Functional Study Of Orf146 Of Autosrapha Californica Multiple Nucleopolyhedroviruses

Autographa californica multiple nuclecopolyhedrovirus(AcMNPV)belongs to the genus Alphabaculovirus of baculovirus and is the most in-depth baculovirus currently studied.As an important model species for molecular studies of baculovirus and common viruses,the mechanism of capsid assembly and virus particle formation of AcMNPV is still unknown.Earlier researches demonstrated that lack of ac146 gene in AcMNPV may result in the formation of empty polycapsids without nucleocapsids in the cells,and the formation of viral particles may be blocked.In this study,experiments were carried out for ac146 genes to find what might affect the formation of AcMNPV virus particles:Firstly,in order to explore the effect of ac146 on the formation of virus particles,the vAcPH-GFP(wild-type virus),vAc146KO-PH-GFP(the deletion recombinant virus)and vAc146Rep-PH-GFP(Recovery type recombinant virus)were transfected into Sf9 cells.Compared to the retroviruses vAc146Rep-PH-GFP and wild-type vAcPH-GFP,the Sf9 cells transfected with vAc146KO-PH-GFP showed no fluorescence diffusion at 96 h after transfection,suggesting that the normal BV virus particles could not be produced after knock out the ac146,resulting in the inability of the virus to spread between cells.The cell supernatants of the vAcPH-GFP,vAc146KO-PH-GFP,vAc146Rep-PH-GFP transfected for 120 hours were infected with Sf9 cells respectively,and the fluorescence in the cells was observed.The results showed that the cells that had been infected with the supernatants of the cells transfected with the reverting recombinant virus and the wild type virus all exhibited fluorescencet.It was demonstrated that normal infectious BV particles were produced in the supernatants of both transfected cells,but no fluorescence was observed in the cells infected with the supernatants of the cells transfected with the deletion recombinant virus,further demonstrating that the infectious BV particles could not be formed after the deletion of ac146.Secondly,based on Virginia L,etc.detected by Western-blot Ac146 protein has two molecular forms: 23 k Da and 34 k Da,but did not pass the mass spectrometry and other technologies to prove.This study combined with Western-blot and mass spectrometry to further explore the existence of Ac146 protein.Sf9 cells were transfected with vAc146Rep-PH-GFP,and the immunohistochemical detection of Ac146 protein was carried out using the HA-tag fused to the end of ac146 gene.Two protein mixtures were extracted for immunoblot detection of the desired protein,one was post-infection whole cell protein,and the other was purified ODV whole protein and was identified by mass spectrometry.The results of mass spectrometry identification showed that Ac146 protein was detected in the 23 k Da band of the whole cell protein sample,but Ac146 protein was not detected in the purified ODV total protein.This result suggests that the Ac146 protein may only have a molecular form of 23 k Da.Finally,due to virus particles do not form properly because of the lack of ac164,yeast two-hybrid technology was used to identify protein members that interacted with Ac146 protein in order to reveal the structural role of Ac146 protein on the viral nucleocapsid.Construction of yeast recombination bait vector with ac146 and yeast recombination capture vector with 14 capsid proteins involved in virus particle formation(ac66,ac69,ac77,ac80,ac89,ac92,ac98,ac100,ac101,ac103,ac104,ac141,ac142,ac146).Through auxotrophy screening,X-?-gal coloration and PCR validation,the final results showed that two proteins can interact with Ac146 protein(Ac103 protein and Ac141 protein).