Mechanistic investigations of HIV restriction by Serincs

项目总结：胎盘通过以下方式形成了HIV传播的物理屏障： 分离母体和胎儿的血液和功能屏障， 限制因子来抑制病毒复制。然而，艾滋病毒的垂直传播确实会发生 表明这些防御机制并不完善这与最近 描述了病毒限制因子Serinc，它在胎盘中高度表达， 掺入出芽的病毒颗粒中以抑制它们的感染能力。然而，Serinc是一个 不完全限制因子，因为它被病毒辅助蛋白Nef灭活， HIV表面蛋白Env.我们计划研究Serinc 限制HIV感染，以更好地了解宿主-病原体相互作用 发生在胎盘，并希望能够开发新的抗- 可以利用与Serinc相同的病毒弱点，但对Serinc不敏感的病毒。 陷阱 丝氨酸是在选择的组织中表达的5种人质膜蛋白的家族。同种型 Serinc3和Serinc5限制HIV增殖，而Serinc2掺入病毒颗粒，但不 限制。Serinc3和Serinc5降低感染性的确切机制尚不完全清楚， 已知在细胞进入前的一步阻断感染。出现了两种主要的理论：1）Serinc 通过引起病毒中Env三聚体的构象和分布的变化来灭活Env 或2）丝氨酸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"水泡"的发展作为病毒的模型， 膜融合，以测试Serinc.我们将评估Serinc- 含有或缺乏HIV假病毒，通过冷冻- 电子断层扫描和亚断层图像平均（目标1）。我们还将评估Serincs是否阻止 通过观察HIV假病毒与水泡融合时含有或缺乏Serincs来观察膜融合 具有高分辨率低温电子断层扫描快照（aim 2.1）和更高分辨率的模型膜 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而阻断膜融合，但不影响 Env分布或病毒颗粒进行膜融合的速度。有了这两个目标，我们 将能够区分关于丝氨酸限制的机制的竞争假设， 艾滋病毒感染，更好地了解艾滋病毒如何在胎盘中传播，并可能使 开发新的抗病毒药物。

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.