Defining the role of phosphatidylserine in hemorrhagic fever virus replication

定义磷脂酰丝氨酸在出血热病毒复制中的作用

• 批准号: 10328452
• 负责人: Melinda Ann Brindley
• 金额: $ 7.37万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-12 至 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10328452
• 关键词: Affinity; Africa; Anabolism; Antiviral Agents; Apoptosis; Apoptotic; Arenavirus; Binding; Biological Assay; Calcium Signaling; Cell Line; Cell membrane; Cell surface; Cells; Cellular Membrane; Chinese Hamster Ovary Cell; Data; Diagnosis; Disease; Disease Outbreaks; Ebola; Ebola virus; Environment; Enzymes; Face; Future; Glycoproteins; Haploidy; Health; Human; Infection; Knock-out; Lassa virus; Link; Lipids; Lymphocytic choriomeningitis virus; Mediating; Membrane; Molecular Chaperones; Oxides; Pathway interactions; Periodicity; Pharmaceutical Preparations; Phosphatidylserine Synthase; Phosphatidylserines; Phospholipids; Plasma Cells; Play; Process; Production; Proteins; Recombinants; Role; Stains; Surface; Vaccines; Vesicular stomatitis Indiana virus; Viral; Virion; Virus; Virus Diseases; Virus Replication; Virus-like particle; base; experimental study; hemorrhagic fever virus; lipidome; lipidomics; mimicry; overexpression; particle; phosphatidylserine receptor; potential biomarker; prevent; receptor binding; recombinant virus; virus envelope

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.

项目摘要 当细胞表达磷脂酰丝氨酸（PS）时，埃博拉病毒（EBOV）和拉沙病毒（LASV）感染增强 结合细胞表面的受体。PS是一种细胞脂质，通常局限于内小叶或细胞质 细胞质膜（PM）的表面。PS和其他磷脂限制在内叶 在健康细胞中产生高度不对称的膜。PS被翻转到细胞的外小叶， 在钙信号传导和细胞凋亡过程中，细胞膜上的细胞凋亡，分别将它们标记为活化或死亡细胞。 称为翻转酶和乱序酶的细胞酶负责翻转膜中的PS。PS是 当EBOV和LASV颗粒适当时， 作为保护膜的宿主细胞质膜的一部分。为了接合PS受体，PS 病毒包膜上的标签必须翻转到外页。我们的目标是阐明病毒 包膜获得适当取向的PS，以及足以与PS受体相互作用的PS量。我们 他们使用一组缺乏PS翻转酶或PS乱序酶的人类单倍体（HAP 1）细胞系来检测 这些蛋白质在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BSL 2条件下。当这些重组病毒在HAP 1敲除细胞系中生长时，我们 确定了VSV粒子有效传播所需的一种翻转酶和一种扰频酶， LASV-GP或EBOV-GP，但不是VSV-G。这一数据表明，改变PS的水平， 细胞PM的小叶抑制病毒复制周期中的一个或多个步骤。病毒样颗粒类似 发现EBOV或LASV在缺乏乱序酶的细胞中产生时含有较少的表面PS 活性，也支持我们的总体假设，即细胞酶参与生产和定位 将影响EBOV和LASV的进入和传播。我们提出了三个具体目标， PS在病毒复制中的作用：目的1：检查EBOV中细胞扰乱酶活性的要求 目的2：检查EBOV中细胞翻转酶活性的要求 目的3：确定改变的细胞PS对EBOV和LASV复制的影响， 病毒脂质谱。除了病毒样EBOV颗粒之外还使用基于VSV的假颗粒， 淋巴细胞性脉络丛脑膜炎病毒（rLCMV）含有LASV-GP，我们将确定细胞的作用， 翻转酶和乱序酶在病毒复制过程中，并确认我们的结果使用类似的实验， BSL 4实验室里的真实病毒实验还将确定这些病毒的脂质组， 专注于量化病毒颗粒上修饰PS的水平。了解PS和其他脂质的作用 对LASV和EBOV感染性的研究将潜在地为未来的抗病毒药物提供新的靶点。