Functional Study Of Autographa Californica Multiple Nucleopolyhedrovirus Orf132

Baculoviruses are arthropod-specific double-stranded DNA viruses. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the representative species of baculoviruses, also the most studied one. It would appear that there are about150orfs in the AcMNPV genome. Ac132is the the132th ORF of AcMNPV genome. The Ac132protein was identified as being associated with AcMNPV virion from proteomics analysis, however, its function is unknown.In this study, we try to study the functions of Ac132. The main results are showing bellows:Ⅰ. Ac132was expressed in E.coli and purified; and Ac132-specific polyclonal antibodies were prepared.Ⅱ. Time course analysis of Ac132in infected Sf9cells. The Sf9cells infected by AcMNPV were collected at0h、6h、12h、24h and36h、48h、72h、96h post infection. And the Ac132was detected by western blot with Ac132-specific antibodies. The results showed that Ac132first detected at12h, and rechead peak level at72h post infection, showing typical characteristics of late gene expression.Ⅲ. Ac132was shown to be structural protein, and localized on the nucleocapsid of BV.Ⅳ. Construction of Ac132-null and repaired viruses. AcMNPV bacmid bMON14272was used to generate the Ac132knockout bacmid by homologous recombination in Escherichia coli using the λ Red system. A390bp sequence of Ac132was replaced by Chloramphenicol acetyltransferase gene (cat). We constructed Ac132knockout bacmid vAcac132ko successfully. Ac132-null (vAcac132ko-PH-gfp), repaired (vAcac132ko-rep-PH-gfp) and wild type AcMNPV (vAcPH-gfp1) viruses with polh gene and gfp gene were constructed through Bac-to-Bac system.V. Transfected-infection assays on Ac132-null and repaired viruses in Sf9cells. Bacmids of vAcac132ko-PH-gfP、vAcac132ko-reP-PH-gfp、vAcPH-gfp1were transfected into Sf9cells. Transfection supernatants were used to infect fresh Sf9cells, TCID50were used to determine the BV titres, virus growth-curve analysis was performed. Experimental results show that the number of infectious BV increased significantly after transfection with vAcac132ko-rep-PH-gfp) and vAcPH-gfp1; BV production was almost equivalent. But in vAcac132ko-PH-gfp_transfected cellS, no infectious By was detected from cells.Ⅵ. Effects of Ac132knockout on virus morphogenesis. Bacmids of vAcac132ko-PH-gfp、vAcac132ko-rep-PH-gfp were transfected into Sf9cells, electron microscopic analysis was shown that nucleocapsid and envelope with a normal appearance were found in vAcac132ko-PH-gfp and vAcac132ko-rep-PH-gfp bacmid-transfected cells. The nucleocapsids or viral particles arranged in clusters were found in vAcac132ko-rep-PH-gfp bacmid-transfected cells, while in vAcac132ko-PH-gfp bacmid-transfected cells, only a single virus particle was found.Ⅶ. Screening of the proteins interacting with Ac132. A bait vector pGBKT7-Ac132was used as a bait plasmid to screen the cDNA library above by Yeast two-hybrid assays, to identify cDNA clones encoding proteins interacting with the Ac132. As a result, lots of positive clones were identified. After the genes were classified and sequenced, they could be confirmed lots of gene, respectively encoding40S ribosomal protein receptor for activated protein kinase C、leukotriene A4hydrolase、putative Spectrin alpha chain、 hypothetical protein、putative kinetochore protein、NDC80-like protein、EF-1. This indicates that there are lots of protein-protein interactions between Ac132and Sf9cells.