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），平息和RNA干扰（RNAi）是机制相关的RNA沉默过程，以同源依赖的方式破坏RNA。RNA沉默显然在高等植物中起天然抗病毒防御作用。我们的假设是，RNA沉默在动物王国中也是一种自然的适应性抗病毒防御机制。我们最近发现，在果蝇宿主细胞中，鸡舍病毒（FHV）既是RNA沉默的启动者，也是RNA沉默的靶点，并且FHV感染这些细胞需要FHV编码蛋白B2抑制RNA沉默。在这里，我们提出利用这种FHV/果蝇模型来进一步表征动物王国中RNA沉默抗病毒途径。第1部分旨在确定病毒诱导的RNA沉默是否导致细胞间沉默传播，是否可以定向到果蝇细胞中的靶细胞基因和异源病毒。第2部分对果蝇细胞中B2抑制RNA沉默的机制进行了详细的分子表征，并确定B2 (i)是否影响Dicer和RISC活性，（ii）是否直接与Dicer、Argonaute2或sirna相互作用，以及（iii）是否干扰sirna诱导的RNA沉默或微RNA的积累。我们最近构建了一个携带绿色荧光蛋白（GFP）报告基因的传染性FHV cDNA克隆，结果表明，在培养的果蝇细胞中，重组FHV的GFP表达和积累都依赖于B2抑制RNA沉默或RNAi抑制AGO2表达。利用重组FHV基因组作为报告基因，我们将筛选（Part 3）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活性，（Part 4）果蝇基因参与(i)这种新型RNA沉默抗病毒途径，（ii）其上行和下行调控，（iii）其被B2抑制。从本研究中鉴定的基因将为其哺乳动物同源基因的鉴定、克隆和功能注释提供便利。此外，了解动物病毒诱导和抑制RNA沉默的机制可以进一步改进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.