The role of the host Ca/Mn pump in emergence of novel viral RNA recombinants

描述（由申请人提供）：新病毒的出现是对我们社会的持续威胁。正链RNA病毒的进化通常是由RNA重组驱动的，RNA重组是一个将不连续的RNA片段连接在一起的过程，产生基因或调节RNA序列的新组合。RNA病毒在所有类型的生物体中的复制对于克服免疫力、跨越物种和改变病毒致病性是重要的。病毒RNA重组的研究对于理解重组过程和特定宿主基因的作用至关重要，其在重组过程中的作用目前尚不清楚。研究人员将使用番茄丛矮病毒（TBSV），这是目前最先进的病毒系统，以快速了解RNA重组的机制和宿主基因的作用。研究人员的研究开辟了一个全新的领域，强大的TBSV-酵母模型系统，这导致了全基因组筛选首次识别抑制RNA重组的宿主基因。TBSV复制酶蛋白质与重要病原体的蛋白质相似的额外优点，所述重要病原体例如丙型肝炎病毒（HCV）、登革热病毒、西尼罗河病毒和其它黄病毒和瘟病毒。总的来说，TBSV的主要进展使研究人员能够对宿主因素的作用进行实验，这些实验目前只适用于TBSV，但将为研究重要的人类病原体的RNA重组/进化开辟新的方法。这一建议的重点是抑制RNA重组的关键宿主因子之一。实验将剖析Pmr 1 p Ca ~（2+）/Mn ~（2+）泵在病毒RNA重组中的作用。pmr 1 p是真核生物中普遍存在的保守蛋白。Pmr 1 p的人类同源物已被证明在遗传疾病如Hailey-Hailey病和Darier病中起关键作用。在不存在Pmr 1 p的情况下，TBSV RNA重组率极高，表明宿主在病毒RNA重组中的关键作用。为了表征Pmr 1 p在RNA重组中的作用，研究人员将使用先进的酵母遗传学工具与生物化学方法相结合。在无细胞系统和酵母模型宿主中获得的结果也将在天然植物宿主中得到证实。此外，在体外实验将进行与丙型肝炎病毒，一个重要的人类病原体的RdRp，以扩大我们的知识的可能作用的Ca 2 +/Mn 2+泵在丙型肝炎病毒RNA重组。以下是该提案的主要优点：（i）病毒RNA重组对于病毒克服免疫力、跨越物种和改变所有类型生物体的致病性显然是极其重要的。(ii)研究者开发的酵母和体外系统的组合是目前世界上唯一适合研究宿主因子参与病毒RNA重组机制的系统。(iii)这项研究有望在病毒RNA重组方面取得突破性的新发现。研究者开发的高度易处理的体外和体内TBSV系统可以证明对其他不太适合的RNA病毒的研究非常有益。这项研究有望在病毒重组/进化、宿主-病毒相互作用和新病毒的出现等领域取得突破性成果，从而造福社会。公共卫生相关性：新病毒的出现是对我们社会的持续威胁。由于RNA重组，新的正链RNA病毒频繁出现。对于RNA病毒来说，变性对于克服免疫力、跳跃到新的宿主物种或改变所有类型生物体中症状的严重程度是重要的。由于缺乏易处理的病毒-宿主系统，目前对宿主在病毒重组中的作用知之甚少。研究人员将使用番茄丛矮病毒（TBSV），这是目前最先进的病毒系统来研究RNA重组和宿主基因的作用。该建议通过剖析Ca 2 +/Mn 2+泵在病毒RNA重组中的作用，关注抑制RNA重组的关键宿主因素之一。这项研究有望在病毒重组/进化、宿主-病毒相互作用和新病毒的出现等领域取得突破性成果，从而造福社会。

DESCRIPTION (provided by applicant): Emergence of new viruses is a continuing threat to our society. Evolution of plus- stranded RNA viruses is frequently driven by RNA recombination, a process that joins noncontiguous RNA segments together, creating novel combinations of genes or regulatory RNA sequences. Recombination is important for RNA viruses to overcome immunity, jump species and change viral pathogenicity in all types of living organisms. Studies on viral RNA recombination are critical to understand the recombination process and the role of specific host genes, whose roles in the recombination process are currently unknown. The investigator will use Tomato bushy stunt virus (TBSV), which is currently the most advanced among viral systems to progress rapidly in understanding the mechanism of RNA recombination and the role of the host genes. The investigator's research opens up a completely new area with the powerful TBSV-yeast model system, which has led to genome-wide screens identifying host genes suppressing RNA recombination for the first time. Additional advantage of the similarity of TBSV replicase proteins to proteins of important pathogens, such as Hepatitis C virus (HCV), dengue virus, West Nile virus and other Flaviviruses and Pestiviruses. Collectively, the major advances with TBSV allow the investigator to conduct experiments on the roles of host factors that are currently only feasible with TBSV, but will open new approaches for studying RNA recombination/evolution for important human pathogens. This proposal focuses on one of the key host factors suppressing RNA recombination. The experiments will dissect the role of Pmr1p Ca2+/Mn2+ pump in viral RNA recombination. Pmr1p is a ubiquitous and conserved protein in eukaryotes. The human homologs of Pmr1p have been shown to play critical roles in genetic diseases, such as Hailey- Hailey Disease and Darier disease. In the absence of Pmr1p, the rate of TBSV RNA recombination is extremely high demonstrating the critical role of the host in viral RNA recombination. In order to characterize the role of Pmr1p in RNA recombination, the investigator will use the advanced genetics tools available for yeast in combination with biochemical approaches. The results obtained in cell-free systems and in yeast model host, will also be confirmed in a native plant host. In addition, in vitro experiments will be conducted with the RdRp of HCV, an important human pathogen, to expand our knowledge on the possible role of Ca2+/Mn2+ pumps in HCV RNA recombination. The following are major strengths of the proposal: (i) Viral RNA recombination is clearly of immense importance for viruses to overcome immunity, jump species and change pathogenicity in all types of living organisms. (ii) The combination of yeast and in vitro systems developed by the investigator is currently the only one in the world suitable for studying the mechanism of host factors involvement in viral RNA recombination. (iii) This research is expected to lead to groundbreaking new discoveries in viral RNA recombination. The highly tractable in vitro and in vivo TBSV system developed by the investigator could prove highly beneficial to studies of other, less amenable RNA viruses. The research holds promise of benefiting society by leading to groundbreaking results in the area of virus recombination/evolution, host-virus interaction and the emergence of new viruses. PUBLIC HEALTH RELEVANCE: Emergence of new viruses is a continuing threat to our society. New plus-stranded RNA viruses emerge frequently due to RNA recombination. Recombination is important for RNA viruses to overcome immunity, jump to new host species or change the severity of symptoms in all types of living organisms. The roles of the host in virus recombination are currently poorly understood due to the lack of tractable virus-host systems. The investigator will use Tomato bushy stunt virus (TBSV), which is currently the most advanced among viral systems to study RNA recombination and the role of the host genes. This proposal focuses on one of the key host factors suppressing RNA recombination by dissecting the role of a Ca2+/Mn2+ pump in viral RNA recombination. The research holds promise of benefiting society by leading to groundbreaking results in the area of virus recombination/evolution, host-virus interaction and the emergence of new viruses.