Study On The Pathway Mechanismsofgrass Carp Reovirus: Antagonizing RNA Interference Of Host Cell

Grass carp reovirus (GCRV), the type strain of Aquareovirus C, is the mostimportant pathogen of grass carp hemorrhagic disease. The viral genome is composedof11segments of double stranded (ds) RNA, and encodes7structural proteins and5non-structural proteins.The interactions between GCRV and its host cells consist of cellmembrane permability, apoptosis and stress granule formation.Genomic dsRNAfragments of GCRV can survival from Dicer-initiated RNA interference (RNAi)pathway in host cells.RACE technology, Real-time PCR, Transfection were employed in the study. Thethree results were as follows:1. The molecular cloning and expression of CiDicer in grass carpDicer protein plays a key role in RNA interference (RNAi) pathway. In this study，the Dicer gene (designated as CiDicer) was cloned and identified from grass carpCtenopharyngodon idella. The complementary DNA (cDNA) of CiDicer contained anopen reading frame (ORF) of5646nucleotides (nts) encoding a putative protein of1881amino acids (aa). The deduced Dicer protein contained all known functional domainsidentified in other organisms. Tissue tropism analysis indicated that CiDicer is abundantin brain, gill, head kidney, liver, spleen, heart,muscle, intestine. In theCtenopharyngodon idella kidney (CIK) cells, messenger RNA (mRNA) expression ofCiDicer was significantly up-regulated at24h (6.36fold, P <0.01) after grass carpreovirus (GCRV) infection, and its transcriptional expression level was also transientlyinduced to a high level (6.54fold, P <0.01) at2h post the stimulation of syntheticdouble stranded polyinosinic-polycytidylic potassium salt [poly (I:C)]. In vivo analysisfurther showed that, the expression level of CiDicer mRNA in the liver was induced to asignificantly high level at12h (8.46fold, P<0.01), and then dropped to normal level at72h post challenge with GCRV. The transcriptional expression pattern of CiDicer in thespleen tissue was similar to that of liver tissue upon GCRV challenge.Meanwhile, Dicerprokaryotic expression, protein purification and polyclonal antibody preparation wereoperated.2. To identify potential RNAi suppressor(s) encoded by GCRVEach of the12viral ORFs was cloned to pEGFP-N1vector for its expression as an EGFP-fusion protein. Screening was performed by monitoring cellular fluorescencesignal after co-transfecting CIK cells with the recombinant plasmid and a shRNA thatspecifically silences EGFP. Real-time PCR for quantitating the mRNA level of theEGFP-fusion proteins， as well as fluorescence imaging for monitoring expression levelof the fusion protein, was employed to identify NS31protein as virus-encodedsuppressor of RNAi.In the study,CiDicerwere successfully cloned and could make response afterGCRV infection or Poly(I:C) stimulation. This study was established anewidea for thevirus resistance mechanism to grass carp hemorrhage disease. NS31by GCRV encodedispreliminarily identified an inhibitor of RNAi in host cells, which deepened anunderstanding about the mechanisms ofinfection and viral replication strategy.