The Protein-protein Interaction Ofhost Cellular ESCRT-Ⅲ/Vps4 And AcMNPV BV-associated Proteins

In eukaryotic cells,the endosomal sorting complex required for transport(ESCRT)is composed of ESCRT-0,-I,-II,-Ⅲ,and Vps4(vacuolar protein sorting protein 4)complexes and the accessory factors,including Alix.ESCRT is a membrane “scissor”,which is required for multivesicular bodies formation,autophagy,cytokinesis,plasma membrane wound repair,and nuclear membrane remodeling process.In addition,host cellular ESCRT is necessary for many enveloped viruses budding.Baculoviruses are a group of large,double-strand DNA,enveloped viruses.In the infection cycle,baculoviruses usually produce two types of virions: budded virions(BV)and occlusion-derived virions(ODV).Recent studies demonstrated that overexpression of the dominant-negative forms of cellular ESCRT-Ⅲ components or Vps4 significantly reduced the infectious Autographa californica multiple nucleopolyhedrovirus(AcMNPV)BV production and inhibited the progeny nueclocapsid egress from nuclear membrane.However,the detailed mechanism related with the roles of cellular ESCRT in AcMNPV infection is largely unknown.In this study,we constructed the transient expression vectors for Spodoptera frugiperda Vps4 and ESCRT-Ⅲ components Vps2 B,Vps20,Vps24,Snf7,Vps46,and Vps60,and seven conserved(core)genes of AcMNPV(ac11,ac76,ac78,ac80(gp41),ac103,ac142,and ac146).In transfected Sf9 cells,bimolecular fluorescence complementation(BiFC)assay showed that the components of ESCRT-Ⅲ and Vps4 interacted with each other.Co-immunoprecipitation analysis combined with BiFC assay revealed that the 7 AcMNPV BV-associated proteins,in a different range,interacted with the components of ESCRT-Ⅲ and Vps4.Among these interactions,Ac76,Ac78,and Ac146 interacted with almost all of the components of ESCRT-Ⅲ(5-6 components),whereas Ac103 and Ac142 only interacted with few subunits of ESCRT-Ⅲ.Additionally,Ac103 and GP41 interacted with Vps4 and its two mutants that abolished the ATPase activity.We propose that the complicated protein-protein interaction network between BV-associated proteins and cellular ESCRT-Ⅲ/Vps4 might form an egress complex required for the budding process of AcMNPV budded virions.