Persistent RNA virus infection of lepidopteran cell lines: Interactions with the RNAi machinery |
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| Institution: | 1. Insect Molecular Genetics and Biotechnology, Institute of Biosciences & Applications, National Centre for Scientific Research “Demokritos”, Aghia Paraskevi Attikis, Athens, Greece;2. Department of Animal Physiology and Neurobiology, KU Leuven, Leuven, Belgium;3. Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium;1. Department of Biology, University of Bergen, P.O. Box 7803, 5006 Bergen, Norway;2. Sea Lice Research Centre, Department of Biology, University of Bergen, P.O. Box 7803, 5006 Bergen, Norway;3. Sea Lice Research Centre, Department of Informatics, University of Bergen, P.O. Box 7803, 5006 Bergen, Norway;1. Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium;2. Laboratory for Pharmaceutical Biotechnology, Faculty of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460, B-9000 Ghent, Belgium;1. Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, 9000 Ghent, Belgium;2. Insect Molecular Genetics and Biotechnology, Institute of Biosciences & Applications, NCSR “Demokritos”, Aghia Paraskevi, Athens, Greece;1. Insect Molecular Genetics and Biotechnology, Institute of Biosciences and Applications, National Centre for Scientific Research “Demokritos”, P. Grigoriou & Neapoleos Str, Aghia Paraskevi Attikis, 153 42 Athens, Greece;2. Guangdong Engineering Research Center of Subtropical Sericulture and Mulberry Resources Protection and Safety, Guangdong Provincial Key Lab of Agro-animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou 510642, People’s Republic of China;3. Department of Biology, University of Crete, Voutes University Campus, 700 13 Heraklion, Crete, Greece;4. Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium;1. Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China;2. Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou, Hainan 571101, China;3. School of Biological Sciences, University of Missouri-Kansas City, 5007 Rockhill Road, Kansas City, MO 64110, USA;4. College of Life Sciences, Shanghai University, Shanghai 200444, China |
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| Abstract: | RNAi is broadly used as a technique for specific gene silencing in insects but few studies have investigated the factors that can affect its efficiency. Viral infections have the potential to interfere with RNAi through their production of viral suppressors of RNAi (VSRs) and the production of viral small RNAs that can saturate and inactivate the RNAi machinery. In this study, the impact of persistent infection of the RNA viruses Flock house virus (FHV) and Macula-like virus (MLV) on RNAi efficiency was investigated in selected lepidopteran cell lines. Lepidopteran cell lines were found to be readily infected by both viruses without any apparent pathogenic effects, with the exception of Bombyx-derived Bm5 and BmN4 cells, which could not be infected by FHV. Because Sf21 cells were free from both FHV and MLV and Hi5-SF were free from FHV and only contained low levels of MLV, they were tested to evaluate the impact of the presence of the virus. Two types of RNAi reporter assays however did not detect a significant interference with gene silencing in infected Sf21 and Hi5-SF cells when compared to virus-free cells. In Hi5 cells, the presence of FHV could be easily cleared through the expression of an RNA hairpin that targets its VSR gene, confirming that the RNAi mechanism was not inhibited. Sequencing indicated that the B2 RNAi inhibitor gene of FHV and a putative VSR gene from MLV were intact in persistently infected cell lines, indicating that protection against RNAi remains essential for virus survival. It is proposed that infection levels of persistent viruses in the cell lines are too low to have an impact on RNAi efficiency in the lepidopteran cell lines and that encoded VSRs act locally at the sites of viral replication (mitochondrial membranes) without affecting the rest of the cytoplasm. |
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| Keywords: | RNA virus Flock house virus Macula-like virus RNA interference Lepidopteran cell lines Persistent infection |
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