Role of an essential RNA chaperone in virus replication

描述（由申请人提供）：RNA复制是正链RNA病毒感染循环的中心步骤，正链RNA病毒在人类、动物和植物中引起许多疾病。尽管它的重要性，病毒RNA复制的机制是不完全理解。易于处理的模型系统，如tombusviruses，可以极大地促进我们对病毒RNA复制的理解。由于依赖于RNA的RNA聚合酶（RdRp）是复制过程中的关键酶，在Tombusviruses和许多其他重要的人类和动物病毒中是相似的（丙型肝炎病毒，黄病毒，包括西尼罗河病毒和登革热病毒，瘟病毒）和植物病毒（黄病毒和卡莫病毒），理解绒毛伞病毒中RNA复制的机制有望有助于研究重要的，但不太顺从的人类RNA病毒，动植物。为了进一步了解病毒复制的机制，研究者已经开发了一种有效的体外RNA复制试验的基础上纯化的番茄丛矮病毒RdRp从酵母。此外，研究者还建立了一种新的基于RNA复制子的酵母体内复制系统。这些进展使Tombusviruses非常适合研究RNA复制的机制。由tombusviruses编码的两种必需复制酶蛋白之一，称为p33，是RNA伴侣蛋白，其影响感染期间的许多过程，包括基因组RNA和Dl RNA的复制、亚基因组RNA合成、RNA重组和Dl RNA形成。本申请的目的是确定p33在毛丛矮病毒复制中的功能，例如：（i）将病毒RNA模板和RdRp靶向复制位点;（ii）模板选择性;（iii）细胞中毛丛矮病毒RNA的稳定化;（iv）增强正链和负链RNA的合成;和（v）RNA结构解旋和链分离。研究人员将使用酵母和整株植物进行生物化学、细胞实验，以确定p33在tombusvirus感染期间的多种功能。总的来说，对tombusvirus复制的研究可能会推进主要的科学领域，包括：（i）RNA复制和干扰的机制;（ii）模板和RdRp募集到复制位点;（iii）通过必需蛋白质因子调节RNA合成;（iv）复制期间RNA-蛋白质和蛋白质-蛋白质相互作用;（v）病毒复制酶的模板选择;（v）RNA复制和干扰的机制。和（vi）开发新的抗病毒剂。

DESCRIPTION (provided by applicant): RNA replication is the central step in the infection cycles of plus-strand RNA viruses, which cause many diseases in humans, animals and plants. In spite of its significance, the mechanism of viral RNA replication is incompletely understood. Tractable model systems, such as tombusviruses, can contribute greatly to our understanding of viral RNA replication. Since the RNA-dependent RNA polymerase (RdRp), the key enzyme in replication, is similar in tombusviruses and numerous other important human and animal viruses (hepatitis C virus, flavivirus, including West Nile virus and dengue virus, pestiviruses) and plant viruses (luteo- and carmoviruses), understanding the mechanism of RNA replication in tombusviruses is expected to help studies on important, but less amenable RNA viruses of humans, animals and plants. In order to advance understanding of the mechanism of virus replication, the investigator has developed an efficient in vitro RNA replication assay based on purified tombusvirus RdRp from yeast. In addition, the investigator has established a novel RNA replicon based in vivo replication system in yeast. These advances make tombusviruses exceptionally suitable for studying the mechanism of RNA replication. One of the two essential replicase proteins coded by tombusviruses, termed p33, is an RNA chaperone protein, which influences many processes during infection, including replication of genomic RNA and Dl RNA, subgenomic RNA synthesis, RNA recombination and Dl RNA formation. The aims of this application is to determine the functions of p33 in tombusvirus replication, such as: (i) targeting the viral RNA template and the RdRp to the site of replication; (ii) template selectivity; (iii) stabilization of tombusvirus RNAs in cells; (iv) enhancement of synthesis of plus- and minus-strand RNAs; and (v) RNA structure unwinding and strand separation. The investigator will perform biochemical, cellular experiments using yeast and whole plants in order to define the multiple functions of p33 during tombusvirus infection. Overall, studies on tombusvirus replication will likely advance major scientific areas, including: (i) mechanism of RNA replication and interference; (ii) template and RdRp recruitment to the site of replication; (iii) regulation of RNA synthesis by essential protein factors; (iv) RNA-protein and protein-protein interactions during replication; (v) template selection by the viral replicase; and (vi) development of novel antiviral agents.