Deletion and functional analysis of ac146 of the baculovirus Autographa californica nucleopolyhedrovirus

• Main Author: Dickison, Virginia L
• Language: English
• Published: University of British Columbia 2010
• Online Access: http://hdl.handle.net/2429/20123

Autographa californica Multiple Nucleopolyhedrovirus (AcMNPV) ac146 is a highly conserved gene in the alpha- and betabaculovirus genera that has an unknown function. To characterize ac146 Northern blot analysis and transcriptional mapping confirmed the prediction that ac146 is transcribed at late times post infection as a 1.2 Kb mRNA that is detected from 12 hpi through to 48 hpi. To determine the role of ac146 in the baculovirus life cycle, AcMNPV bacmids were used to generate a series of four increasingly larger ac146 deletion viruses (AcBACac146KO¹⁻⁴) by recombination in Escherichia coli. Transfection and plaque assays were completed to visualize the movement of ac146 deletion and repair viruses under fluorescence and light microscopy. The results showed that all the ac146 deletions produced a single cell phenotype indicating that no infectious budded virus (BV) was produced. Lack of BV production was confirmed by titrating the virus utilizing both qPCR and TCID₅₀. Within cells transfected by AcBACac146KO¹⁻⁴ the infection preceded to late times post-infection as evidenced by the development of apparently normal occlusion bodies (OB). AC146 was detected at 18 hpi through to 96 hpi and located in both nuclear and cytoplasmic fractions at 24 and 48 hpi. Purification of BV and occlusion derived virus (ODV) revealed that AC146 is associated with both forms of the virus. AC146 was located in the nucleocapsid fractionation of BV but not in the envelope fraction. Therefore in conclusion, this study has shown that ac146 is a late gene that is essential for the virus life cycle and is required for the production of BV.