Role of the viral AlkB homologs in RNA repair

AlkB proteins that repair DNA via reversing methylation damage are conserved in a broad range of prokaryotes and eukaryotes including plants. Surprisingly, AlkB-domains were discovered in the genomes of numerous plant positive-strand RNA viruses, majority of which belong to the family Flexiviridae. The major goal of this research was to reveal the AlkB functions in the viral infection cycle using a range of complementary genetic and biochemical approaches. Our hypotheses was that AlkB is required for efficient replication and genetic stability of viral RNA genomes The major objectives of the research were to identify the functions of GVA AlkB domain throughout the virus infection cycle in N. benthamiana and grapevine, to investigate possible RNA silencing suppression activity of the viral AlkBs, and to characterize the RNA demethylation activity of the mutated GVA AlkBs in vitro and in vivo to determine methylation status of the viral RNA. Over the duration of project, we have made a very substantial progress with the first two objectives. Because of the extreme low titer of the virus particles in plants infected with the AlkB mutant viruses, we were unable to analyze RNA demethylation activity and therefore had to abandon third objective. The major achievements with our objectives were demonstration of the AlkB function in virus spread and accumulation in both experimental and natural hosts of GVA, discovery of the functional cooperation and physical interaction between AlkB and p10 AlkB in suppression of plant RNA silencing response, developing a powerful virus vector technology for grapevine using GLRaV-2-derived vectors for functional genomics and pathogen control in grapevine, and in addition we used massive parallel sequencing of siRNAs to conduct comparative analysis of the siRNA populations in grape plants infected with AlkB-containing GLRaV-3 versus GLRaV-2 that does not encode AlkB. This analysis revealed dramatically reduced levels of virus-specific siRNAs in plants infected with GLRaV-3 compared to that in GLRaV-2 infection implicating AlkB in suppression of siRNA formation. We are pleased to report that BARD funding resulted in 5 publications directly supported by BARD, one US patent, and 9 more publications also relevant to project. Moreover, two joint manuscripts that summarize work on GVA AlkB (led by Israeli PI) and on viral siRNAs in grapevine (led by US PI in collaboration with University of Basel) are in preparation.