Intracellular functions and mechanisms of alphavirus ion channel 6K

甲病毒离子通道6K的细胞内功能和机制

基本信息

批准号：
10727819
负责人：
Joyce Jose
金额：
$ 23.63万
依托单位：
PENNSYLVANIA STATE UNIVERSITY, THE
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-01 至 2025-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10727819
关键词：
2019-nCoV Acute Address Affect Alphavirus Alphavirus Infections Americas Amiloride Antiviral Agents Arboviruses Arthralgia Arthritis Biological Assay CRISPR library CRISPR/Cas technology Cell Membrane Permeability Cell membrane Cell physiology Cell surface Cells Chikungunya fever Chikungunya virus Chimera organism Communities Confocal Microscopy Defect Disease Outbreaks Drug Design E protein Eastern Equine Encephalitis Virus Encephalitis Family Fluorescence-Activated Cell Sorting Fluorescent Probes Foundations Geographic Locations Glycoproteins Golgi Apparatus HIV-1 Hepatitis C virus Homeostasis Human Immunofluorescence Immunologic In Vitro Indian Ocean Islands Influenza A virus Integral Membrane Protein Intracellular Membranes Ion Channel Ion Channel Protein Ion Transport Ions Knock-out Knowledge Life Cycle Stages Mediating Membrane Methods Microscopy Mission Modification Molecular Molecular Biology Morbidity - disease rate Morphogenesis Mutation National Institute of Allergy and Infectious Disease Nature Pathway interactions Persons Pharmaceutical Preparations Plaque Assay Play Polyarthritides Process Proteins Public Health Publishing Quantitative Reverse Transcriptase PCR RNA Viruses Reporting Reunion Island Risk Role Secretory Vesicles Series Sindbis Virus Structural Protein Structure Surface Testing Therapeutic Intervention Time Togaviridae United States Vacuole Vesicle Viral Virion Virus Western Blotting Work chikungunya confocal imaging drug repurposing env Gene Products enzootic experimental study global health inhibitor knowledge of results member mortality mosquito-borne mutant new outbreak new therapeutic target novel pathogen premature prevent protein transport proteoliposomes reverse genetics stable cell line tool trafficking vaccine access vector

项目摘要

Mosquito-borne alphaviruses such as chikungunya, Mayaro, and Eastern equine encephalitis viruses cause high morbidity and mortality in their mammalian hosts. Alphaviruses are globally distributed arthropod-borne viruses that are enzootic in nature with the potential to disseminate to new geographical regions due to vector adaptations causing new outbreaks. A 2019 outbreak of 38 human cases of Eastern equine encephalitis virus occurred in the United States, raising concerns about its reemergence. In 2005–2006, a chikungunya outbreak started in the Indian Ocean Island of Réunion had spread around the world, infecting millions of people. Chikungunya fever is characterized by debilitating joint pain that can last up to 2–3 years, causing arthritis-like conditions. No effective antiviral strategies or vaccines are available against any of these pathogens. Studying these viruses to gain a molecular understanding of their lifecycle is essential to discovering novel targets for therapeutic intervention. Specifically, the poorly understood intracellular mechanisms that drive alphavirus assembly and budding represent promising antiviral targets. We reported for the first time that the alphavirus- encoded ion channel protein 6K plays an essential part in virus budding by enabling the formation of cytopathic vacuoles-II and envelope spike protein transport to the plasma membrane. The defects due to the deletion of 6K can be restored to varying levels by the expression of a functional HIV-1 Vpu and influenza A virus M2 ion channel. We also demonstrated that ion channel inhibitors could be utilized as antivirals. Building on these observations, in Aim 1, we will characterize chikungunya and Sindbis virus 6K ion channels and their activity by reverse genetics, transport assays in proteoliposomes and fractionated intracellular membrane vesicles, and live-confocal imaging of virus-infected cells with ion-specific fluorescent probes. In Aim 2, by expressing virus- encoded ion channels, including the SARS-CoV-2 E protein in cells and from an alphavirus, we will determine if ion channel-based Golgi remodeling is a standard mechanism used by enveloped RNA viruses for membrane modification and efficient virus budding. With new reverse genetics tools and CRISPR-Cas9 methods, we will investigate the functional involvement of host-encoded ion channels in alphavirus budding. By completing these aims, we will define a novel mechanism by which ion channel proteins modify the secretory pathway for virus budding and how this process can be exploited as an antiviral target. We will also generate new reverse genetics tools that will be useful to the scientific community. The critical knowledge gaps we will address are 1) what are the ions transported by alphavirus 6K? 2) how does 6K participate in virus budding? and 3) an understanding of a general mechanism involving virus and host ion channels utilized by alphaviruses for efficient budding.

蚊媒甲病毒，如基孔肯雅病毒、马亚罗病毒和东部马脑炎病毒，可导致高发病率甲病毒是全球分布的节肢动物传播的病毒。本质上是地方性的，有可能由于病媒传播到新的地理区域 2019 年爆发了 38 例东部马脑炎病毒人类病例。 2005-2006 年，基孔肯雅热在美国爆发，引发了人们对其再次爆发的担忧。起源于印度洋留尼汪岛的病毒已传播到世界各地，感染了数百万人。基孔肯雅热的特点是使人衰弱的关节疼痛，可持续长达 2-3 年，类似关节炎目前还没有针对这些病原体的有效抗病毒策略或疫苗。从分子角度了解这些病毒的生命周期对于发现新的靶标至关重要具体来说，人们对甲病毒驱动的细胞内机制知之甚少。组装和出芽代表了有希望的抗病毒靶标。编码的离子通道蛋白 6K 通过促进细胞病变的形成，在病毒出芽中发挥重要作用。液泡-II 和包膜刺突蛋白转运至质膜的缺陷是由于 6K 缺失造成的。可以通过功能性 HIV-1 Vpu 和甲型流感病毒 M2 离子的表达恢复到不同水平我们还证明离子通道抑制剂可以用作抗病毒药物。观察，在目标 1 中，我们将通过以下方式表征基孔肯雅病毒和辛德比斯病毒 6K 离子通道及其活性：反向遗传学、蛋白脂质体和分级细胞内膜囊泡的转运测定，以及在目标 2 中，通过表达病毒，使用离子特异性荧光探针对病毒感染的细胞进行实时共聚焦成像。编码的离子通道，包括细胞中的 SARS-CoV-2 E 蛋白和甲病毒，我们将确定是否基于离子通道的高尔基体重塑包膜是 d RNA 病毒用于膜的标准机制借助新的反向遗传学工具和 CRISPR-Cas9 方法，我们将实现修饰和高效的病毒出芽。通过完成这些研究，研究宿主编码的离子通道在甲病毒出芽中的功能参与。目标是，我们将定义一种新的机制，离子通道蛋白可以通过该机制改变病毒的分泌途径萌芽以及如何利用这一过程作为抗病毒靶点我们还将产生新的反向遗传学。我们将解决的关键知识差距是：1）什么是对科学界有用的工具。甲病毒 6K 的离子运输？ 2) 6K 如何参与病毒出芽？涉及甲病毒有效出芽的病毒和宿主离子通道的一般机制。