Intracellular functions and mechanisms of alphavirus ion channel 6K

蚊媒甲型病毒，如基孔肯雅、马雅罗和东部马脑炎病毒导致高 其哺乳动物宿主的发病率和死亡率。甲型病毒是全球分布的节肢动物传播的病毒 由于媒介有可能传播到新的地理区域的地方性传染病 导致新的疫情爆发的适应。2019年爆发38例人感染东部马脑炎病毒 发生在美国，引发了人们对其重新出现的担忧。2005-2006年间，基孔肯雅热暴发 始于印度洋留尼汪岛的埃博拉病毒蔓延到世界各地，感染了数百万人。 基孔肯雅热的特征是虚弱的关节疼痛，可持续2-3年，引起关节炎样症状。 条件。目前还没有针对这些病原体的有效抗病毒策略或疫苗。学习 这些病毒对其生命周期的分子理解对于发现新的靶点是必不可少的 治疗性干预。具体地说，对驱动甲型病毒的细胞内机制知之甚少 组装和萌芽是很有希望的抗病毒靶点。我们第一次报道了甲型病毒- 编码的离子通道蛋白6K通过使细胞病变的形成而在病毒萌发过程中发挥重要作用 液泡-II和包膜尖峰蛋白向质膜运输。6K的缺失带来的缺陷 可通过表达功能性HIV-1VPU和甲型流感病毒M2离子恢复到不同的水平 频道。我们还证明了离子通道抑制剂可以用作抗病毒药物。建立在这些基础上 观察，在目标1中，我们将通过以下方式表征基孔肯雅和辛德比斯病毒6K离子通道及其活性 反向遗传学、蛋白脂质体和分离的细胞膜囊泡中的转运分析，以及 用离子特异性荧光探针对感染病毒的细胞进行实时共聚焦成像。在目标2中，通过表达病毒- 编码的离子通道，包括细胞和甲型病毒中的SARS-CoV-2E蛋白，我们将确定 基于离子通道的高尔基体重塑是囊膜RNA病毒用于膜的标准机制 修改和高效的病毒萌发。有了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型病毒萌发中的功能参与。通过完成这些 目的，我们将定义一种新的机制，通过它离子通道蛋白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目标。我们还将产生新的反向遗传学 将对科学界有用的工具。我们要解决的关键知识差距是1)什么是 甲型病毒6K转运的离子？2)6K如何参与病毒萌发？3)了解 甲型病毒利用病毒和宿主离子通道进行有效萌发的一般机制。

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.