Study On The Viral Replication And Transcription Of Bombyx Mori Nucleopolyhedrovirus DNA-binding Protein GP88

As is well known to us all, it is a very effective means to study the function of baculovirusgenes by gene knockout, especially in the studies on viral replication and transcriptionalregulation related genes. GP88of BmNPV is a DNA-binding protein and it was reported to be aregulatory factor in BmNPV. However, the regulatory mechanism of GP88on virusproliferation, genome replication, and gene transcription are still unknow. In this study,BmNPV gp88gene was deleted by the means of Red recombination system to constructe thegp88deletion Bacmid, gp88-ko-Bacmid. Morever, gp88-repaired Bacmid, gp88-re-Bacmid wascontructed by Bac to Bac system. And then three kind of Bacmid (wtBacmid, gp88-ko-Bacmidand gp88-re-Bacmid) were transfected into BmN cells, respectively. According to the results ofviral titer determination, the TCID50of gp88-knock out virus was zero, however, the TCID50ofgp88-repaired was similar to wild type virus (p>0.05), indicating that gp88-ko-Bacmid can notassemble virus particles. In order to know more about the biological function of GP88, thetranscriptional level of virus early gene, late gene, very late gene and viral replication level ofvirus genome in BmN cells were analyzed by real time q-PCR. The results showed that thereplication level of virus genome DNA was lower than gp88-repaired virus and wild type virusobviously (p<0.05). The transcriptional level of gp88-ko-Bacmid early gene lef-3, ie-1, dnapol,late gene vp39and very late gene p10were lower than gp88-re-Bacmid and wild type virussignificantly (p<0.05). The results presented in Western blot indicated that the lacking of gp88gene woule lead to low or no expression of lef3, vp39and p10. However, the correspondingproteins could be detected in the gp88-repaired virus as well as wild type virus. Furthermore,the electron microscopic analysis showed that there was no mature virus particles in the BmNcells infected with gp88-knock out virus. However, a large number of mature virus particleswere found in the BmN cells infected with gp88-repaired virus and wild type virus. Inconclusion, gp88was an essential gene for viral genome replication; lacking of gp88wouldlead to lower transcriptioal level of early gene, late gene and very late gene of BmNPV.Furthermore, the protein expression level of these genes were significantly reduced or evendisappeared, leading to non-mature infectious virus particles formation.