Roles Of Host ESCRT-Ⅲ Components CHMP7,IST1 And Vps2A In Infection Of Budded Virions Of AcMNPV

In eukaryotic cells,the endosomal sorting complex required for transport(ESCRT)machinery includes five biochemically distinct protein complexes designated as ESCRT-0,ESCRT-I,ESCRT-II,ESCRT-III and Vps4.Mammals have a total of twelve ESCRT-III proteins named as CHMPs(charged MVB proteins)which can be divided into eight subfamilies(CHMP1-CHMP7 and IST1)in accordance with the eight yeast proteins(Did2,Vps2,Vps24,Snf7,Vps60,Vps20,Chm7 and IST1).Both CHMP1 and CHMP2 have two isoforms,however,CHMP4 has three isoforms.The ESCRT machinery is required for formation of multivesicular bodies(MVBs),cytokenetic abscission,plasma membrane repair,nuclear envelope reassembly,as well as enveloped virus entry and/or egress budding.Baculoviruses are a large family of enveloped double-strand DNA viruses.Autographa californica multiple nucleopolyhedrovirus(AcMNPV)is a type species of the virus family Baculovirdae.Recent studies revealed that overexpression of GFP-tagged cellular ESCRT-III subunits Snf7,Vps2 B,Vps20,Vps24,Vps46,Vps60 and Vps4 dominant-negative(DN)constructs or knockdown the expression of these cellular genes using dsRNA-based RNAi significantly reduced the entry of AcMNPV into host cells and also decreased the production of infectious AcMNPV.However,the roles of other ESCRT-III components including CHMP7,IST1,and Vps2 A in AcMNPV infection are not clear.In this study,we initially cloned CHMP7,IST1 and Vps2 A genes from Spodoptera frugiperda cells Sf9.Quantitavie RT-PCR analysis showed that the transcription level of CHMP7,IST1 and Vps2 A were significantly up-regulated at the early stage(0-12 h)of AcMNPV infection,implying that these components might be involved in AcMNPV infection.By contructing GFP-tagged CHMP7,IST1 and Vps2 A transient expression plasmids,we found that transient overexpression of these GFP-tagged cellular proteins significantly reduced the production of infectious budded viruses.Futher analysis showed that overexpression of GFP-tagged CHMP7,IST1 or Vps2 A significantly decreased the expression of two reporter genes LacZ and GUS,which were separately controlled by theAcMNPV immedietaly early gene ie1 promoter and the late gene p6.9 promoter,suggesting that these cellular proteins might be required for efficient entry of budded virions of AcMNPV into Sf9 cells.To avoid the poteintial effect of overexpression of these cellular proteins on entry of AcMNPV,we transfected Sf9 cells with recombinant AcMNPV bacmids that expressing GFP-tagged CHMP7,IST1 or Vps2 A.We found that the infectious virus release was significantly decreased.Addtionally,using a mCherry-based bimolecular fluorescence complementation assay,we found that CHMP7,IST1 and Vps2 A interact with several core(or conserved)baculovirus proteins(Ac11,Ac76,Ac78,Ac80(GP41),Ac93,Ac103,Ac142 and Ac146).We proposed that CHMP7,IST1 and Vps2 A coordinate with other ESCRT-III components and viral proteins to regulate the efficient entry and egress of budded virions of AcMNPV in insect cells.