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.

蚊子传播的α病毒，例如基孔肯雅，玛雅罗和东马脑炎病毒引起高 其哺乳动物宿主的发病率和死亡率。 α病毒是全球分布的节肢动物传播病毒 本质上是enzootic的，由于向量而有可能传播到新的地理区域 改编导致新爆发。 2019年爆发了38例东马脑炎病毒病例 发生在美国，引起人们对其重生的担忧。在2005 - 2006年，chikungunya爆发 始于印度洋岛的Réunion群岛已经蔓延到世界各地，影响了数百万人。 Chikungunya Fever的特征是可衰弱的关节疼痛，可能持续2 - 3年，导致类似关节炎 状况。对于任何这些病原体，都没有有效的抗病毒策略或疫苗。研究 这些病毒以获得对生命周期的分子理解，对于发现新的目标至关重要 治疗干预。具体而言，驱动α病毒的细胞内机制知之甚少 组装和萌芽代表有希望的抗病毒靶标。我们首次报道了α-病毒 - 编码的离子通道蛋白6K通过启用细胞病的形成在病毒萌芽中起着至关重要的作用 Vacuumoles-II和包膜尖峰蛋白传输到质膜。由于删除6K而导致的缺陷 可以通过功能性HIV-1 VPU和影响病毒M2离子的表达来恢复到变化的水平 渠道。我们还证明了离子通道抑制剂可以用作抗病毒药。建立在这些基础上 在AIM 1中，我们将表征Chikungunya和Sindbis病毒6K离子通道及其活性 反向遗传学，蛋白质脂质体中的转运测定和细胞内膜蔬菜的分离，以及 具有离子特异性荧光问题的病毒感染细胞的活体成像。在AIM 2中，通过表达病毒 - 编码的离子通道，包括细胞中的SARS-COV-2 E蛋白和α病毒，我们将确定是否确定是否是否 基于离子通道的高尔基重塑是包裹的RNA病毒用于膜的标准机制 修饰和有效的病毒萌芽。使用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α病毒芽中的功能受累。通过完成这些 目的，我们将定义一种新的机制 萌芽以及如何将该过程作为抗病毒靶标探索。我们还将生成新的反向遗传学 对科学界有用的工具。我们要解决的关键知识差距是1） α6K运输的离子？ 2）6K如何参与病毒萌芽？ 3）对 涉及病毒和宿主离子通道的一种通用机制，该通道是通过α-用于有效萌芽的。