Host factors involved in viral RNA recombination

描述(申请人提供)：宿主不仅在正链RNA病毒的复制中发挥作用，而且在病毒RNA重组中也发挥作用，尽管宿主因素在重组中的作用(S)目前尚不清楚。RNA重组是正链RNA病毒的重要机制，因为它有助于新的致病或耐药病毒和毒株的出现。因此，大量的人、动物和植物病毒，包括脊髓灰质炎病毒、登革热病毒、传染性支气管炎病毒、甲型流感病毒、汉坦病毒、丁型肝炎病毒、人类呼吸道合胞病毒、冠状病毒、Rota-、Flavi-、Calici-、Arteri-、Astro-、Entero-和甲型病毒，都发生了RNA重组。为了加深对寄主在病毒RNA重组中作用的了解，PI最近开发了一种基于酵母的重组系统，用于模式植物病毒黄瓜坏死病病毒(CNV)。CNV复制酶类似于重要的人和动物病毒病原体的复制酶，如丙型肝炎病毒、黄病毒和害虫病毒。CNV特别适合于研究RNA重组，因为大量的重组体可以容易和快速地回收：(I)在酵母细胞中，(Ii)在整个植物中，(Iii)在体外CNV RNA依赖的RNA聚合酶(RdRp)试验中。在这项工作中检验的中心假设是宿主因素可能影响病毒RNA重组。PI将使用(目标1和目标2)可用于模式宿主酵母的先进遗传学工具。在有关Tombus病毒复制子在酵母中有效复制和重组的初步数据的鼓舞下，PI将使用高通量筛选来测试每个宿主基因对病毒RNA重组的影响，基于酵母缺失菌株文库(~5,500株)，其中每个酵母基因被单独删除。对文库的部分筛选已经确定了影响RNA重组的三个宿主基因，从而证实了该方法的可行性。将对已识别的影响病毒RNA重组的宿主基因进行进一步测试(目标3)，以确定它们对病毒重组体的影响。本文描述的研究将有助于更好地理解宿主基因在病毒RNA重组中的作用以及宿主在病毒进化中的作用。通过提供限制病毒感染和开发更安全的疫苗的手段，重组的知识有望带来数不清的健康益处。

DESCRIPTION (provided by applicant): The host plays a role not only in replication of plus-stranded RNA viruses, but also in viral RNA recombination, although the role(s) of host factors in recombination is currently unknown. RNA recombination is an important mechanism for plus-strand RNA viruses, because it contributes to the emergence of new pathogenic or drug-resistant viruses and strains. Accordingly, RNA recombination has been demonstrated to occur for a large number of human, animal and plant viruses, including poliovirus, dengue virus, infectious bronchitis virus, influenza A virus, hantavirus, hepatitis D virus, Human Respiratory Syncytial Virus, Corona-, Rota-, Flavi-, Calici-, Arteri-, Astro-, Entero-; and Alphaviruses. In order to advance understanding of the role of the host in viral RNA recombination, the PI has recently developed a yeast-based recombination system for a model plant virus, Cucumber necrosis tombusvirus (CNV). The CNV replicase is similar to the replicases of important human and animal viral pathogens, such as hepatitis C virus, Flaviviruses and Pestiviruses. CNV is exceptionally suitable for studying RNA recombination because numerous recombinants can easily and rapidly be recovered: (i) in yeast cells, (ii) in whole plants, and (iii) in the in vitro CNV RNA-dependent RNA polymerase (RdRp) assay. The central hypothesis tested in this work is that host factors may affect viral RNA recombination. The PI will use (Aim 1 and 2) the advanced genetics tools available for yeast, a model host. Encouraged by preliminary data on the efficient replication and recombination of a Tombusvirus replicon in yeast, the PI will use a high-throughput screen to test the effect of each host gene on viral RNA recombination based on the yeast deletion strain library (~ 5,500 strains), in which each yeast gene was separately deleted. A partial screen of the library has already identified three host genes affecting RNA recombination, thus confirming the feasibility of the approach. The identified host genes affecting viral RNA recombination will be further tested (Aim 3) to characterize their effect on viral recombinants. The research described here will lead to better understanding of the role of host genes in viral RNA recombination and the role of the host in virus evolution. Knowledge of recombination promises untold health benefits by providing means to limit virus infections and to develop safer vaccines.