The Interaction Between Autographa Californica Multiple Nucleopolyhedrovirus GP41and Two Host Proteins

Autographa californica multiple nucleopolyhedrovirus (AcMNPV), a representative species of baculovirus, is a double-stranded, circular DNA virus. gp41is one of the baculovirus core genes, and its homologues present in all the baculovirus genomes sequenced to data. GP41is located between the envelope and nucleocapsid. It has been reported that GP41was required for the egress of BV nucleocapsids from the nucleus, but its specific mechanism is unclear yet. In this study, we attempt to explore its function by investigating interactions between GP41and host proteins.At first, we inserted the gp41gene from AcMNPV genome into the plasmid pGBKT7to build bait plasmid pGBKT7-gp41, which was used as a bait to screen a cDNA library of Sf9cells by yeast two-hybrid assays, and identified three cDNA clones encoding proteins that interact with AcMNPV GP41. Sequence analysis showed that two of the clones encode a protein homologuous to9’, and another one encodes a homologue of12.To verify the interaction between GP41and9’/12, we constructed recombinant viruses, which respectively expressed GST-GP41and HIS-9’or12fusion proteins in infected Sf9cells, by using a BAC-to-BAC system. Result from GST-pull down experiments showed that the HIS-9’was co-purified with GST-GP41by GST-bind resin, confirming the interaction between the AcMNPV GP41and the9’protein of Sf9cells.Two recombinant viruses separately expressing GP41-EGFP and9’-RFP or GP41-EGFP and12-RFP fusion proteins were constructed and used to infect Sf9cells, and the GP41-EGFP,9’-RFP and12-RFP expressd in the infected cells were viewed by fluorescence microscope. It was shown that GP41-EGFP was distributed throughout the cells at18hpi; the fusion proteins gradually gathered to the cell nucleus at24hpi and36hpi; they mostly gathered in the nucleus at48hpi.9’-RFP located in the cytoplasm at each time point, and12-RFP located around the nuclear membrane. When GP41-EGFP and12-RFP were expressed simultaneously in Sf9cells, GP41-EGFP still partialy located around the nuclear membrane at48hpi. GP41-EGFP and12-RFP obviously co-localized around the nuclear membrane at that time point. According to the results, the interaction between GP41and12may affect trafficking of GP41into the nucleus.In order to identify the interaction sites between the GP41and9’/12, we constructed16truncated gp41mutants and cloned the truncated mutants into the plasmid pGBKT7to construct bait plasmids which express the truncated GP41. The interactions between GP41truncated mutants and9’/12were retested in yeast. The identified interaction site between GP41and9’ is in the region of161-210AA. And the interaction site between GP41and12is81-330AA.AcMNPV bacmid bMON14272was used to generate the gp41knockout bacmid by homologous recombination in Escherichia coli using the λ-Red system. A500bp sequence of gp41was replaced by chloramphenicol resistance gene (cat).gp41knocked out (vAcgp41KO-PH-Pp10-egfp)) and wild type (vAcPH-Pp10-egfp) viruses contained polh and egfp genes were constructed through BAC-to-BAC system. After transfection-infection assays, we found that gp41was essential for BV production. Knockout of gp41eleminated production of infectious BV, whicle formation of occlusion bodies was not affected.