Zhongjian Guo

Characterization of a late gene, ORF67 from Bombyx mori nucleopolyhedrovirus

Open reading frame 67 of Bombyx mori nucleopolyhedrovirus (BmORF67) is a homologue of Autographa californica multiple NPV ORF81. The gene is conserved among all baculoviruses and is thus considered a baculovirus core gene. The transcript of BmORF67 was detected at 18–72 h post‐infection (p.i.). Polyclonal antiserum raised to a His‐BmORF67 fusion protein recognized BmORF67 in infected cell lysates from 24 to 72 h p.i., suggesting that BmORF67 is a late gene. BmORF67 was not detected either in budded viruses or occlusion‐derived virus. Immunofluoresence analysis showed that the protein located in the cytoplasm and interacted with host protein actin A3. In conclusion, BmORF67 is a late protein localized in the cytoplasm of infected cells that interacts with host protein.

Characterization of Helicoverpa armigera nucleopolyhedrovirus orf33 that encodes a novel budded virion derived protein, BV-e31

Summary.Homologues of Helicoverpa armigera nucleopolyhedrovirus (HearSNPV) orf33 are found in all 22 completely sequenced members of the lepidopteran nucleopolyhedroviruses and granuloviruses, but so far their functions are unknown. In this report, we describe the characterization of HearSNPV orf33 (ha33). Northern blot analysis showed a single transcript of ha33 of approximately 0.7 kb was transcribed beginning at 3 h post-infection in infected Helicoverpa zea cells (HzAM1) and the gene product could be detected as early as 6 h post-infection by western blot analysis using a rabbit derived polyclonal antibody, suggesting it was an early gene. Western blot analysis also demonstrated the ha33 protein in infected cells was a 31 kDa protein, larger than the theoretical size of 28.4 kDa, and located in the envelope fraction of budded virions (BVs). The results suggested that HearSNPV ha33 gene is a functional gene that encodes a novel structural protein of baculovirus BVs, BV-e31.

Open reading frame 60 of the Bombyx mori nucleopolyhedrovirus plays a role in budded virus production.

Open reading frame 60 (bm60) of the Bombyx mori nucleopolyhedrovirus (BmNPV) is a conserved gene among group I and some group II NPVs. bm60 encodes a late expressed protein that localizes to both the cytoplasm and nucleus of infected cells. This paper describes the characterization of a BmNPV mutant (vbm60-Null) lacking functional bm60. It was observed that the production of budded virus (BV) was reduced by nearly an order of magnitude relative to wt virus in vbm60-Null-infected BmN cells and B. mori larvae. Quantitative real-time PCR assay showed that the viral DNA replication was affected in infected cells due to disruption of bm60. Larval bioassays showed that the speed of kill of vbm60-Null virus was greatly reduced, as it took approximately 28-36 h longer to kill the fifth instar B. mori larvae. These results suggest that BmNPV bm60 is not essential for viral replication, but required for efficient BV production.

Expression of non-structural protein NS3 gene of Bombyx mori densovirus (China isolate).

The invertebrate parvovirus Bombyx mori Densonucleosis Virus type 3 (China isolate), named BmDNV-3, is a kind of bidensovirus. It is a new type of virus with unique replication mechanisms. To investigate the effects of the NS3 gene during viral DNA replication, a pair of primers was designed for amplifying NS3 gene of Bombyx mori densovirus (China isolate). Gene NS3 amplified was cloned into a prokaryotic expression vector pET-30a and the donor plasmid pFastBacHTe, respectively. The NS3 protein was expressed in Escherichia coli BL21. The pFastBacHTe-NS3 was transformed to E. coli DH10Bac. The recombinant bacmid baculoviruses (rBacmid-EGFP-NS3) isolated from the white colonies were transfected into BmN-4 cells using a transfection reagent. BmN-4 cells were infected with recombinant virus to express fusion proteins. The expression of fusion protein around 30 kDa in E. coli BL21 was identified by SDS-PAGE, Western blotting, and mass spectrometry. The expressed NS3 protein by B. mori nucleopolyhedrovirus bacmid system was confirmed by Western blotting using an anti-NS3 polyclonal antibody. And about 45 kDa protein was found. The expressed fusion protein was smaller than the expected size of EGFP-NS3, 55 kDa. Western blotting analysis indicated that EGFP-NS3 protein was expressed in infected larvae with smaller molecular size.

Identification of a novel functional nuclear localization signal in the protein encoded by open reading frame 47 of Bombyx mori nucleopolyhedrovirus.

