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节段负链（NNS）RNA病毒（分类学目的为单负链病毒目）中病毒基因组复制和基因表达的宿主因子。该目包含多种人类和动物病毒，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，并且包括许多基本上未鉴定的宿主蛋白因子。我们目前对宿主蛋白在病毒复制中的贡献的理解非常有限，迫切需要开发新的系统方法来鉴定宿主因子，这些因子应作为有前途的抗病毒靶点。拟议的试点实验将集中在一个原型单负性病毒目，水泡性口炎病毒（VSV，弹状病毒）。第一个目标是测试酿酒酵母作为支持VSV基因组复制和基因表达的实验宿主的可行性。如果它的工作，该系统应该是适合于有效的，成本效益高，高通量的系统鉴定宿主蛋白质的重要因素的病毒RNA合成VSV和其他单负性病毒。第二个目标将采用新开发的“细胞质”酵母双杂交系统，用于鉴定与VSV蛋白质物理相互作用的细胞蛋白质。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成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，以研究非节段负链RNA病毒（分类目的单负链RNA病毒目）中的病毒-宿主相互作用。该目包含许多致命的人类病原体，包括埃博拉病毒和马尔堡病毒，它们被疾病控制和预防中心归类为A类生物武器制剂，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。尽管在发达国家，通过各种疫苗接种和治疗策略，由许多单负性病毒目引起的感染已在很大程度上得到控制，但它们仍然引起许多临床破坏性疾病（包括许多新兴疾病），这显著地导致了世界范围的发病率和死亡率。确定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很重要，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.