Characterization Of ORF67 And Three Other Genes From Bombyx Mori Nucleopolyhedrovirus

The family Baculoviridae is a highly selective pathogen in arthropods, mainly in insects of the order Lepidoptera. So far, the genomes of 42 baculoviruses, including 33 nucleopolyhedroviruses and 9 granuloviruses, have been sequenced. By comprehensive comparisons of all the sequenced baculovirus genomes, 30 genes are revealed as baculovirus core genes, 62 genes are conserved in Lepidoptera baculoviruses. Bombyx mori nucleopolyhedrovirus is one of most studied baculoviruses; some genes have not been characterized. In this study, two baculovirus core genes (orf67 and p33), and two conseved genes (p26 and orf122) in Lepidoptera baculoviruses are characterized. The gene transcription, gene expression, sub-cellular location, and gene knock-out were adopted to characterize these four genes. The object of this study is to enhance our understanding of baculovirus molecular biology. The results are as following:1. Functional analysis of BmNPV orf67 (Bm67)The Bm67 is located at 61190-61892nt in the genome of BmNPV T3 strain. Its ORF are 705bp in length and is predicted to encode a 234 amino acid peptide with a deduced molecular weight of 27.0kDa. A baculovirus consensus late transcriptional start motif ATAAG is found at 57nt upstream of the start codon ATG. Temporal transcription of Bm67 was examined by RT-PCR using total RNA isolated from BmNPV-infected BmN cells. Its transcripts was detected as early as 6 h p.i.. The antiserum against Bm67 was prepared by fusion protein GST-Bm67 expressed in Escherichia.coli. The temporal expression of Bm67 protein was analyzed by Western blot using anti-Bm67 serum, a positive band was detected as early as 12 h p.i.. From above data, we conclude that Bm67 is a baculovirus late expression gene. The sub-cellular localization of the Bm67 protein was investigated by immmunofluorescence. The fluorescence was both detected within the nucleoplasm and the cytoplasm. To further investigate the role of Bm67 in BmNPV infection cycle, we generated a Bm67 knockout virus. Furthermore, a Bm67 repair bacmid was constructed by transposing the Bm67 native promoter-promoted Bm67 ORF into the polyhedrin locus of the Bm67 knockout bacmid. After these recombinant bacmids were transfected into BmN cells, the Bm67 knockout bacmid caused defects in the production of infectious budded viruses. However, the Bm67 repair bacmid could rescue the defect, and the budded virus titers could reach wild type (wt) levels. Quantitative real-time PCR analysis indicated that Bm67 is required for normal levels of DNA synthesis or for the stability of nascent viral DNA at the early stage. Electronmicroscopic analysis revealed that the formation of normal-appearing nucleocapsids is decreased in Bm67 knockout bacmid-transfected cells, and nucleocapsids are rarely found in the cytoplasm. The presence of "enveloped" nucleocapsids at the nucleoplasm bilayer indicated that they are abnormally enveloped. These results indicated that Bm67 is required for the production of infectious budded viruses and assembly of envelope and nucleocapsids.2. Characterization of BmNPV p33 (p33)The p33 is located at 70487-71264nt in the genome of BmNPV T3 strain. Its ORF are 780bp in length, and is predicted to encode a 259 amino acid peptide with a deduced molecular weight of 30.9kDa. Three baculovirus consensus late transcriptional start motif TAAG are found at 184-181nt, 153-150nt and 125-122nt upstream of the start codon ATG, respectively. Temporal transcription of p33 was examined by RT-PCR using total RNA isolated from BmNPV-infected BmN cells. Its transcripts was detected as early as 12 h p.i.. The antiserum against p33 was prepared by fusion protein GST-p33 expressed in Escherichia.coli. The expression of p33 protein was analyzed by Western blot using anti-p33 serum, and the result demonstrated that a positive band was detected as early as 18 h p.i.. From above data, we conclude that p33 is a baculovirus late expression gene. Additionally, GFP fused p33 was expressed in BmN cells using Bac to Bac expression system, the results indicated that p33 protein was accumulated in cytoplasm, and which was confirmed by immmunofluorescence. Furthermore, Western blot was performed to determine if p33 is a structural protein, and no immuno-reactive band was detected both in ODVs and BVs. These results indicate that p33 is not a virus structural protein.3. Characterization of BmNPV p26 (p26)The p26 is located at 107702- 108422nt in the genome of BmNPV T3 strain. Its ORF are 723bp in length, and is predicted to encode a 240 amino acid peptide with a deduced molecular weight of 27.3kDa. A TATA box is found at 21-24nt upstream of the start codon ATG. Temporal transcription of p26 was examined by RT-PCR. Its transcripts was detected as early as 3 h p.i., and remain detectable at 96 h p.i.. The antiserum against p26 was prepared using fusion protein His-p26. The temporal expression of p26 protein was analyzed by Western blot using anti-p26 serum, and a positive band was detected as early as 18 h p.i.. GFP fused p26 was expressed in BmN cells using Bac to Bac expression system; the results indicated that p26 protein was displayed both in cytoplasm and nucleoplasm. However, immmunofluorescence indicated that p26 was displayed in cytoplasm.4. Characterization of BmNPV orfl22 (Bm122)The Bm122 is located at116325-116928nt in the genome of BmNPV T3 strain. Its ORF are 606bp in length, and is predicted to encode a 201 amino acid peptide with a deduced molecular weight of 22.9kDa. A baculovirus consensus earyly transcription motif CAGT and a TATA box are found at 240nt and 189nt upstream of the start codon ATG, respectively. Its transcript was detected at 3 h p.i. and remained detectable at up to 96 h p.i. Cytosine arabinoside (Ara-C) inhibitor assay revealed that its transcription was initiated at 2.5 h p.i.. Temporal transcription analysis indicated that Bm122 is transcribed by host RNA polymerase. The size of the translational product of the Bm122 in Tn5B-1-4 cells was approximately 23 kDa, which is in agreement with the predicted value of 22.9 kDa, suggesting that no major posttranslational modification occurred in the primary protein product. The subcellular localization of Bm122 was studied using EGFP-Bm122, which revealed that Bm122 protein was accumulated within the nuclear region of virus-infected BmN cells. All these results suggest that Bm122 is an early gene encoding a protein that functions in the nucleus.