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

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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.