机制的相互竞争的假说 艾滋病毒感染，更好地了解艾滋病毒如何在胎盘中传播，并有可能使 新的抗病毒药物的开发。