Gene silencing as an antiviral defense in animal cells

描述（由申请人提供）：转录后基因沉默（PTGS）、抑制和RNA干扰（RNAi）是以同源依赖性方式破坏RNA的机制相关RNA沉默过程。RNA沉默在高等植物中显然是一种天然的抗病毒防御。我们的假设是，RNA沉默在动物界也是一种自然的适应性抗病毒防御机制。我们最近发现，鸡舍病毒（FHV）是果蝇宿主细胞中RNA沉默的启动子和靶标，并且FHV感染这些细胞需要通过FHV编码的蛋白B2抑制RNA沉默。在这里，我们建议利用这种FHV/果蝇模型，以进一步表征RNA沉默抗病毒途径在动物王国。第一部分的目的是确定是否病毒诱导的RNA沉默导致细胞间的沉默传播，并可以针对果蝇细胞中的靶细胞基因和异源病毒。第2部分提出了B2抑制果蝇细胞RNA沉默的机制的详细分子表征，并确定B2是否（i）影响Dicer和RISC活性，（ii）直接与Dicer，Argonaute 2或siRNA相互作用，（iii）干扰siRNA诱导的RNA沉默或micro-RNA的积累。我们最近构建了一个感染性的FHV cDNA克隆，携带一个绿色荧光蛋白（GFP）报告基因，并表明GFP表达和重组FHV在培养的果蝇细胞中的积累依赖于B2抑制RNA沉默或RNAi抑制AGO 2表达。使用这种重组FHV基因组作为报告基因，我们将筛选（第3部分）由其他相关和不相关的无脊椎动物和脊椎动物病毒编码的沉默抑制子活性，以及（第4部分）参与（i）这种新型RNA沉默抗病毒途径，（ii）其上调和下调以及（iii）B2抑制的果蝇基因。从这项研究中确定的基因将有助于鉴定，克隆和功能注释的哺乳动物同源物。此外，对动物病毒诱导和抑制RNA沉默的机制的理解可能会导致RNAi技术在实际应用中的进一步改进。

DESCRIPTION (provided by applicant): 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.