Protein composition analysis of polyhedra matrix of Bombyx mori nucleopolyhedrovirus (BmNPV) showed powerful capacity of polyhedra to encapsulate foreign proteins.

Polyhedra can encapsulate other proteins and have potential applications as protein stabilizers. The extremely stable polyhedra matrix may provide a platform for future engineered micro-crystal devices. However, the protein composition of the polyhedra matrix remains largely unknown. In this study, the occlusion-derived virus (ODV)-removed BmNPV polyhedra matrix fraction was subjected to SDS-PAGE and then an LC-ESI-MS/MS analysis using a Thermo Scientific Q Exactive mass spectrometer. In total, 28 host and 91 viral proteins were identified. The host components were grouped into one of six categories, i.e., chaperones, ubiquitin and related proteins, host helicases, cytoskeleton-related proteins, RNA-binding proteins and others, according to their predicted Pfam domain(s). Most viral proteins may not be essential for polyhedra assembly, as evidenced by studies in the literature showing that polyhedra formation occurs in the nucleus upon the disruption of individual genes. The structural role of these proteins in baculovirus replication will be of significant interest in future studies. The immobilization of enhanced green fluorescent protein (eGFP) into the polyhedra by fusing with the C-terminus of BM134 that is encoded by open reading frame (ORF) 134 suggested that the polyhedra had a powerful capacity to trap foreign proteins, and BM134 was a potential carrier for incorporating proteins of interest into the polyhedra.

Characterization of aggregate/aggresome structures formed by polyhedrin of Bombyx mori nucleopolyhedrovirus.

Virus infections often lead to formation of aggregates and aggresomes in host cells. In this study, production of aggregates and aggresomes by the highly expressed protein polyhedrin of Bombyx mori nucleopolyhedrovirus (BmNPV) at 24 h postinfection (p.i.) was detected with a fluorescent molecular dye, and verified by colocalization of polyhedrin with aggresomal markers, GFP-250 and γ-tubulin. Polyhedrin aggregates showed hallmark characteristics of aggresomes: formation was microtubule-dependent; they colocalized with heat shock cognates/proteins of the 70-kDa family (HSC/HSP70s), ubiquitinated proteins and recruited the mitochondria. Aggregated polyhedrin protein gradually gained its active conformation accompanying progress of BmNPV infection. At 48 h p.i. recovered polyhedrin bound directly to Bombyx mori microtubule-associated protein 1-light chain 3 (BmLC3), an autophagosome marker, and was colocalized with BmLC3 to the isolation membrane of autophagosome, implying the involvement of polyhedrin in cellular autophagy. Inhibition of autophagy by 3-methyladenine (3-MA) dramatically resulted in decrease of polyhedrin expression and polyhedra particle production. These observations suggested that highly expressed polyhedrin forms aggregate to get involved in cellular autophagy then play an important role in polyhedra production.

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.

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.

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.

[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 a late gene, ORF60 from Bombyx mori nucleopolyhedrovirus.

Open reading frame 60 of Bombyx mori nucleopolyhedrovirus (Bm60) is located between 56,673 and 57,479 bp in the BmNPV genome which encodes 268 amino acid residues with predicted molecular weight of 31.0 kDa. Bm60 and its homologues have been identified in 11 completely sequenced lepidopteran NPVs. The transcript of Bm60 was detected by RT-PCR at 18-72 h post-infection (p.i.), while the corresponding protein could be detected at 24-72 h p.i. in BmNPV-infected BmN cells by Western blot analysis using a polyclonal antibody against Bm60. The expression of Bm60 was inhibited in the presence of Ara-c, an inhibitor of viral DNA synthesis. These results together indicated that Bm60 was a late gene. The size of Bm60 product was found to be a 31 kDa in BmNPV-infected BmN cells, consistent with predicted molecular weight. Immunofluoresence analysis showed that the Bm60 product was first detected in the cytoplasm at 24 h p.i and also located in nucleus during later infection. In conclusion, the available data suggest that Bm60 is a functional ORF of BmNPV and encodes a 31kDa protein expressed in the later stage of infection cycle.

