Mechanistic analyses of RNA silencing suppression by cucumoviral 2b protein

RNA silencing has been established as a natural antiviral defense mechanism in both plants and animals. In plants, RNA silencing not only operates at the site where it is triggered but also produces mobile signal to silence homologous genes in the neighbouring cells and in the distant tissues. The cell-to-cell movement of RNA silencing is mediated by DCL4-produced 21-nt siRNA. Spread of RNA silencing into distant tissue is mediated by a yet unknown signal traveling via phloem tissue and is termed systemic RNA silencing.; To counteract the RNA silencing immunity, many viruses have evolved a variety of strategies. The cucumoviral 2b protein is one of the first characterized viral suppressors of RNA silencing (VSR). The 2b protein facilitates virus movement in many host species and 2b suppression of RNA silencing has been demonstrated in silencing reversal assay, infiltration assay and grafting assay. It was hypothesized that 2b might directly interact with and subsequently inactivate systemic RNA silencing signal.; In this study, gel mobility shift assay was first conducted to test the potential interaction between 2b and various species of RNAs involved in the RNA silencing pathway. The results demonstrated that 2b protein is a dsRNA binding protein and binds dsRNA without size-selection. Mutational analyses revealed that the dsRNA binding domain of 2b is within the conserved N-terminal region overlapped with the viral replicase protein 2a. The results also suggest that different domains of the 2b protein are involved in binding the 21-nucleotide (nt) siRNA and the long dsRNA.; Infiltration assay revealed an essential role for the 21-nt siRNA binding activity in suppression of intracellular silencing by 2b. A correlation between inactivation of the phloem-dependent systemic signal and binding to 25-nt siRNA and the longer dsRNA by 2b was observed. This observation supports a previous finding that longer class of siRNAs is associated with systemic RNA silencing. Moreover, transitive RNA silencing was triggered in transgenic plants expressing CMV-GFP chimeric genes infected with cucumber mosaic viruses (CMV) and was suppressed by 2b in the early stage of infection.; Genetic studies in collaboration with Dr. Juan Diaz-Pendon revealed production of virus-specific siRNAs of 21, 22, and 24-nt, by DCL4, DCL2, and DCL3, repectively, in Arabidopsis plants infected with CMV. These studies also indicated that expression of 2b inhibits the production of the three species of viral siRNAs.; The RNA binding property of B2 proteins encoded by Flock House virus and Nodamura virus was investigated using the experimental framework established for the cucumoviral 2b. The results demonstrated that B2 binds both siRNA and long dsRNA and has a higher affinity for long dsRNA. In vitro biochemical analyses in collaboration with Dr. Hongwei Li demonstrated that B2 inhibits the production of siRNAs from synthetic dsRNA by Drosophila extracts.; In summary, studies described in this dissertation established a new class of plant and animal VSRs that binds both siRNA and longer dsRNA and may act by inhibiting the production of antiviral siRNAs in infected host cells.