Developing a yeast system to study virus-host interactions in Mononegavirales

描述（由申请人提供）：本申请的主要目的是开发和优化一种独特的酵母系统，用于系统鉴定和表征非分段负链（NNS） RNA病毒（分类学目Mononegavirales）中病毒基因组复制和基因表达的宿主因子。这一目包含各种各样的人类和动物病毒，包括许多致命的人类病原体。NNS RNA病毒的病毒RNA聚合酶由两个病毒亚基组成，并包括许多大部分未识别的宿主蛋白因子。我们目前对宿主蛋白在病毒复制中的作用的了解非常有限，迫切需要开发新的系统方法来鉴定宿主因子，这些因子应该作为有希望的抗病毒靶点。拟议的试点实验将集中于一种典型的单病毒，水疱性口炎病毒（VSV，一种横纹肌病毒）。第一个目的是测试酵母作为支持VSV基因组复制和基因表达的实验宿主的可行性。如果有效，该系统将适用于高效、经济、高通量的系统鉴定VSV和其他单核苷酸病毒中对病毒RNA合成重要的宿主蛋白因子。第二个目标将采用新开发的“细胞质”酵母2杂交系统来鉴定与VSV蛋白物理相互作用的细胞蛋白。病毒RNA复制和转录的宿主因子的鉴定将为理解病毒RNA合成的分子机制和开发通过控制宿主组分来对抗病毒感染的新方法提供新的线索。公共卫生相关性：本申请的主要目的是开发一种新的基于酵母的实验系统来研究非分段负链RNA病毒（单链病毒目）中病毒与宿主的相互作用。这一目包含许多致命的人类病原体，包括被疾病控制和预防中心列为A类生物武器制剂的埃博拉病毒和马尔堡病毒，以及高度流行的人类病原体，如呼吸道合胞病毒和副流感病毒。尽管发达国家通过各种疫苗接种和治疗策略已在很大程度上控制了许多单病毒引起的感染，但它们仍然引起许多临床毁灭性疾病（包括许多新出现的疾病），这些疾病对世界范围内的发病率和死亡率有重大影响。定义这些病毒繁殖的基本机制，特别是病毒与宿主的相互作用，仍然是重要的，最终希望这些知识不仅有助于对病毒生物学的基本理解，而且还将导致新的或更好的治疗药物。

DESCRIPTION (provided by applicant): The major objective of this application is to develop and optimize a unique yeast system for systematic identification and characterization of host factors of viral genome replication and gene expression in nonsegmented negative-strand (NNS) RNA viruses (taxonomic order Mononegavirales). This order contains a wide variety of human and animal viruses, including many lethal human pathogens. The viral RNA polymerase of NNS RNA viruses consists of two viral subunits, and includes a number of largely unidentified host protein factors. Our current understanding of the contributions of host proteins in viral replication is very limited, and there is an urgent need to develop new systematic approaches to the identification of host factors, which should serve as promising antiviral targets. The proposed pilot experiments will be focused on a prototypic Mononegavirales, vesicular stomatitis virus (VSV, a rhabdovirus). The first aim will test the feasibility of the yeast Saccharomyces cerevisiae as an experimental host supporting genome replication and gene expression of VSV. If it works, this system should be suitable for efficient, cost-effective, and high-throughput systematic identification of host protein factors important for viral RNA synthesis in VSV and other Mononegavirales. The second aim will employ a newly-developed "cytoplasmic" yeast 2-hybrid system for the identification of cellular proteins physically interacting with VSV proteins. The identification of host factors of virus RNA replication and transcription should provide new clues for understanding the molecular mechanisms of viral RNA synthesis and for developing new ways to combat viral infections via control of host components. PUBLIC HEALTH RELEVANCE: The major objective of this application is to develop a new yeast-based experimental system to study virus- host interactions in nonsegmented negative-strand RNA viruses (taxonomic order Mononegavirales). This order contains many lethal human pathogens, including the Ebola and Marburg viruses which are classified as category A bioweapon agents by the Centers for Disease Control and Prevention, and highly prevalent human pathogens, such as the respiratory syncytial and parainfluenza viruses. Although infections caused by many Mononegavirales have been largely controlled in the developed world through a variety of vaccination and therapeutic strategies, they still cause many clinically devastating diseases (including many emerging diseases), which contribute significantly to world-wide morbidity and mortality. It continues to be important to define the basic mechanisms of reproduction of these viruses, especially virus-host interactions, with the ultimate hope that this knowledge will contribute not only to the basic understanding of virus biology, but will also lead to new or better therapeutic agents.