Suppression Of BmCPV Infection On BmN Cells RNAi System And Function Of BmCPV Segment 8

RNA interference(RNAi)is a gene expression regulation mechanism in ubiquitous eukaryotes,it's also plays a key role in antiviral defense mechanism.To counteract the antiviral RNAi pathway and promote viral infection,insect viruses encode RNAi suppressors during infection.Bombyx mori cytoplasmic polyhedrosis virus(BmCPV)is a typical species of Reoviridae.It is important for understanding of interaction between virus and host to verify the supression of BmCPV infection on BmN cells RNAi system and function of BmCPV Segment 8,it is also possible to provide antiviral molecular targets.Previous studies have reported that the BmCPV genome can be a target to RNAi pathway in Bombyx mori midgut cells,but it is unknown whether BmCPV can inhibit RNAi system by encoding RNAi suppressors to promote viral infection during infecting BmN cells.The functional study of VP8 encoded by BmCPV segment 8 focused on conjecture by sequencing alignment,and the function of VP8 were poorly understood.In this study,to verify the inhibition of virus invasion and proliferation in BmN cells by exogenous RNA interference(exo-RNAi)pathway,we identified the viral small RNAs(vs RNAs)originating from BmCPV in BmN cells infected with BmCPV by deep-sequencing.The analysis of deep sequence data showed that there was a large number of vs RNA originating from BmCPV in BmN cells infected with BmCPV.Analysis of vs RNA size revealed that the 19-nt class is the predominant class among the vs RNAs for all viral segments,which was assumed production of viral ds RNA segments cleaveged by Dicer-2 of the host cell.The distribution of vs RNA reads mapping to viral segments and different strands of showed that most vs RNAs was originate from S3 and S4 of virus genome,and there was a strong positive-strand bias in all vs RNAs.In particular,the distributions of vs RNAs revealed distinct hot spot patterns which was clearly observable for segments 3,4 and 10,suggesting a nonrandom origin and segment bias of vs RNAs distributions.Analysis of cleavage sites showed a conservative degenerate motif at the cleavage sites of the vs RNAs.It has been reported that antiviral exo-RNAi pathway was used to resist virus invasion and proliferation in host cells of insects during viral infection,meanwhile,insect viruses encode RNAi suppressors to counteract the antiviral RNAi pathway and promote viral infection.The effect of BmCPV infection on RNAi system of BmN cells was evaluated by dual-luciferase reporter assay,which was by co-transfecting both the luciferase luc gene expression plasmid and reference plasmid p RL-TK and special si RNA(or ds RNA)of luc gene into BmN cells.The dual-luciferase reporter assay was also applied to screen viral suppressors of RNAi(VSRs)encoded by BmCPV genomes by co-transfecting dual-luciferase expression plasmid and special si RNA(or ds RNA)of luc gene into transgenic BmN cells.The results showed that BmCPV infection can suppress si RNA-induced RANi in BmN cells,and VP8,encoded by BmCPV segment 8,can functionally as a RNAi suppressor for its inhibition function on si RNA-induced RANi.VP8,encoded by BmCPV segment 8,has been found to suppress si RNA-induced RANi in BmN cells,but it is unclear how dose it work.The BmCPV genome consists of ten ds RNA fragments(S1~S10),and VP8 is a non-structural protein encoded by segment 8.In order to investigate the function of VP8,we constructed the transgenic cells with overexpression of S8.The result of q PCR and Western blotting showed that the multiplication of BmCPV was increased in S8 overexpression cells.The subcellular localization of VP8 was in the cytoplasm of both BmN cells and yeast cells.The result of q PCR showed that the relative expression of Dcr2,Apa and Cyt were significantly decreased in S8 overexpression cells.The results of flow cytometry showed that overexpression of S8 could promote the proliferation of BmN cells.Co-IP was used to screen the interaction proteins of VP8 in BmN cells,there were Mitochondrial prohibitin complex protein 2,Endonuclease-reverse transcriptase,Vasa intronic protein and TCTP and factors or enzymes related to transcription and translation and so on identified by mass spectrometry.The test of yeast cell growth showed that BmCPV VP6-VP10(polyhedrosis protein)had different inhibitory effects on yeast cell growth,and VP8 protein was the most toxic to yeast cells.The result of immunofluorescence assay showed that VP8 protein was co-located with Dcp2 protein in yeast cells and Ago2 protein in BmN cells.