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

基本信息

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
项目类别：
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

项目摘要

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.

项目概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) 感染会增强结合细胞表面的受体。 PS 是一种细胞脂质，通常限制在内部小叶或细胞质中细胞质膜（PM）的表面。 PS 和其他磷脂对内叶的限制在健康细胞中产生高度不对称的膜。 PS翻转至细胞外层钙信号传导和细胞凋亡过程中的膜，分别将它们标记为激活或死亡细胞。称为翻转酶和扰乱酶的细胞酶负责翻转膜中的 PS。 PS是在病毒出芽期间，当 EBOV 和 LASV 颗粒适当地结合到病毒膜中时，宿主细胞质膜的一部分作为保护膜。为了与 PS 受体结合，PS 病毒包膜上的病毒必须翻转到外层传单。我们的目标是阐明病毒的机制包膜获得正确定向的 PS，以及足以与 PS 受体相互作用的 PS 量。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) 细胞系来检查这些蛋白质在 EBOV 和 LASV 的复制中发挥着重要作用。我们生产水疱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 敲除细胞系中生长时，我们鉴定出一种翻转酶和一种扰乱酶，它们是有效传播包含以下物质的 VSV 颗粒所必需的： LASV-GP 或 EBOV-GP，但不是 VSV-G。该数据表明改变外层中的 PS 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。类似病毒的颗粒当在缺乏扰乱酶的细胞中产生时，EBOV 或 LASV 被发现含有较少的表面 PS 活性，也支持我们的总体假设，即细胞酶参与生产和定位 PS 的影响将影响 EBOV 和 LASV 的进入和传播。我们提出了三个具体目标来进一步研究 PS 在病毒复制中的作用：目标 1：检查 EBOV 中细胞扰乱酶活性的要求 LASV 进入和病毒体产生；目标 2：检查 EBOV 细胞翻转酶活性的要求和 LASV 进入；目标 3：确定改变的细胞 PS 对 EBOV 和 LASV 复制的影响以及病毒脂质谱。除了病毒样 EBOV 颗粒和重组体之外，还使用基于 VSV 的伪颗粒含有 LASV-GP 的淋巴细胞性脉络膜脑膜炎病毒 (rLCMV)，我们将确定细胞的作用病毒复制过程中的翻转酶和扰乱酶，并使用类似的实验证实了我们的结果 BSL4 实验室中的真实病毒。实验还将定义这些病毒的脂质组，方法将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 LASV 和 EBOV 感染性的研究可能为未来的抗病毒药物提供新的靶点。