| Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the typical member of the NPVs, is the first sequenced baculovirus. Two viral forms, budded virus (BV) and occlusion-derived virus(ODV), are produced during the viral infection. BVs and ODVs are identical in genetic information, but the composition of their envelopes is different to accommodate their respective functions in an infection cycle. Odv-e25is one of ODV envelop protein, and it is also one of the37core genes of baculovirus, however its function is still unknown. The object of the present study is to investigate the role of odv-e25in Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infection cycle. The results were presented as the followings:1. Expression and location analysis of odv-e25Homologs of odv-e25are found in all baculovirus genomes, indicating that odv-e25plays an important role in baculovirus infection cycle. The5’RACE analysis indicated that the odv-e25mRNA initiated at the fourth A of a late promoter ATAAG elements located at positions-217upstream of the ATG, indicating that odv-e25is an late gene. The results were further proved by reverse transcription PCR and Western blotting. Immunofluorescence microscopy analysis revealed that ODV-E25was predominantly localized in the intranuclear ring zone, agreed with the results that ODV-E25plays a role in intranuclear microvesicle formation. Western blotting analyses showed that ODV-E25was associated with fractions of both BV and ODV.2. Function analysis of AcMNPV odv-e25To investigate the role of odv-e25in the baculovirus life cycle, an odv-e25knockout AcMNPV was constructed via homologous recombination in Escherichia coli. Fluorescence and light microscopy showed that the odv-e25mutant had defects in virus infection, and titration assays confirmed a defect in budded virus (BV) production. However, odv-e25deletion did not affect viral DNA replication. Electron microscopy showed that nucleocapsids were observed in cells transfected with the odv-e25-knockout virus. Virus-induced intranuclear microvesicles as well as occlusion-derived virions (ODVs) were also not detected in the transfected cells.3. The formation of ODV was affected by the expression level of ODV-E25.The viral genes are systematically expressed in its infection cycle and abnoral expression of the genes usually affected the phenotype of the virus. It is unclear that whether the expression level of ODV-E25affected the viral infection process, odv-e25was expressed under three different promoters, separately, and three constructs were generated. The three promoters were immediately eary-1(ie-1) promoter, odv-e25native promoter, and the polyhedrin promoter. Western blotting analysis showed that the production of ODV-E25under its native promoter was lower than that under polyhedrin promoter but higher than that under ie-1promoter. Viral replication, budded virus production, and viral titer assays demonstrated that the defects caused by the deletion of odv-e25were not rescued by the expression of odv-e25under ie-1promoter or polyhedrin promoter. Electron microscopy revealed that intranuclear microvesicles were detected when odv-e25expressed under ie-1promoter, but ODVs were never observed. However, both intranuclear microvesicles and ODVs were detected when ODV-E25was expressed under polyhedrin or its native promoter, and ODVs were occluded into polyhedra. Western blotting analysis revealed that ODV-E25in BV and ODV could be improved by increasing its expression level. |
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