Dissecting the mechanism of herpesvirus genome packaging

项目摘要 人类疱疹病毒可导致新生儿先天性出生缺陷和人类多种疾病， 对免疫功能低下的个体具有特别破坏性的影响。卡波济肉瘤相关 疱疹病毒（KSHV）是卡波西肉瘤和其他癌症的病原体，在亚健康人群中发病率很高。 撒哈拉非洲。没有治愈任何疱疹病毒;大多数现有的抗病毒药物针对单一的病毒过程， 会导致抗药性突变最近开发的疱疹病毒包装抑制剂突出了 病毒基因组包装机制是新型抗病毒药物的有吸引力的靶点。疱疹病毒包装 机器在概念上类似于噬菌体，但它具有额外的复杂性，包括 未知功能的因素。在这个建议中，我概述了我的愿景，以了解分子机制， 疱疹病毒包装。我将确定疱疹病毒包装中一个重要因素的功能， 使用最先进的蛋白质组学和显微镜方法长期回避机械解剖。我也会 开发一种新的策略，将深度突变扫描方法应用于疱疹病毒蛋白。与此同时，我将 确定KSHV末端酶的结构，KSHV末端酶是将病毒基因组包装成 新生的衣壳然后，我将重组KSHV包装机械的基本组成部分， 使用这些来开发体外包装测定。该检测将用于筛选新的抗病毒药物， 确定针对包装机械的现有药物的分子基础。拟议 研究将为解决DNA病毒包装中长期存在的基本问题奠定基础， 了解抗疱疹病毒药物的作用机制。

PROJECT SUMMARY Human herpesviruses can cause congenital birth defects in neonates and a wide range of disease in humans, with particularly devastating effects in immunocompromised individuals. Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma and other cancers, with high prevalence in sub- Saharan Africa. There is no cure for any herpesvirus; most existing antivirals target a single viral process and can lead to resistance mutations. The recent development of a herpesviral packaging inhibitor highlights the viral genome packaging machinery as an attractive target for novel antivirals. The herpesviral packaging machinery is conceptually similar to that of bacteriophages, yet it has additional complexities that include factors of unknown function. In this proposal, I outline my vision to understand the molecular mechanism of herpesvirus packaging. I will determine the function of an essential factor in herpesvirus packaging that has long evaded mechanistic dissection using state-of-the-art proteomics and microscopy approaches. I will also develop a new strategy to apply deep mutational scanning approaches to herpesviral proteins. In parallel, I will determine the structure of the KSHV terminase, the viral molecular motor that packages the viral genome into the nascent capsid. I will then reconstitute the essential components of the KSHV packaging machinery and use these to develop an in vitro packaging assay. This assay will be leveraged to screen for new antivirals and determine the molecular underpinnings of existing drugs that target the packaging machinery. The proposed studies will build a foundation to address long-standing, fundamental questions in DNA virus packaging and understand the mechanism of anti-herpesvirals.