BM47 is encoded by open reading frame 47 of Bombyx mori nucleopolyhedrovirus (BmNPV). BM47 was localized in the nucleus of BmNPV-infected cells. In the present study, we investigated a novel nuclear localization signal (NLS) for BM47 transport and accumulation in the nucleus. By expressing various regions of BM47 fused to enhanced green fluorescent protein (EGFP), we demonstrated that residues 117–148 are necessary for mediating nuclear localization of BM47. Site-directed mutation analysis showed that the two basic residue clusters at positions 117–120 (117RKRR) and 144–148 (144RKR-K) constitute an authentic NLS for BM47 localization. Finally, we observed that two clusters of basic residues were conserved in BM47 homologues of group-I nucleopolyhedroviruses.

Characterization of a late expression gene, Open reading frame 128 of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus

Summary.Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HearSNPV) is a single embedded NPV pathogenic to the bollworm, which is a major agricultural pest in many areas around the world. Ha128 homologues have been identified in all completely sequenced lepidopteran NPV’s, but no homologue has been found in a granulovirus (GV) and it is thus considered as a lepidopteran NPV-specific gene. In the HearSNPV-C1 genome Ha128 is located between 120,252 and 121,052 bp and encodes a putative protein of 266 amino acid residues with a predicted molecular weight of 30.5 kDa. Ha128 transcripts in HearSNPV-infected HzAM1 cells could be detected from 24 to 120 h post-infection (p.i.) by Northern blot. The Ha128 protein was detected at 24 h p.i. and remained detectable until 120 h p.i. by western blot using an anti-GST-Ha128 antiserum. The expression of Ha128 was inhibited in the presence of Ara-C, an inhibitor of viral DNA synthesis. These results together indicated that Ha128 was a late gene. The product of Ha128 was found to have an Mr of about 31 kDa, in agreement with the predicted molecular weight. Immunoflorescence using anti-GST-Ha128 serum showed that Ha128 was located in cytoplasm. GST pull-down and co-immunoprecipitation showed that at least two potential host proteins interacted with Ha128. In conclusion, Ha128 is a late protein localized in the cytoplasm of infected cells that may interact with host proteins.

Discovery of anti-viral molecules and their vital functions in Bombyx mori

The silkworm Bombyx mori (B. mori), a lepidopteran model organism, has become an important model for molecular biology researches with its genome completely sequenced. Silkworms confront different types of virus diseases, mainly including those caused by Bombyx mori nucleopolyhedrovirus (BmNPV), Bombyx mori densovirus type 1 (BmDNV-1), Bombyx mori bidesovirus (BmBDV) which was termed as Bombyx mori densovirus type 2 (BmDNV-2) or Bombyx mori parvo-like virus (BmPLV) before in sericulture. B. mori offers excellent models to study the molecular mechanisms of insect innate immune responses to viruses. A variety of molecules and pathways have been identified to be involved in the immune responses in the silkworm to viruses, such as the antimicrobial peptides, prophenoloxidase-activating system, apoptosis, ROS, small RNA and related molecules. Here in this review, we summarize the current research advances in molecules involved in silkworm anti-virus pathways. Moreover, taking BmNPV as an example, we proposed a schematic model of molecules and pathways involved in silkworm immune responses against virus infection. We hope this review can facilitate further study of antiviral mechanisms in silkworm, and provide a reference for virus diseases in other organisms.

Rapid disruption of Bombyx mori nucleopolyhedrovirus orf60 by red recombination system

BmNPV bacmid constructed recently and Red recombinant system were used to rapidly disrupted Bombyx monri nucleopolyhedrovirus (BmNPV) orf60 in Escherichia coli (E. coli) BW25113. BmNPV bacmid isolated from E. coli BmDH10Bac was electroporated into E. coli BW25113, which harbors plasmid pKD46 encoding lamda Red recombinase,to produce E. coli BW25113-Bac, which could be used for gene deletion of BmNPV. A linear fragment was amplified by PCR from plasmid pKD3 (containing a chloramphenicol acetyltransferase gene cat) using a pair of primers with length of 63bp,which had 45 bp homologous to the orf60 gene and 18bp homologous to cat sequences. The linear fragment was electroporated into E. coli BW25113-Bac and homologous recombination occurred between the linear fragment and orf60 with the help of lamda Red recombinase. Three specific primer pairs were used to confirm the replacement of orf60 by cat gene. Western blot analysis showed that orf60 was not expressed in BmN cells infected with knockout bacmid.

Characterization of Bombyx mori nucleopolyhedrovirus with a knockout of Bm17

Open reading frame 17 (Bm17) gene of Bombyx mori nucleopolyhedrovirus is a highly conserved gene in lepidopteran nucleopolyhedroviruses, but its function remains unknown. In this report, transient-expression and superinfection assays indicated that BM17 localized in the nucleus and cytoplasm of infected BmN cells. To determine the role of Bm17 in baculovirus life cycle, we constructed a Bm17 knockout virus and characterized its properties in cells. Analysis of the production and infection of budded virions, the level of viral DNA replication revealed showed that there was no significant difference among the mutant, the control, and the Bm17 repaired virus strains. These results suggest that BM17 is not essential for virus replication in cultured cells.