Interaction Of The Chitin-binding Domain-containing Proteins Of The Baculovirus AcMNPV And Their Knockout Effects On Virus Infection

Baculoviruses are large,double-stranded circular DNA enveloped viruses.In the life cycle,they usually produce two distinct types of virions: budded virions(BV)and occlusion-derived virions(ODV).In the environment,when the larvae ingests the occlusion bodies(OB)which contians ODV virions contaminated food,OB enter into the intestine of insects and they will be lysised under the alkaline condition of the midgut and release ODV.Then,ODV virions penetrate through the peritrophic matrix(PM)of midgut and attach to the epithelial cells.After replication in epithelial cells,the progeny BV virions are released into the hemolymph and trachel system of infected insects and spread to infect other tissues.During this infection process,how ODV virions passage through the PM is a critical step for initiating the viral infection.Earlier studies revealed that,Autographa californica multiple nucleopolyhedrovirus(AcMNPV),a type species of baculoviruses,encodes three chitin-binding domain containing protein(CBDP),including Ac64/GP37,Ac145,and Ac150.Single deletion of these CBDP genes had no significant effect on infectious BV production.However,double knockout ac145 and ac150 dramatically reduced the oral infectivity of AcMNPV.In addition,these three viral proteins were found to be associated with BV and ODV,and GP37 and Ac145 also have chitin-biding activities.Based on these prior studies,we hypothized that these CBDP proteins may form a complex to regulate BV and ODV infection.By using yeast two-hybrid(Y2H)and bimolecular fluorescent complementation(BiFC),multiple gene knockout and transfection-infection assays,we assessed the interaction of the three viral proteins and determined the effect of double or triple knockout of these genes on infectious BV production.The main results are listed as following:1.Y2H screen and BiFC in living Sf9 cells indicated that GP37,Ac145,and Ac150 associated with themselves,and they also interacted with each other,suggesting that they can form a protein complex.2.By using multiple antibiotics as the screen marker,we constructed double or triple knockout AcMNPV bacmids,that including double gene knockout bacmids gp37/145 Ko,gp37/150 Ko and Ac145/150 Ko,and a triple knockout bacmid gp37/145/150 Ko.3.Double knockout of the three CBDP genes,such as gp37+Ac145,gp37+Ac150 or Ac145+Ac150 had no significant effect on infectious BV production.However,together knockout of gp37,Ac145,and Ac150 significantly reduced the production of infectious AcMNPV BV.In conclusion,we found the three CBDP proteins of AcMNPV can self-associate and form a protein complex.The negative effect of knockout of these genes together on infectious BV production suggest that they may play an important role in BV infection.The impact of deletion of these viral genes on oral infection of AcMNPV is still under investigating.