Grapevine rupestris stem pitting-associated virus as a model system for the study of Betaflexiviridae, a new family of unique viruses that infect woody fruit crops

我们的研究计划的长期目标是建立一个基于葡萄藤岩蔷薇茎痘相关病毒（GRSPaV）的模型实验系统，以推进对这组重要而独特的病毒的研究。β-柔曲病毒科是一个新建立的病毒科，包括七个属的正链单链RNA病毒。它含有许多重要的病毒病原体，对水果生产造成相当大的经济损失。该病毒家族在其生物学特性和遗传特征方面表现出几个独特的特征。例如，该科的大多数成员感染多年生木本植物作物。此外，β-柔曲病毒科病毒编码具有高度模块化结构的复制酶多蛋白。这些复制酶多聚蛋白不仅含有在正链单链RNA病毒的甲病毒样超组中保守的充分表征的功能结构域，其包括甲基转移酶、解旋酶/ATP酶和RNA依赖性RNA聚合酶，它们还含有OTU超家族的烷基化B（Alk B）结构域和推定的半胱氨酸蛋白酶结构域。AlkB和OTU样蛋白酶结构域都是最近才通过生物信息学在少数植物RNA病毒中鉴定的，这些病毒的浓度很高，来自β-柔曲病毒科。这些特征表明，Betaflexiviridae家族的病毒与已经研究的那些病毒有很大不同，并且由于与木本植物宿主的长期相互作用和适应，可能采用新的机制在其木本宿主中进行复制、运动和长期存活。不幸的是，研究木本植物的高度困难严重阻碍了我们对该病毒家族的生物学、复制周期和感染过程的了解。这些困难可归因于许多因素，包括繁殖和生长木本植物所需的长时间、抑制核酸和蛋白质的分离和纯化的复杂次级代谢产物（多酚和多糖）的存在以及单一植物被多种病毒混合感染的复杂性。在过去的15年里，我们的研究项目在葡萄藤病毒研究方面一直处于世界领先地位。本研究将在已有的研究基础上，围绕以下三个方面展开：（1）研究GRSPaV复制酶复合物的膜结构和组成;（2）阐明新发现的AlkB结构域的功能;以及（3）通过与一种病毒进行基因交换，将GRSPaV的宿主范围扩大到天然木本宿主以外，以包括高效模式植物本塞姆氏烟草，自然感染N.本萨米亚纳利用这种实验宿主将是非常有益的，因为它的基因组序列和大量的EST（表达序列标签）数据集是可用的。我们将使用我们已经建立的体外和体内实验系统以及分子生物学，细胞生物学，生物化学和蛋白质组学方法来解决这些基本问题。凭借我们已经建立的良好基础，并通过国际合作，我们相信我们将实现这些目标。从这项研究中获得的信息将揭示控制该病毒家族、其他木本植物病毒的复制和感染周期的不同方面的机制，以及它们与木本宿主的相互作用。反过来，所获得的信息可能导致有效控制它们引起的疾病的新战略。

The long-term goal of our research program has been to develop a model experimental system based on Grapevine rupestris stem pitting-associated virus (GRSPaV), a member of the genus Foveavirus genus (family Betaflexiviridae), to advance studies on this group of important and unique viruses. Betaflexiviridae is a newly established virus family and comprises seven genera of positive-strand, single-stranded RNA viruses. It contains numerous important viral pathogens responsible for considerable economic loss to fruit production. This family of viruses exhibit several unique features with regards to their biological properties and genetic characteristics. For example, most members of this family infect woody perennial plant crops. Furthermore, Betaflexiviridae viruses encode a replicase polyprotein with a highly modular structure. These replicase polyproteins not only contain well characterized functional domains that are conserved the Alphavirus-like supergroup of positive-strand, single-stranded RNA viruses, which include a methyl-transferase, a helicase/ATPase and a RNA-dependent RNA polymerase, they also contain an alkylation B (AlkB) domain and a putative cysteine protease domain of the OTU superfamily. Both the AlkB and OTU-like protease domains were identified only recently through bioinformatics in a small number of plant RNA viruses, with a high concentration of these viruses from the Betaflexiviridae. These features suggest that viruses of the Betaflexiviridae family are substantially different from those that have been studied and may employ novel mechanisms for replication, movement, and long-term survival in their woody hosts as a result of the long-term interaction and adaptation to woody plant hosts. Unfortunately, the high levels of difficulties in working with woody plants have severely impeded our understanding of the biology, replication cycle and infection processes of this family of viruses. These difficulties can be attributed to a number of factors, which include the long time required to propagate and grow woody plants, presence of complex secondary metabolites (polyphenolics and polysaccharides) that inhibit isolation and purification of nucleic acids and proteins, and complexity of mixed infection by multiple viruses of a single plant. Our research program has been on the leading front in the world in research on grapevine viruses in the past 15 years. In the proposed research, we will build upon the solid foundation we have established and aim to address three objectives: (1) to dissect the membrane association and composition of the replicase-associated complex of GRSPaV; (2) to elucidate the function of the newly identified AlkB domain; and (3) to widen the host range of GRSPaV beyond the natural woody host to include the highly efficient model plant Nicotiana benthamiana through gene swapping with a virus that naturally infects N. benthamiana. Tapping into this experimental host would be highly beneficial as its genome sequence and a large dataset of ESTs (expressed sequence tags) are available. We will be using both in vitro and in vivo experimental systems that we have established and molecular biology, cell biology, biochemistry as well as proteomics approach to address these fundamental questions. With the excellent foundation we have built and through international collaboration, we are confident that we will achieve these objectives as proposed. Information derived from this research will shed light on mechanisms that govern different aspects of the replication and infection cycle of this family of viruses, other woody plant viruses, as well as their interactions with the woody hosts. In turn, the information obtained may lead to novel strategies for the effective control of diseases they cause.