Identification Of Putative Viral Suppressors Of RNAi In BmNPV

RNA interference(RNAi)is a highly conserved gene silencing mechanism triggered by double-strand RNA(ds RNA).In plants and insects,RNAi mediated by small interfering RNA(si RNA)plays the major role of antiviral immune response.si RNA 21-24 nucleotides(nt)in length were derived from ds RNA after processing by Dicer proteins,a type III nuclease.RNA induced silencing complex(RISC)then recruit si RNA as a template to guide Argonaute protein(AGO2)'s specific degradation of viral RNA.Synchronously,virus has evolved viral suppressor of RNAi(VSR)to counteract with the antiviral defense.VSRs are generally proteins critical for viral infection,but they share less identity in gene sequence and protein structure.Moreover,host factors involved in RNAi pathway that VSRs target to are also diverse.For example,the B2 protein of Flock House virus(FHV)and the 1A protein of Drosophila C virus(DCV)abolish the accumulation of si RNA by binding to ds RNA to avoid slicing by Dicer.B2 protein could also sequester si RNA to provent its binding with AGO proteins.This kind of VSRs always contain a ds RNA-binding motif.Some proteins,like 1A protein of Cr PV and P0 of Poleoviruses,interact with AGO proteins to disturb the assembly of RISC or distroy its slicing activity.Recently,several novel VSRs are reported to inhibit RNAi by interacting with AGO proteins through a conserved motif---GW/WG motif.It's the PIWI domain of AGO proteins that holds the binding capacity with GW/WG motifs.Bombyx mori nucleopolyhedrovirus(Bm NPV)is the most threatening pathogen to silkworm,causing great damage to the sericulture every year.To take a further understanding about how exactly does Bm NPV infect the silkworm to help figure out more strategies to confront it,we conducted this research.We mainly explored whether Bm NPV suppress the RNAi of silkworm or not,and made a prediction of putative VSRs of Bm NPV,then identified the suppression to RNAi of several VSR candidates.Here are the key results:1.Identification of the suppression to RNAi of Bm NPV in Bm N4-SID1 and Bm E cellsTwo ds RNA(ds Fluc1 and ds Fluc2)different in length but both target firefly luciferase were synthesized in vitro,and were transfected to Bm N4-SID1 cells together with dual luciferase reporter system containing a firefly luciferase(Fluc)expressing plasmid and an internal reference plasmid expressing renilla luciferase(RLuc).ds Fluc2 showed stronger inhibition of RNAi than ds Fluc1,hence ds Fluc2 was chosen for the experiment.dsFluc2 subdue the expression of firefly luciferase in BmN4-SID1 cells,and the silencing value is 4.7 times more than that caused by ds Red.However,BV infection led to almost total return of the gene silencing,indicating that Bm NPV suppress the RNAi of Bm N4-SID1 cells.The silencing in Bm E cells caused by ds Fluc2 is 27 times more than the silencing caused by ds Red.BV infection also totally returned the gene silencing,suggesting Bm NPV also restrain the RNAi in Bm E cells.These results concluded that Bm NPV encodes VSRs.2.The prediction of putative VSRs of Bm NPVBioinformatic analyses of the proteome of TCV,SPMMV and To RSV were done using blastx in NCBI.In TCV,two GW/WG motifs-containing proteins were identified,while Five GW/WG motifs-containing proteins were identified both in SPMMV and in To RSV.The known VSR P38,P1 and CP of each virus was all among them,indicating this method could be used for the identification of VSR.16 proteins containing GW/WG motifs are identified in Bm NPV using Blastx.The 16 proteins were classified into 4 categories based on their functions---structure proteins,gene replication and expression related proteins,host bodies modulating proteins and others.These proteins play important roles in the viral DNA replication,assembly of mature virions,virus' s entrance,progeny virus' s release and other critical process of virus infection.Envelope proteins GP64/67,ODV-E66 and capsid proteins orf1629,38 K,VP39 are the main component of virions,GP64/67 and ODV-E66 induce the entrance of virus to cells and also the formation of BV.P47,DNA Polymerase and DNA Helicase function in the replication of viral DNA.Cystein Protease,Chitinase and p35 govern the host bodies.Cystein Protease and Chitinase works together to cause the death and dicomposition of cells after the multiplication of virus is finished,and also the liquefaction of bodies,then promotes the transmission of virions.Infected cells will initiate the "self killing program"---cell apoptosis to protects their neighbor cells,as a counteract,p35 works to prevent cells' apoptosis to help virus produce more progenies,and the ortholog of p35 in Ac MNPV was reported to exhibiting the inhibition of RNAi.P33?orf4 and P48 are critical for the proliferation of BV,the assembly of ODV and production of mature offspring virus.At the same time,we predicted VSRs of Bm NPV through a VSRs prediction website: http://viralzone.expasy.org/all_by_protein/891.html.5 proteins were chosen to be candidates refer to the Prediction score.In total,19 proteins were predicted.3.Identification of the suppression to RNAi of Bm NPV-VSR candidates in Bm E cells and expression of PIWI domain14 candidates of putative VSRs,Bm NPV-p35/LEF-3/IE1/GP64/Chitinase/Cystein protease/PIF1/orf1629/P47/P43/P48/orf4/VP39 were chosen to be verified.Those genes were cloned to T5 vectors,then recombinated to overexpressing vector p SLfa1180[Hr3-A4-SV40] after sequencing certification,double enzymes digestions confirm that the vectors are successfully conducted.Based on the silencing of firefly luciferase induced by ds Fluc2,the suppression effect of gene silencing under virus genes expressing were detected.Quantitative real-time PCR confirmed that vector are successfully expressed in Bm E cellls in the RNA level.9 genes,Bm NPV-p35/LEF-3/IE1/orf1629/P47/P43/P48/orf4/VP39,were chosen to be verified primarily and Western Blot showed that all of the 9 genes were expressed.Detection of fluorescence displayed that orf4/P48/P47/VP39 repressed the RNA silencing value in a extent of 67%,67%,53% and 64%,indicating orf4/P48/P47/VP39 may function as VSR.However,other genes did not show obvious down-regulatation of RNAi,meaning that they do not play the role of VSR.We also constructed recombinant expression plasmids of PIWI domain,which could interact with GW/WG-containing VSRs,with Flag and HA tags respectively.They could be used to hook up GW/WG-containing VSRs of Bm NPV through pull-down or Immunoprecipitation(IP).