Defining the pathway of alphavirus capsid assembly into viral particles

项目摘要/摘要 甲型病毒是一种小的、包膜的正链RNA病毒，包括严重的人类病原体，如 如脑炎病毒和基孔肯雅病毒。尽管它们对人类健康构成风险，但我们缺乏明确的 了解甲型病毒如何组装成病毒颗粒，这是病毒生命周期中定义不清的一个阶段 对于大多数被包膜的病毒。具体地说，核衣壳(NC)组装和并入的机制 甲型病毒颗粒在很大程度上是未知的。NC由衣壳蛋白(CP)的球状晶格组成。 围绕病毒RNA基因组(GRNA)，其组装是生产传染性病毒所必需的。 由于存在几种不同的CP/NC，确定NC组装的机制一直很困难 在感染期间形成的水池。在感染期间产生的总CP中只有一小部分被合并到 子代病毒，目前尚不清楚使用的是哪个池/S，也不清楚这是如何发生的。在这个项目中，我将调查 通过鉴定哪些CP/NC池被整合到病毒中而将CP组装成α病毒NC的途径， 确定CP如何运输到质膜上的病毒萌发部位，并确定CP如何 在NC装配过程中选择性地包装gRNA。为此，我建立了一个生物素化系统来跟踪 CP在感染期间使用链霉亲和素在生化和成像技术中。我把优化后的生物素 在CP(CP-AVI)上的允许部位的受体多肽(AVI标签)，并表明AVI标签不干扰 CP的生物学。我已经建立了在特定的亚细胞位置稳定表达Bira生物素连接酶的细胞系。 结果表明，在显微镜和蛋白质印迹研究中，可以很好地检测到Bira对CP-AVI的生物素化作用。我 已经启动了生物素脉冲追逐实验，目标Bira细胞系被生物素冲击并被追逐 在无生物素的介质中，在感染后的特定时间诱导特定的CP-AVI池的生物素化。我会追踪的 CP-AVI池通过评估其随时间的本地化和传输，并确定哪些CP池起作用 通过对释放的病毒颗粒中生物素化的CP-AVI进行评分来成熟病毒。我会确定CP-AVI传输 通过一种主动的运输机制，可能通过与病毒E2的共同运输，发生到病毒萌发地点 跨膜糖蛋白，通过测试囊泡转运抑制剂如何影响CP-AVI定位和通过 随着时间的推移，CP-E2结合与它们在病毒萌芽部位的共定位相关。最后，我将决定如何 CP通过使用PAR-CLIP(可光激活的核糖核苷增强的交联剂)来特地包装gRNA 和免疫沉淀)时间进程实验，以确定CP在NC过程中与gRNA的接触 集合。我将识别CP特定识别的gRNA位点，并将CP-gRNA接触者与 释放出病毒颗粒。这一项目的结果将为我们提供有关分子机制的重要见解。 甲型病毒NC组装，提高了我们对包膜病毒退出的基本认识。

Project Summary/Abstract Alphaviruses are small, enveloped plus strand RNA viruses and include serious human pathogens such as the encephalitis viruses and Chikungunya virus. Despite the risk they pose to human health, we lack a clear understanding of how alphaviruses assemble into viral particles, a stage in the viral lifecycle that is ill-defined for most enveloped viruses. Specifically, the mechanism of nucleocapsid (NC) assembly and incorporation into alphaviral particles is largely unknown. The NC is made up of a spherical lattice of capsid protein (Cp) surrounding the viral RNA genome (gRNA), and its assembly is required for the production of infectious virus. Determining the mechanism of NC assembly has been difficult due to the presence of several distinct Cp/NC pools that form during an infection. Only a fraction of total Cp produced during an infection is incorporated into progeny virus and it is unclear which pool/s is used and how this occurs. In this project, I will investigate the pathway of Cp assembly into alphavirus NC by identifying which Cp/NC pools are incorporated into the virus, defining how Cp is transported to virus budding sites at the plasma membrane, and determining how Cp selectively packages gRNA during NC assembly. To do this, I have established a biotinylation system to track Cp during an infection using Streptavidin in biochemical and imaging techniques. I inserted the optimized biotin acceptor peptide (AVI tag) at a permissive site on Cp (Cp-AVI) and showed that the AVI tag does not perturb Cp’s biology. I have created cell lines that stably express the BirA biotin ligase at specific subcellular locations and shown that biotinylation of Cp-AVI by BirA is robustly detectable in microscopy and western blot studies. I have initiated biotin pulse-chase experiments where targeted BirA cell lines are pulsed with biotin and chased in biotin-free media to induce biotinylation of specific Cp-AVI pools at defined times post-infection. I will track Cp-AVI pools by assessing their localization and transport over time, and determine which Cp pools contribute to mature virus by scoring for biotinylated Cp-AVI in released viral particles. I will determine if Cp-AVI transport to virus budding sites occurs through an active transport mechanism, possibly by co-trafficking with the viral E2 transmembrane glycoprotein, by testing how vesicular transport inhibitors affect Cp-AVI localization and by correlating Cp-E2 binding with their co-localization to virus budding sites over time. Lastly, I will determine how Cp specifically packages gRNA by using PAR-CLIP (photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation) time course experiments to define the contacts Cp makes with the gRNA during NC assembly. I will identify gRNA sites Cp specifically recognizes and compare Cp-gRNA contacts to those in the released viral particle. The results from this project will provide critical insights on the molecular mechanism of alphavirus NC assembly and improve our fundamental understanding of the exit of enveloped viruses.