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

• 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
• 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

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、马亚罗病毒和东方马脑炎病毒， 发病率和死亡率。甲病毒属是全球分布的节肢动物传播病毒 具有地方性，由于病媒原因，有可能传播到新的地理区域 适应导致新的爆发。2019年爆发38例东方马脑炎病毒人间病例 在美国发生，引起了人们对其重新出现的担忧。2005-2006年，基孔肯雅热爆发， 始于印度洋留尼旺岛的埃博拉病毒已经蔓延到世界各地，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2-3年，引起关节炎样 条件没有有效的抗病毒策略或疫苗可以对抗这些病原体。研究 从分子水平了解这些病毒的生命周期对于发现新的靶点至关重要， 治疗干预具体来说，驱动甲病毒的细胞内机制知之甚少， 组装和出芽代表有希望的抗病毒靶点。我们第一次报道了甲病毒- 编码的离子通道蛋白6 K通过使细胞病变的形成在病毒出芽中起重要作用。 vacuoles-II和包膜刺突蛋白转运到质膜。由于删除6 K的缺陷 通过表达功能性HIV-1 Vpu和甲型流感病毒M2离子， 频道我们还证明了离子通道抑制剂可以用作抗病毒药物。根据这些 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毕斯病毒6 K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中的转运测定，以及 用离子特异性荧光探针对病毒感染的细胞进行活体共聚焦成像。在目标2中，通过表达病毒- 编码的离子通道，包括SARS-CoV-2 E蛋白在细胞和从甲病毒，我们将确定， 基于离子通道的高尔基体重构是包膜RNA病毒用于膜重构的标准机制。 修饰和高效的病毒出芽。通过新的反向遗传学工具和CRISPR-Cas9方法，我们将 研究宿主编码的离子通道在甲病毒出芽中的功能参与。通过完成这些 目的：阐明离子通道蛋白修饰病毒分泌途径的新机制 以及如何将这一过程作为抗病毒靶点。我们还将产生新的反向遗传学 这些工具对科学界很有用。我们将解决的关键知识差距是：1） 甲病毒6 K携带的离子2)6 K如何参与病毒萌芽？（3）理解 一种涉及病毒和宿主离子通道的一般机制，被甲病毒用于有效出芽。