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. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI052447-04
Application #
7014489
Study Section
Genetics Study Section (GEN)
Program Officer
Cassetti, Cristina
Project Start
2003-02-15
Project End
2008-01-31
Budget Start
2006-02-01
Budget End
2007-01-31
Support Year
4
Fiscal Year
2006
Total Cost
$405,098
Indirect Cost
Name
University of California Riverside
Department
Other Basic Sciences
Type
Schools of Earth Sciences/Natur
DUNS #
627797426
City
Riverside
State
CA
Country
United States
Zip Code
92521
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Li, Yang; Basavappa, Megha; Lu, Jinfeng et al. (2016) Induction and suppression of antiviral RNA interference by influenza A virus in mammalian cells. Nat Microbiol 2:16250
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Han, Yan-Hong; Luo, Ying-Jun; Wu, Qingfa et al. (2011) RNA-based immunity terminates viral infection in adult Drosophila in the absence of viral suppression of RNA interference: characterization of viral small interfering RNA populations in wild-type and mutant flies. J Virol 85:13153-63

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