Nonstructural Protein P6 Of Southern Rice Black-streaked Dwarf Virus Is A Key Factor For Viral Replication

The southern rice black streaked dwarf virus(SRBSDV)is transmitted by white-backed planthopper(WBPH),but can't be transmitted into insect offspring.The non-structural proteins P5-1,P6 and P9-1 of SRBSDV are the components of the viroplasm,which is the factory for viral replicate and assemble of progeny virions.The three proteins play the important role in the viral replication.Because P6 can interact with P5-1 and P9-1,while P5-1 can't interact with P9-1,we deduce that one of non-structural proteins may play the key important role in the viroplasm formation.In our experiments,we observed the subcellular distribution of P5-1?P6 and P9-1 of SRBSDV by using immunofluorescent assayover a time course of viral infection in culture cells in of WBPH.At 18 h postinfection(hpi),we found P6 was expressed slightly higher than that of P5-1 in VCMs,but didn't found P9-1.At this time,P5-1 and P6 formed discrete,punctuate inclusions.At 36 hpi,P5-1 and P6 were abundantly expressed,and the formed viroplasm inclusions were largee.P9-1 began to express and formed some small granular inclusions.With the extension of viral infection,P5-1,P6 and P9-1 were significantly increased.P5-1,P6 and P9-1 were all expressed in the cytoplasm at 72 hpi after infection.P5-1 was scattered throughout the cytoplasm.P6 and P9-1 were aggregated in the cytoplasm to form large granular inclusions.Therefore,the expression order was P6,P5-1 and P9-1 in the VCMs.Then,we used RNA interference(RNAi)by transfection of dsRNAs targeting to P5,P6 or P9-1 genes(dsP5,dsP6 or dsP9-1)to knock down the expression of P5-1,P6 and P9-1,respectively,and found that the expression levels of P5-1,P6,and P9-1 were decreasd compared to the control by immunofluorescent microscopy.Meanwhile,after treatment of dsP5-1,the expression levels of P5-1 and P9-1 were decreased obviously,but,there was a similar expression quantity of P6 compared to the control group;after treatment of dsP6,the expression levels of these three proteins expression levels were decreased;after treatment of dsP9-1,the expression level of P9-1 was decreased,but the expression levels of P5-1 or P6 were not affected.Quantitative reverse transcription PCR(RT-qPCR)and Western blot results further verified the findings:dsRNA decreased the transcription and expression levels of target proteins,and the knockdown of P6 had a largest effect on viralproliferation in insect vector cells.Finally,we constructed the recombinant baculovirus expression vectors with N-terminal fusion of e-GFP or mCherry.When SRBSDV P5-1-eGFP?P6-mCherry and P9-1-mCherry were expressed in Sf9 cells alone,P5-1-eGFP was formed distributed viroplasm throughout the cytoplasm,while P6 and P9-1 were formed granular inclusions.When they were co-expressed in the Sf9 cells,P6-mCherry could bind with P5-1-eGFP and P9-1-eGFP,and form large granular inclusions in the cytoplasm.However,P5-1-eGFP couldn't interact with P9-1-mCherry.These results were consistent with the baculovirus expression vectors Bacmid-SRBSDV-P5-1,P6 and P9-1 infected Sf9 cells.They also illustrated that P6 play the irreplaceable role in the formation of viroplasm of SRBSDV.Comprehensive the above results,by observing the expression order of the three non-structural proteins in the VCMs after the viral infection by immunofluorescent microscopy,we know that P6 was first expressed and it may paly the important regulation role in the viral replication.We then confirm that P6 is the pivotal protein in the viral replication with the help of dsRNA-induced RNAi,RT-qPCR and Western blot.P6 can initiate the formation of viroplasm and recruit P5-1 and P9-1 to form viroplasm.However,P5-1 and P9-1 are involved in the viral genome replication and virion assembly.Therefore,through the in-depth study of target genes,we can find effective means to control rice virus disease and provide some suggestions for biological control.