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.

An innovative technique for inoculating recombinant baculovirus into the silkworm Bombyx mori using lipofectin.

The insect baculovirus expression system is one of the most effective eukaryotic expression systems known, and has been widely used to produce numerous recombinant proteins. The current traditional inoculation method consists of injecting recombinant baculovirus directly into insects, thus causing potential contamination to the environment due to virus diffusion during the inoculation process. In the present experiment, we directly introduced baculovirus DNA into silkworms using a cationic lipofectin reagent instead of directly injecting the virus. This new method produced the same infection results as the traditional method. A new safe infection technique without direct use of the virus was thus developed.