Post-transcriptional gene silencing (PTGS), quelling and RNA interference (RNAi) are mechanistically related RNA silencing processes that destroy RNA in a homology-dependent manner. RNA silencing clearly acts as a natural antiviral defense in higher plants. Our hypothesis is that RNA silencing also functions as a natural adaptive antiviral defense mechanism in the animal kingdom. We have recently shown that flock house virus (FHV) is both an initiator and a target of RNA silencing in Drosophila host cells and that FHV infection of these cells requires suppression of RNA silencing by an FHV-encoded protein, B2. Here we propose to exploit this FHV/Drosophila model to further characterize the RNA silencing antiviral pathway in the animal kingdom. Part 1 aims to determine if virus-induced RNA silencing leads to intercellular silencing spread and can be directed to target cellular genes and heterologous viruses in Drosophila cells. Part 2 proposes a detailed molecular characterization of the mechanism involved in the B2 suppression of RNA silencing in Drosophila cells and determine if B2 (i) influences the Dicer and RISC activities, (ii) interacts directly with Dicer, Argonaute2 or siRNAs, and (iii) interferes with the siRNA-induced RNA silencing or the accumulation of micro-RNAs. We have recently constructed an infectious FHV cDNA clone that carries a green fluorescent protein (GFP) reporter and showed that both GFP expression and accumulation of the recombinant FHV in cultured Drosophila cells were dependent on B2 suppression of RNA silencing or RNAi inhibition of AGO2 expression. Using this recombinant FHV genome as a reporter, we will screen for (Part 3) silencing suppressor activities encoded by other related and unrelated invertebrate and vertebrate viruses and (Part 4) Drosophila genes involved (i) in this novel RNA silencing antiviral pathway, (ii) in its up- and down regulations and (iii) in its suppression by B2. Genes identified from this study will facilitate the identification, cloning and functional annotation of their mammalian homologues. In addition, a mechanistic understanding of induction and suppression of RNA silencing by animal viruses could lead to further improvements in the practical application of the RNAi technology. ? ?
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