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)是机械上相关的RNA沉默过程，以同源依赖的方式破坏RNA。在高等植物中，RNA沉默显然是一种天然的抗病毒防御。我们的假设是，在动物界，RNA沉默也是一种天然的适应性抗病毒防御机制。我们最近发现，家禽病毒(FHV)既是果蝇宿主细胞中RNA沉默的启动者，也是RNA沉默的靶点，而FHV感染这些细胞需要通过FHV编码的蛋白质B2来抑制RNA沉默。在这里，我们建议利用这种FHV/果蝇模型来进一步表征动物王国中的RNA沉默抗病毒途径。第一部分的目的是确定病毒诱导的RNA沉默是否会导致细胞间的沉默传播，并可以针对果蝇细胞中的细胞基因和异源病毒进行靶向。第二部分对B2抑制果蝇细胞RNA沉默的机制进行了详细的分子表征，并确定了B2是否(I)影响Dier和RISC的活性，(Ii)直接与Dier、ArgAerte2或siRNAs相互作用，以及(Iii)干扰siRNA诱导的沉默或microRNAs的积累。我们最近构建了一个携带绿色荧光蛋白(GFP)报告基因的感染性FHV cDNA克隆，并表明GFP的表达和重组FHV在培养的果蝇细胞中的积累都依赖于B2抑制RNA沉默或RNAi抑制AGO2的表达。利用这个重组的FHV基因组作为报告，我们将筛选(第3部分)由其他相关和无关的无脊椎动物和脊椎动物病毒编码的沉默抑制活性和(第4部分)果蝇基因涉及(I)这一新的RNA沉默抗病毒途径，(Ii)其上调和下调调节以及(Iii)其被B2抑制。本研究确定的基因将有助于其哺乳动物同源物的鉴定、克隆和功能注释。此外，从机制上理解动物病毒诱导和抑制RNA沉默可能会进一步改进RNAi技术的实际应用。

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.