Defining the role of phosphatidylserine in hemorrhagic fever virus replication

项目摘要 当细胞表达磷脂酰丝氨酸(PS)时，埃博拉病毒(EBOV)和拉萨病毒(LASV)的感染增加 结合细胞表面的受体。PS是一种细胞脂，通常局限于内部小叶，或细胞质 细胞质膜(PM)的表面。PS等磷脂对内叶的限制作用 在健康细胞中产生高度不对称的膜。PS被翻转到细胞的外层小叶 钙信号转导和细胞凋亡过程中的细胞膜，分别将它们标记为激活或死亡的细胞。 被称为翻转酶和打乱酶的细胞酶负责翻转膜中的PS。PS是 在病毒萌发期间，当EBOV和LASV颗粒适当时，被整合到病毒膜上 作为保护膜的宿主细胞质膜的一部分。为了激活PS受体，PS 病毒信封上的病毒必须翻到外面的传单上。我们的目标是阐明病毒感染的机制 包膜获得定向正确的PS，以及足够数量的PS与PS受体相互作用。我们 正在使用一组缺乏PS翻转酶或PS扰乱酶的人类单倍体(HAP1)细胞株来研究 这些蛋白在EBOV和LASV的复制中发挥作用。我们生产了水泡性口炎病毒 含有其天然糖蛋白(G)、LASV-gp或EBOV-gp，使我们能够进行实验 在BSL2条件下。当这些重组病毒在HAP1基因敲除细胞系中生长时，我们 确定了VSV颗粒有效扩散所需的一种翻转酶和一种加扰酶，其中包括 LASV-GP或EBOV-GP，但不是VSV-G。这一数据表明，外周PS水平发生了变化 细胞质膜的小叶抑制病毒复制周期中的一个或多个步骤。类似病毒的颗粒类似于 EBOV或LASV在缺乏扰乱酶的细胞中产生时，发现含有较少的表面PS 活性，也支持了我们的总体假设，即细胞酶参与了生产和定位 将影响EBOV和LASV的进入和传播。我们提出了三个具体目标，以进一步审查 PS在病毒复制中的作用：目标1：研究EBOV对细胞扰乱酶活性的要求 和LASV进入和病毒粒子的产生；目标2：检查EBOV对细胞翻转酶活性的要求 和LASV进入；目标3：确定改变细胞PS对EBOV和LASV复制的影响以及 病毒型脂蛋白。在病毒样EBOV颗粒和重组EBOV颗粒之外使用基于VSV的伪颗粒 淋巴细胞性脉络膜脑膜炎病毒(RLCMV)含有LASV-gp，我们将确定其在细胞内的作用 病毒复制过程中的翻转酶和扰乱酶，并用类似的实验证实了我们的结果 BSL4实验室中的真实病毒。实验还将确定这些病毒的脂体，方法将 重点是量化病毒颗粒上修饰的PS的水平。理解PS和其他脂质的作用 利用LASV和EBOV的传染性可能为未来的抗病毒药物提供新的靶点。

Project Summary Ebola virus (EBOV) and Lassa virus (LASV) infection is enhanced when cells express phosphatidylserine (PS) binding receptors on the cell surface. PS is a cellular lipid normally restricted to the inner leaflet, or cytoplasmic face, of the cellular plasma membrane (PM). The restriction of PS and other phospholipids to the inner leaflet produces a highly asymmetric membrane in healthy cells. PS is flipped to the outer leaflet of the cellular membrane during calcium signaling and apoptosis, marking them as activated or dying cells, respectively. Cellular enzymes termed flippases and scramblases are responsible for flipping the PS in the membrane. PS is incorporated into the viral membrane during virus budding, when EBOV and LASV particles appropriate a portion of the host cell's plasma membrane as a protective envelope. In order to engage PS receptors, the PS on a viral envelope must be flipped to the outer leaflet. We aim to elucidate the mechanism by which viral envelopes obtain properly oriented PS, as well as the amount of PS sufficient to interact with PS receptors. We are using a panel of human haploid (HAP1) cell lines lacking PS flippases or PS scramblases to examine the role these proteins play in the replication of EBOV and LASV. We produced vesicular stomatitis virus containing either its native glycoprotein (G), LASV-GP, or EBOV-GP, enabling us to perform experiments under BSL2 conditions. When these recombinant viruses were grown in the HAP1 knock-out cell lines, we identified one flippase and one scramblase that are required for efficient spread of VSV particles containing either the LASV-GP or EBOV-GP, but not VSV-G. This data suggests altering the levels of PS in the outer leaflet of the cellular PM inhibits one or more steps in the viral replication cycle. Virus-like particles resembling either EBOV or LASV were found to contain less surface PS when produced in cells deficient in scramblase activity, also supporting our overall hypothesis that cellular enzymes involved in the production and localization of PS will impact EBOV and LASV entry and spread. We have proposed three specific aims to further examine the role PS plays in viral replication: Aim 1: Examine the requirements for cellular scramblase activity in EBOV and LASV entry and virion production; Aim 2: Examine the requirements for cellular flippase activity in EBOV and LASV entry; Aim 3: Determine the effects of altered cellular PS to EBOV and LASV replication and the viral lipid profile. Using VSV-based pseudoparticles in addition to virus-like EBOV particles and recombinant lymphocytic choriomeningitis virus (rLCMV) containing the LASV-GP, we will determine the role of cellular flippases and scramblases during viral replication, and confirm our results using similar experiments with authentic virus in a BSL4 lab. Experiments will also define the lipidome of these viruses and approaches will focus on quantifying the levels of modified PS on viral particles. Understanding the role PS and other lipids play on LASV and EBOV infectivity will potentially provide new targets for future antivirals.