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

描述(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重组推动的，RNA重组是将不连续的RNA片段连接在一起，创建新的基因组合或调节的RNA序列的过程。重组对于RNA病毒克服免疫、跨越物种和改变病毒在所有类型生物体中的致病性具有重要意义。对病毒RNA重组的研究对于了解重组过程和特定宿主基因在重组过程中的作用至关重要，目前这些宿主基因在重组过程中的作用尚不清楚。研究人员将利用目前病毒系统中最先进的番茄丛状矮化病毒(TBSV)来快速了解RNA重组的机制和宿主基因的作用。这位研究人员的研究开辟了一个全新的领域，使用了强大的TBSV-酵母模型系统，这导致了首次在全基因组范围内筛选出抑制RNA重组的宿主基因。TBSV复制酶蛋白与丙型肝炎病毒、登革热病毒、西尼罗河病毒以及其他黄病毒和害虫病毒等重要病原体的蛋白相似的另一个优势。总体而言，TBSV的重大进展使研究人员能够对宿主因素的作用进行实验，这些实验目前只在TBSV上可行，但将为研究重要人类病原体的RNA重组/进化开辟新的途径。这项建议的重点是抑制RNA重组的关键宿主因素之一。本实验将剖析Pmr1p钙/锰泵在病毒RNA重组中的作用。Pmr1p是真核生物中普遍存在的一种保守的蛋白质。Pmr1p的人类同源物已被证明在遗传性疾病中发挥关键作用，如Hailey-Hailey病和Darier病。在没有Pmr1p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。为了确定Pmr1p在RNA重组中的作用，研究人员将结合生物化学方法，使用可用于酵母的先进遗传学工具。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，我们还将利用人类重要病原体丙型肝炎病毒的RdRp进行体外实验，以扩大我们对钙/锰泵在丙型肝炎病毒RNA重组中可能扮演的角色的认识。该建议的主要优点如下：(I)病毒RNA重组显然对病毒克服免疫力、跳跃物种和改变所有类型生物的致病性具有极其重要的作用。(Ii)研究人员开发的酵母菌和体外系统相结合，是目前世界上唯一适合研究宿主因素参与病毒RNA重组的机制的系统。(Iii)这项研究有望在病毒RNA重组方面带来开创性的新发现。研究人员开发的高度易处理的体外和体内TBSV系统可能对其他不太容易驯化的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-病毒相互作用和出现新病毒方面取得开创性成果，从而造福社会。与公共卫生相关：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。重组对于RNA病毒克服免疫、跳转到新的寄主物种或改变所有类型生物中症状的严重程度都是重要的。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。研究人员将使用目前病毒系统中最先进的番茄丛生矮化病毒(TBSV)来研究RNA重组和宿主基因的作用。这项建议通过剖析钙/锰泵在病毒RNA重组中的作用，重点讨论了抑制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-病毒相互作用和出现新病毒方面取得开创性成果，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.