Host factors involved in viral RNA recombination

描述（由申请人提供）：宿主不仅在正链RNA病毒的复制中起作用，而且在病毒RNA重组中也起作用，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病毒的一个重要机制，因为它有助于产生新的致病或耐药病毒和毒株。因此，RNA重组已被证明发生在许多人类、动物和植物病毒中，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、Corona-、Rota-、Flavi-、Calici-、Arteri-、Astro-、Entero-；和再。为了进一步了解宿主在病毒RNA重组中的作用，PI最近开发了一种基于酵母的模式植物病毒黄瓜坏死瘤病毒（CNV）重组系统。CNV复制酶与重要的人类和动物病毒病原体（如丙型肝炎病毒、黄病毒和鼠疫病毒）的复制酶相似。CNV特别适合于研究RNA重组，因为许多重组可以很容易和快速地恢复：(i)在酵母细胞中，（ii）在整个植物中，以及（iii）在体外CNV RNA依赖的RNA聚合酶（RdRp）测定中。本研究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）酵母（一种模型宿主）可用的先进遗传学工具。受到酵母中Tombusvirus复制子有效复制和重组的初步数据的鼓舞，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，该筛选基于酵母删除菌株文库（约5,500株），其中每个酵母基因分别被删除。该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。已鉴定的影响病毒RNA重组的宿主基因将被进一步测试（目标3），以表征它们对病毒重组的影响。本文所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。重组的知识通过提供限制病毒感染和开发更安全的疫苗的手段，承诺了无数的健康益处。

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.