Mechanisms Of RNA Replication Of Insect Virus And Antiviral Innate Immune

Insect RNA viruses are a large group of viruses that infect insects. Thus, the studies of insect viruses can be used to protect beneficial insects or control pests. Moreover, many insect viruses can be used as experimental models to extend our understanding to a variery of basic aspects of virology.Nodaviruses are a group of positive-stranded (+) RNA viruses with a bipartite genome of RNAs that belong to the family Nodaviridae. This family includes two genera, insect-infecting Alphanodavirus and fish-infecting Betanodavirus. My studies are mainly focused on Wuhan nodavirus (WhNV), which is the first isolated nodaivirus in China and identified by our group, Flock House virus (FHV), which is the best characterized nodavirus. Both WhNV and FHV infect insects.In nodaviruses, genomic RNA1encodes protein A, which is recognized as an RNA-dependent RNA polymerase (RdRP) and functions as the sole viral replicase protein responsible for its RNA replication, whereas RNA2encodes capsid precursor protein a. A subgenomic RNA3(sgRNA3), which is synthesized during RNA1replication, encodes nonstructural protein B2that functions to suppress host RNA interference (RNAi) antiviral immunity. Although the mechanism of nodaviral replication has been extensively studied, the biochemical features and RNA synthesis initiation mechanism(s) employed by nodaviral RdRPs have never been determined. In this study, we expressed and purified recombinant WhNV protein A in E. coli and characterized its RdRP activity in detail. Our study revealed that this nodaviral protein A can initiate RNA synthesis via a primer-independent de novo mechanism, and this initiation mechanism could be independent of other viral or cellular factors. We further showed that WhNV protein A can add ribonucleotides to the3’end of template RNAs, and this terminal nucleotidyl transferase (TNTase) activity of protein A functions to restore one or two3’-proximal nucleotides of template RNA as a potential mechanism for rescuing3’-terminal nucleotide loss. Furthermore, we also determined the cis-acting elements within positive (+) or negative (-) stranded RNA1template required for RdRP activity or TNTase activity of protein A. Moreover, we showed that both of WhNV and FHV protein A possess RNA unwinding activity, and this unwinding activity is dependent on NTP and divalent metal ions. Similar to some helicases, protein A could only unwind dsRNAs with5’single-stranded overhang but not dsRNAs with3’single-stranded overhang or blunt ends. Our work represents not only an important step toward a better understanding of nodaviral RNA replication, but also a progress in understanding the role of this kind of RdRP-associated enzymatic activities in viral RNA replication.Insects lack acquired immune responses, and completely relies on various innate immune pathways to defend against viruses. Insect innate immune defenses against viruses include five critical pathways, RNA interference (RNAi) pathway, Toll pathway, immune deficiency (Imd) pathway, Jak-STAT pathway and autophagy. Previous studies by several groups showed that RNAi pathway is a major anti-nodaviral immune pathway in Drosophila. Previous studies and we have find that the knockdown of Dcr-2is more effective than the knockdown of AG02in increasing the accumulation of FHV RNAs. Considering that Dcr-2belongs to the RIG-I-like receptor (RLR) family, our data implied that Dcr-2may has additional functions in some antiviral pathways, other than its essential role in RNAi, in Drosophila innate immunity. We further assessed the ability of FHV, and a negative-stranded RNA virus, vesicular stomatitis virus (VSV), to induce other signaling in cultured S2cells. Our study revealed that both FHV and VSV induced Toll signaling and knockdown of Dcr-2inhibited the Toll signaling that was induced by FHV or VSV. Moreover, our finding showed that knockdown of Dcr-2also inhibited the Toll signaling that was induced by Gram+bacterial or PGN. Furthermore, we find that the expression of Toll protein was downregulated by Dcr-2knockdown, demonstrating that Dcr-2is involved in the post-transcriptional or translational regulation of Toll. We also found that Dcr-2can binds to the3’-UTR of Toll mRNA directly and promoting the polyadenylation of Toll mRNA. These data indicate that Dcr-2mediates the induction of Toll immune signaling and Toll translation probably by interacting with the Toll3’-UTR. In summary, our findings in this study showed that Dcr-2has a novel function in the other innate immune signaling pathway.