Characterization Of The Open Reading Frame 4 Of Bombyx Mori Nucleopolyhedrovirus (BmNPV)

Using BLAST in NCBI, we found that open reading frame 4 of BmNPV (Bm4) and open reading frame 11 of AcMNPV share 96% identity in nucleotide sequence, and both of them are 1020 bp in size. Among the 29 sequenced baculoviruses, they have their homologues in three other baculoviruses including Plutella xylostella multiple nucleopolyhedrovirus, Rachiplusia ou multiple nucleopolyhedrovirus and Maruca vitrata MNPV. In the promoter region of Bm4, there is an early promoter motif. However, the function of Bm4 is completely unknown so far.In order to study the subcellular localization of Bm4 product in the context of BmNPV infection, we used EGFP which is fused to 3' terminus of Bm4 as the reporter gene, and used BmNPV bacmid to express Bm4-EGFP fusion protein. Using Confocal scanning microscope, we found that the green fluorescent signal was localized primarily in the nucleus of Bm cells, however, a weaker signal was also observed in the membrane.Polyhedrin promoter was replaced with ie1 promoter or Bm4 promoter by digesting pFastBac1 with SnaBI and BamHI. With the modified pFastBac1, recombinant viruses were constructed expressing foreign genes under the control of ie1 promoter or Bm4 promoter, laying the foundation for the rescue experiment of Bm4-disrupted virus. Meanwhile, by transfecting Bm cells with the constructed pFastbac-4p-EGFP in which EGFP was under the control of Bm4 promoter, we found that EGFP was expressed without the help of viral factors, showing that Bm4 was an immediate early gene.To disrupt Bm4, Bm4 transfer vector was constructed and co-transfected with BmNPV bacmid into Bm cells, and then three passages of viruses in Bm cells was carried out, followed by one round of purification. Subsequently, bacmid DNA was extracted and transformed into competent DH10B cells. Blue colonies grown on plate containing kanamycin, X-gai and IPTG, were selected to be identified by PCR with Bm4 primers. A colony harboring only Bm4-disrupted bacmid DNA was identified, and then Bm4-disrupted bacmid DNA was extracted and transfected into Bm cells. 5 days post infection, cytopathic effects were observed. Supernatant was removed from transfected cells and added to a second group of freshly plated Bm cells, cytopathic effects were also observed, suggesting that Bm4 is not an essential gene for viral growth in cultured Bm cells.To exclude the possibility of second mutations, we constructed rescue viruses that express Bm4 at the polyhedrin locus of Bm4-disrupted bacmid by using Bac-to-Bac/BmNPV baculovirus expression system and the modified pFastBac1 in which the polyhedrin promoter was replaced with ie1 promoter or Bm4 promoter, paving the way for studying the specific function of Bm4.Taken together, the results showed that Bm4 was a non-esseantial immediate early gene whose product was primarily localized in the nucleus.