Bacterial expression and cellular localization of Helicoverpa armigera nucleopolyhedrovirus Orf33 in infected host cells.

Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HearSNPV) is a selective, highly infectious pathogen to H. armigera and has been extensively used for the control of this pest. This study presents the bacterial expression and localization of ha33 in infected host cells. The ha33 protein expressed in Escherichia coli had protein size of 17kDa. Western blot analysis using polyclonal antibody showed that the product of ha33 in infected Helicoverpa zea cells (HzAM1) was a 31kDa protein, larger than the theoretically predicted size of 28.4kDa. The confocal laser scanning microscope observation demonstrated that ha33 gene product was localized in the cytoplasm of infected HzAM1 cells beginning at 6 h p. i. and remained throughout the infection.

Characterization of a unique gene ORF135 from Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus.

The ORF135 of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HearSNPV)(Ha135) is one of the 20 genes that are unique to HearSNPV. Computer-assisted analysis revealed that four potential post translation modification sites, four transcription factor associated domains and a DNA binding protein domain were found in Ha135 amino acid sequence. Northern blot analysis of Ha135 indicated that Ha135 transcript was detected at 12 h.p.i. and remained detectable at up to 122 h.p.i. RT-PCR method was used to understand the temporal regulation of the transcript at earlier stages, the result showed that the Ha135 transcript was detected as early as 3 h p.i. suggesting that Ha135 was an early gene, which is in agreement with the early promoter motifs. The Ha135 protein was also detected at 12 h.p.i and remained detectable until 122 h.p.i. by western blot using an anti-Ha135 antiserum. The product of Ha135 was found to be about 29 kDa, bigger than the predicted 24 kDa molecular weight, suggesting that post translational modification of the Ha135 protein occur in host cells. The subcellular location was studied using EGFP-Ha135, which suggested that the Ha135 protein is primarily localized in the nucleus, which is compatible with several functional domains present in Ha135 amino acid sequence. Together, these results suggest the possibility that HearSNPV ORFI35 might be involved in viral DNA transcription and/or replication.

Characterization of Ha29, a specific gene for Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus.

Open reading frame 29 (ha29) is a gene specific for Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HearSNPV). Sequence analyses showed that the transcription factor Tfb2 motif, bromodomain and Half-A-TPR (HAT) repeat were present at aa 66-82, 4-76, 55-90 of the Ha29 protein respectively. The product of Ha29 was detected in HearSNPV-infected HzAM1 cells at 3 h post-infection. Western blot analysis using a polyclonal antibody produced by immunizing a rabbit with purified GST-Ha29 fusion protein indicates that Ha29 is an early gene. The size of Ha29 product in infected HzAM1 cells was about 25 kDa, which was larger than the presumed size of 20.4 kDa. Tunicamycin treatment of HearSNPV-infected HzAM1 cells suggested that the Ha29 protein is N-glycosylated. Fluorescent confocal laser scanning microscope examination, and Western blot analysis of purified budded virus (BVs), occlusion-derived virus (ODVs), cell nuclear and cytoplasmic fraction, showed that the Ha29 protein was localized in the nucleus. Our results suggested that ha29 of HearSNPV encodes a non-structurally functional protein that may be associated with virus gene transcription in Helicoverpa hosts.

Comparison of the complete genome sequence between C1 and G4 isolates of the Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus.

The complete nucleotide sequence of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus isolate C1 (HearSNPV-C1) was determined and analyzed by comparing with the genome of HearSNPV-G4 isolate. C1 and G4 isolates occurred in the same host species and geographic location but showed different virulence. The HearSNPV-C1 genome consisted of 130,759 bp and 137 putative open reading frames larger than 150 nucleotides were identified. The two genomes shared 98.1% nucleotide sequence identity, with a total number of 555 bp substitutions, 1354 bp deletions, and 710 bp insertions in HearSNPV-C1. Comparison of ORFs and homologous repeat (hr) regions of the two genomes showed that there were four highly variable regions hr1, hr4, hr5, and bro-b, all in repeat regions. These results suggest that baculovirus strain heterogeneity may be often caused by SNPs and changes in the hrs and bro genes.