Functional role of a host metabolic enzyme in viral replication

描述（由申请人提供）：正链（+）RNA病毒感染各种物种，导致人类、动物和植物的许多严重疾病，导致粮食生产的重大损失，对人类健康和福祉构成重大风险。病毒与宿主相互作用中最不为人所知的方面是宿主细胞的作用。由于RNA病毒的基因组较小，在复制过程中很大程度上依赖于宿主蛋白。尽管宿主蛋白在病毒发病机制中具有重要意义，但我们目前对宿主因子参与病毒复制的认识却令人惊讶地不完整。这一领域的进展相对缓慢主要是由于缺乏可处理的病毒-宿主系统。因此，高度可处理的模型系统的发展，如番茄灌木矮突病毒（TBSV）和酵母模型宿主，促进了我们对宿主因子在病毒复制中的作用的理解取得了快速而显著的进展。该项目将利用目前最先进的病毒系统之一TBSV，在理解RNA复制机制和宿主蛋白的作用方面取得快速进展。事实上，PI的研究使用了系统生物学方法，包括全基因组筛选和蛋白质组学方法，已经产生了一个非常广泛的数据集，其中包含近200种影响瘤病毒复制、进化和发病机制的宿主蛋白质。此外，使用新的无细胞系统与酵母提取物能够支持真正的tombusvirus复制结合细胞生物学方法允许PI剖析鉴定宿主蛋白在tombusvirus复制中的实际功能。PI已经表明，在酵母中发现的发现也适用于原生寄主植物，从而验证了该方法。这些进展使tombusvirus成为RNA病毒的前沿选择系统，以进一步了解RNA病毒如何“重新编程”感染细胞。PI的工作可能会导致对RNA病毒复制和病毒发病机制的重大新见解。TBSV复制酶蛋白与丙型肝炎病毒（HCV）、登革热病毒、西尼罗河病毒以及其他黄病毒和鼠疫病毒等重要病原体的蛋白相似的另一个优势。总的来说，TBSV的主要进展使PI能够对宿主因子在TBSV中可行的作用进行实验，并将刺激研究RNA复制和重要人类病原体宿主-病原体相互作用的新方法的发展。这一建议的重点是病毒复制酶复合体中存在的一个关键宿主因子，即GAPDH（甘油醛-3-磷酸脱氢酶）代谢蛋白，它与病毒RNA结合，是酵母和原生寄主植物中tombusvirus复制所必需的。这项工作将揭示GAPDH在病毒复制酶中的多种功能。PI的这些研究必将导致宿主在RNA病毒复制、进化和病毒-宿主相互作用中的重要作用进一步取得重大进展。因此，GAPDH已被证明可以结合多种RNA病毒中富含au的序列，包括甲型肝炎病毒（HAV）、丙型肝炎病毒和人3型副流感病毒。以下是该建议的主要优势：(i)病毒RNA复制显然对病毒感染所有类型的生物体具有极其重要的意义。（ii） PI开发的酵母和真实无细胞试验相结合是目前研究宿主因子参与病毒RNA复制机制最有效的方法。（iii）这项研究有望在病毒RNA复制方面取得突破性的新发现。PI开发的高度可处理的体外和体内TBSV系统可以证明对其他较难处理的RNA病毒的研究非常有益。这项研究有望在病毒复制、宿主-病毒相互作用和病毒对宿主的适应等领域取得突破性成果，从而造福社会。公共卫生相关性：RNA病毒对人类健康构成重大风险，并对农业造成重大损失，它在受感染细胞中复制时严重依赖宿主因子。由于缺乏可处理的病毒-宿主系统，目前对破坏宿主因子在病毒复制中的作用知之甚少。PI将使用强大的番茄丛矮病毒(TBSV)-酵母模型系统来分析一个关键的增用宿主因子的作用，即GAPDH（甘油醛-3-磷酸脱氢酶）代谢蛋白，它与病毒RNA结合，对酵母和原生寄主植物中的tombusavirus复制至关重要。这项工作将有助于深入了解宿主在RNA病毒感染中的作用，并可能开辟新的抗病毒策略。

DESCRIPTION (provided by applicant): Plus-stranded (+)RNA viruses infect all kind of species and cause many serious diseases in humans, animals and plants, leading to major losses in food production and representing significant risk to human health and welfare. The least understood aspect of virus - host interaction is the contribution of the host cells. Due to their small genome sizes, RNA viruses greatly depend on the co-opted host proteins during replication. In spite of the significance of host proteins in viral pathogenesis, our current knowledge on host factors co- opted for virus replication is surprisingly incomplete. The relatively slow progress in this area is mainly due to the lack of tractable virus - host systems. Therefore, development of highly tractable model systems, such as Tomato bushy stunt tombusvirus (TBSV) and the yeast model host has promoted rapid and significant progress in our understanding of the roles of host factors in viral replication. The PI will use TBSV, which is currently one of the most advanced among viral systems to progress rapidly in understanding the mechanism of RNA replication and the role of the host proteins. Indeed, the PI's research, using a systems biology approach, including genome-wide screens and proteomics approaches, has led to a remarkably extensive data set containing close to 200 host proteins affecting tombusvirus replication, evolution and pathogenesis. Moreover, using novel cell-free system with a yeast extract capable of supporting authentic tombusvirus replication in combination with cell biology approach allows the PI to dissect the actual functions of the identified host proteins in tombusvirus replication. The PI has shown that the discoveries made in yeast are also relevant in the native host plant, thus validating the approach. These advances have put tombusviruses into the forefront of RNA viruses as a system of choice to further our understanding how RNA viruses "re-program" the infected cells. The PI's work will likely lead to major new insights into RNA virus replication and viral pathogenesis. 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 PI to conduct experiments on the roles of host factors that are feasible with TBSV, and will stimulate development of new approaches for studying RNA replication and host - pathogen interactions for important human pathogens. This proposal focuses on a key host factor present in the viral replicase complex, namely GAPDH (glyceraldehyde-3-phosphate dehydrogenase) metabolic protein that binds to the viral RNA and essential for tombusvirus replication in yeast and in the native host plant. The proposed work will unravel the multiple functions of GAPDH in the viral replicase. These studies by the PI will certainly lead to further significant advances on the essential role of the host in RNA virus replication, evolution and virus-host interaction. Accordingly, GAPDH has been shown to bind to AU-rich sequences present in various RNA viruses, including hepatitis A virus (HAV), hepatitis C virus, and human parainfluenza virus type 3. The following are the major strengths of the proposal: (i) Viral RNA replication is clearly of immense importance for viruses to infect all types of living organisms. (ii) The combination of yeast and authentic cell-free assay developed by the PI is currently the most potent for studying the mechanism of host factors involvement in viral RNA replication. (iii) This research is expected to lead to groundbreaking new discoveries in viral RNA replication. The highly tractable in vitro and in vivo TBSV system developed by the PI 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 replication, host-virus interactions and the adaptation of viruses to their hosts. PUBLIC HEALTH RELEVANCE: RNA viruses, which pose significant risks to human health and cause major losses for agriculture, depend heavily on host factors to replicate in infected cells. The roles of the subverted host factors in virus replication are currently poorly understood due to the lack of tractable virus-host systems. The PI will use the powerful Tomato bushy stunt virus (TBSV)-yeast model system to dissect the role of a key co-opted host factor, namely GAPDH (glyceraldehyde-3-phosphate dehydrogenase) metabolic protein that binds to the viral RNA and essential for tombusvirus replication in yeast and in the native host plant. The proposed work will lead to deeper understanding of the role of the host in RNA virus infections and likely open new antiviral strategies.