Molecular and cellular biology of grapevine leafroll-associated virus 3 and its effects on grape gene expression through transcriptomic analyses

葡萄和葡萄酒产业是加拿大和许多其他国家的经济支柱。葡萄和葡萄酒产业，连同相关的旅游业，每年产生44亿美元的经济影响，提供3.7万个就业机会。优质葡萄和葡萄酒可持续生产的主要障碍是由病毒引起的疾病。葡萄卷叶病(GLR)是全球分布的最具破坏性的病毒性病害，威胁着加拿大葡萄和葡萄酒行业的产量、质量、盈利能力和可持续性。轮状病毒科的五种不同的病毒与GLR有关；葡萄卷叶相关病毒3(GLRaV-3)是主要的病原体。GLRaV-3是蛇类病毒属的模式成员，是所有病毒中最大的RNA基因组之一，具有许多独特的特征，如限制韧皮部，靶向线粒体形成VRC(病毒复制复合体)，具有GLRaV-3特有的几个开放阅读框架，以及包含过长的非编码区。不幸的是，对GLRaV-3和其他GLR相关病毒的复制和感染机制的分子和细胞方面的研究仍然不足。这种知识差距是由多种因素造成的，包括为如此大的RNA基因组制作全长cdna克隆的技术挑战，缺乏有效的系统来利用此类克隆在葡萄中发起感染，葡萄生长和建立病毒滴度所需的时间非常长，等等。我们的实验室一直专注于葡萄主要病毒的研究，希望将葡萄病毒学领域提高到分子和细胞水平。在接下来的5年里，我们将集中精力于4个目标：1)通过RNA-Seq和转录组分析从全球范围内阐明GLRaV-3对葡萄基因表达的影响；2)病毒基因和序列决定簇的有丝分裂靶向和VRC形成；3)GLRaV-3基因组3‘端附近5个ORF的功能和亚细胞定位；4)病毒诱导的基因沉默载体的开发和验证。以前对GRSPaV的研究，加上我们最近建立了具有感染性的GLRaV-3克隆和葡萄传染性系统，使得拟议的研究具有很高的可行性。这项研究的结果将使我们对GLRaV-3复制及其感染过程的分子和细胞方面有更深入的了解。这项开创性的研究还将使我们能够建立GLRaV-3作为Ampe病毒属的模式系统。从长远来看，这些信息将帮助我们制定新的战略，以减轻GLRaV-3和其他与GLR相关的病毒造成的疾病的经济影响。此外，拟议研究的跨学科和协作性质将继续为HQP培训提供优秀的平台。

The grape & wine industry is an economic pillar for Canada and many other countries. The grape & wine industry, together with the related tourism, generate an annual economic impact of 4.4 billion and provides 37,000 jobs. A major obstacle to sustainable production of premium grape & wine is disease caused by viruses. Being the most destructive viral disease with a global distribution, grapevine leafroll (GLR) threatens the yield, quality, profitability and sustainability of the Canadian grape & wine sector. Five distinct viruses of the family Closteroviridae are associated with GLR; grapevine leafroll-associated virus 3 (GLRaV-3) is the predominant causal agent. As the type member of the genus Ampelovirus, GLRaV-3 has one of the largest RNA genomes among all viruses, and exhibits numerous unique features such as restriction to the phloem, targeting mitochondria for VRC (viral replication complexes) formation, possessing several open reading frames unique to GLRaV-3, and containing excessively long non-coding genomic regions. Unfortunately, the molecular and cellular aspects of the replication and mechanisms of infection remain understudied for GLRaV-3 and other GLR-associated viruses. This knowledge gap is due to multiple factors, including technical challenges in making full-length cDNA clones for such large RNA genomes, lack of efficient systems to launch infection in grapes with such clones, the very long time period required for vines to grow and to build up viral titer, among other factors. Our lab has been focusing on major grape viruses in hope to advance the grape virology field to the molecular and cellular level. In the next 5 years, we will concentrate our effort on 4 objectives: 1) global elucidation of the impact of GLRaV-3 infection on grape gene expression through RNA-Seq and transcriptome analysis; 2) viral gene and sequence determinants for mito targeting and VRC formation; 3) function and subcellular localization of the five ORFs unique for GLRaV-3 that are located near the 3' end of its genome; 4) the development and validation of virus-induced gene silencing vectors to confer resistance against GLRaV-3. Prior research on GRSPaV (a member of family Betaflexiviridae involved in the rugose woody disease complex), together with our recent establishment of an infectious GLRaV-3 clone and the grapevine infectivity system, make the proposed research highly feasible. Outcome of this research will enable us to gain deeper understanding of the molecular and cellular aspects concerning GLRaV-3 replication and its infection process. This pioneering research will also allow us to establish GLRaV-3 as a model system for the genus Ampelovirus. In the long run, this information will help us devise new strategies to mitigate the economic impact of disease by GLRaV-3 and other GLR-related viruses. Moreover, the interdisciplinary and collaborative nature of the proposed research will continue to serve excellent platforms for HQP training.