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。葡萄和葡萄酒产业以及相关的旅游业每年产生44亿英镑的经济影响，并提供3.7万个就业岗位。优质葡萄和葡萄酒可持续生产的一个主要障碍是由病毒引起的疾病。作为全球最具破坏性的病毒性疾病，葡萄叶卷病（GLR）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。Closteroviridae科的五种不同病毒与GLR相关；葡萄叶相关病毒3 （glav -3）是主要致病因子。作为蛇病毒属的类型成员，glrav3具有所有病毒中最大的RNA基因组之一，并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），具有几个glrav3特有的开放阅读框，以及包含过长的非编码基因组区域。不幸的是，glav -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仍未得到充分研究。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，缺乏有效的系统来用这种克隆在葡萄中发动感染，葡萄生长和建立病毒滴度所需的时间很长，以及其他因素。我们的实验室一直致力于葡萄主要病毒的研究，希望将葡萄病毒学领域推进到分子和细胞水平。未来5年，我们将集中在4个目标上：1)通过RNA-Seq和转录组分析，在全球范围内阐明glrav3感染对葡萄基因表达的影响；2) mitto靶向和VRC形成的病毒基因和序列决定因素；3) glrav3基因组3′端附近的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，以赋予对glav -3的抗性。先前对GRSPaV （Betaflexiviridae家族成员之一，参与榄树木本病复发病）的研究，以及我们最近建立的传染性glrav3克隆和葡萄感染系统，使我们提出的研究具有很高的可行性。本研究结果将使我们对glrav3复制及其感染过程的分子和细胞方面有更深入的了解。这项开创性的研究也将使我们能够建立glrav3作为蛇病毒属的模型系统。从长远来看，这一信息将帮助我们制定新的战略，以减轻由glav -3和其他glr相关病毒引起的疾病对经济的影响。此外，拟议研究的跨学科和协作性质将继续为HQP培训提供良好的平台。

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.