Analysis Of Helicoverpa Armigera Single Nucleopolyhedrovirus ORF33, ORF80, ORF81 And ORF83

Nucleopolyhedrovirus (NPV) is belonged to the family Baculoviridae that infect invertebrates, particularly insects of the order Lepidoptera, and is a natural control agent of insect pests in agriculture and forestry. Nucleopolyhedroviruses are used widely as bio-insecticides to control heliothine insects in cotton and vegetable crops around the world. Two isolates of Helicoverpa armigera single nucleocapsid nucleopolyhedroviriis (HearSNPV), C1 and G4, were cloned and sequenced. Some of the genes of HearSNPV have been determined and most of them remain unknown for their functions. The functions of HearSNPV ORF33, ORF80, ORF81 and ORF83 genes and their homologues have not been reported thus far. We analyzed these four genes of HearSNPV C1 by the means of cloning and expression, preparation of polyclonal antibodies, localization of gene products, time course of gene products in HearSNPV infected host cells and transcriptional analysis. The results were reported as following:1. Analysis results of ORF33ORF33 is 717 nt long and located at 27566bp~28282bp in the HearSNPV genome, encoding a predicted protein of 238 aa with a predicted size of 28.4 kDa. A baculovirus consensus early promoter motif AACAGT and a TATA box are found at 139 and 127 nt upstream of the start codon (ATG), respectively. Ten putative phosphorylation sites are found in the putative ORF33 protein. The ORF33 homologues are identified in 21 completely sequenced lepidopteran NPVs and GVs. Its amino acid sequence shares 100% identity with HzSNPV and 44-55% identities with other 13 NPVs while shares 31-35% identities with seven GVs。 One conserved domain (residues 54-77) related to the family Nudix is detected in ORF33, which is characterized with the pattern of GX5EX7REUXEEXGU. The Nudix domain is conserved in 14 NPVs and seven GVs.Northern blot shows single transcript of approximate 0.7 kb was detected at 3 h p.i. and remained detectable until 48 h p.i., suggesting that ORF33 is transcriptionally early in infection. This is consistent with the baculovirus consensus early promoter motif AACAGT.An ORF33-pGEX-4T2 plasmid was constructed and ORF33 was expressed in the E. coli (BL21). The expressed fusion protein, ORF33-GST, has a less molecular weight than the predicted. It is possibly caused by the minor codons in the frame of ORF33 for E. coli. However, the quantity of expressed fusion protein is as high as 10.1% of total bacterial protein. The expressed protein was purified and used as an immunogen to raise ORF33-specific antiserum in male rabbits. Besides, the ORF33 was expressed in Tn cells by the Bac to Bac system. The expressed fusion protein has a molecular weight as the same of predicted size. The confocal laser scanning microscope detection demonstrated that the fusion protein, ORF33-EGFP was localized in the nuclei of Tn cells.A time course of HearSNPV-infected HzAMl cells were analyzed by western blot using the polyclonal antibody against ORF33. A specific immunoreactive band of approximately 31 kDa was first observed at 6 h p.i. and remained detectable up to 48 h p.i.. This was in tune with the analysis of transcription. The results of transcription and expression suggested that the ha33 protein was synthesized early after infection. The 31 kDa protein of ORF33 was detected in the envelope BVs, indicating that ORF33 encodes a BV-specific structural protein, without association with ODV. We noticed that ORF33 protein detected in BVs or infected cells were larger than the predicted molecular weight (28.4 kDa). The actual size of ORF33 protein suggested that ORF33 might be subjected to eukaryotic post-translational modifications such as phosphorylation. In addition, ORF33 gene product was localized in the cytoplasm of infected HzAM1 cells, detected by the confocal laser scanning microscope using polyclonal antibody and protein G fused with enhanced green fluorescent protein (EGFP).2. Analysis results of ORF80ORF80 is 765 nt long and located at 2915bp ~73697bp in the HearSNPV genome, encoding a predicted protein of 254 aa with a predicted size of 30.8 k...