Host factors involved in viral RNA recombination

描述（由申请方提供）：宿主不仅在正链RNA病毒的复制中发挥作用，而且在病毒RNA重组中发挥作用，尽管宿主因子在重组中的作用目前尚不清楚。RNA重组是正链RNA病毒的重要机制，因为它有助于新的致病性或耐药病毒和毒株的出现。因此，RNA重组已被证明在大量的人、动物和植物病毒中发生，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、人呼吸道合胞病毒、冠状病毒、轮状病毒、黄病毒、杯状病毒、动脉病毒、星状病毒、肠病毒和甲病毒。为了促进对宿主在病毒RNA重组中的作用的理解，PI最近开发了用于模式植物病毒黄瓜坏死番茄丛病毒（CNV）的基于酵母的重组系统。CNV复制酶类似于重要的人类和动物病毒病原体的复制酶，例如丙型肝炎病毒、黄病毒和瘟病毒。CNV特别适合于研究RNA重组，因为许多重组体可以容易且快速地回收：（i）在酵母细胞中，（ii）在整个植物中，和（iii）在体外CNV RNA依赖性RNA聚合酶（RdRp）测定中。在这项工作中测试的中心假设是，宿主因素可能会影响病毒RNA重组。PI将使用（目标1和2）酵母（一种模型宿主）可用的先进遗传学工具。在酵母中番茄丛矮病毒复制子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，基于酵母缺失菌株文库（约5，500株）检测每种宿主基因对病毒RNA重组的影响，其中每种酵母基因分别缺失。对文库的部分筛选已经鉴定出三个影响RNA重组的宿主基因，从而证实了该方法的可行性。将进一步检测鉴定的影响病毒RNA重组的宿主基因（目的3），以表征其对病毒重组体的影响。这里描述的研究将导致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。重组的知识通过提供限制病毒感染和开发更安全疫苗的方法，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.