| In eukaryotic cells,the endosomal sorting complex required for transport(ESCRT)machinery catalyzes membrane scission and is required for multivesicular bodies formation,cytokinesis,plasma membrane repair,nuclear membrane remodeling and enveloped viruses entry and budding and release.As known,ESCRT is composed of five complexes,including ESCRT-0-III,and Vps4,and some accessory proteins.Among these components,ESCRT-0,-I,-II are responsible for cargo sorting and vesicles formation,while ESCRT-III serves as the “scissor” to cut and release vesicles from target membrane.Late in the ESCRT pathway,Vps4 hydrolysis ATP and disassemble the ESCRT-III complexBaculoviruses are a group of large,envelopedviruses with double-stranded genomic DNA.In a typical infection cycle,these viruses usually produce two distinct types of virions: budded virions(BV)and occlusion-derived virions(ODV).Recent studies showed that overexpression of dominant-negative(DN)forms of cellular Vps4 significantly reduced the efficiency of entry and egress of infectious BV of Autographa californica multiple nucleopolyhedrovirus(AcMNPV).However,roles of cellular ESCRT in AcMNPV infection is largely unknown.In this study,we isolated the key components of ESCRT-III,including Vps2 B,Vps20,Vps24,Snf7,Vps46,and Vps60 from Spodoptera frugiperda Sf9 cells.Sequence alignment indicates that the orthologs of these ESCRT-III components are widely distributed in sequenced insect genomes.Confocal microscopy analysis revealed that,in transient transfected Sf9 cells,the GFP-tagged ESCRT-III components were mainly colocalized with mCherry-tagged Vps4 mutated construct E231 Q,suggesting that the GFP-fused ESCRT-III proteins may be localized in the endosomal compartment.By using a viral complementary assay(in which the replication of gp64-knockout AcMNPV is rescued by the transiently expressed GP64),we found that overexpression of GPF-tagged ESCRT-III proteins(serve as the DN constructs)significantly reduced the internalization of BV virions into Sf9 cells.Also,the presence of DN ESCRT-III proteins resulted in the entried virions mainly localizing in cytoplasm and less efficiently transporting to the nucleus for replication.Further analysis of the viral gene expression by using ?-galactosidase and ?-glucuronidase as marker genes and viral genomic DNA accumulation,we found that expression of DN ESCRT-III proteins inhibited the viral replication at an early stage of nfection,suggesting that ESCRT-III is required for efficient entry of AcMNPV.To avoid the negative effect of DN ESCRT-III proteins on virus entry,we constructed recombinant AcMNPV bacmids that contained GFP-fused ESCRT-III components,which were controlled by the AcMNPV ie1 promoter,and transfected Sf9 cells.At 24 h post-transfection(p.t.),the supernants were collected and the infectious AcMNPV production was measured.The results showed that expression of GFP-tagged ESCRT-III proteins significantly reduced infectious AcMNPV production.Transmission electronic microscopy analysis indicated that expression of DN ESCRT-III proteins Vps24 and Snf7,or Vps4 significantly inhibited the egress of progeny nucleocapsid from the nuclear membrane.Collectively,these data suggest that ESCRT-III is involved in efficient egress of BV virions of AcMNPV.Since the efficient egress of BV virions of AcMNPV is also dependent on some viral proteins,such as Ac93(encoded by ORF93 of AcMNPV),we selected Ac93 to analyze the potential interaction of it with ESCRT-III subunits and Vps4.By using co-immunoprecipitation(Co-IP)and bimolecular fluorescence complementation assay(BiFC),we found that Ac93 interacts widely with the ESCRT-III components and Vps4.The association of Ac93 with Vps4 mutated constructs K176 Q and E231 Q suggested that the interaction was not dependent on the ATPase activity of Vps4.Further analysis revealed that Ac93 also interacted with two other viral core proteins Ac76 and Ac103,which both were required for progeny nucleocapsids egress from the nuclear membrane.These data suggest that Ac93 might interact with Ac76,Ac103,and cellular ESCRT-III proteins and Vps4 to form a complex to regulate the egress of BV virions in AcMNPV-infected cells.To further identification of the interaction mechanism of Ac93 and ESCRT-III/Vps4,we initially analyzed the amino acid sequences of Ac93 and its othologs from sequenced baculovirus genomes.By comparison of the sequences of Ac93 proteins and ESCRT-III components,we identified a conserved leucine-rich motif at the C-terminus of Ac93 that might be involved in the interaction of Ac93 with ESCRT-III/Vps4.Using site-directed mutagenesis,we made a series of Ac93 mutated constructs in which the 6 conserved leucine residues were singly or multiply replaced with an alanine residue.Transient expression analysis showed that alanine substitution of leucine residues in Ac93 had no effect on the expression of the mutated proteins.Co-IP and BiFC analysis revealed that single or multiple mutation of leucine resides in Ac93 resulted in a reduction of the interaction of Ac93 and ESCRT-III/Vps4,and viral proteins Ac76 and Ac103.In addition,mutations of these leucine residues resulted in dramatic reduction of infectious BV production of AcMNPV,less efficient egress of nucleocapsids from nuclear membrane,and the defect of intranuclear microvesicles formation.Vps4Additionally,prior studies demonstrated that the efficient infection of AcMNPV is dependent on the activity of cellular fatty acid synthesis enzyme(FAS).To futher analyze the roles of mitochondrion in AcMNPV infection,we obtained the mitochondrion genome sequences of S.frugipera and Trichoplusia ni and analyzed the phylogenetic relationship of them with that from other insect species.In conclusion,our studies demonstrated that ESCRT-III/Vps4 were required for efficient entry and egress of BV virions of AcMNPV.The interaction of Ac93 with ESCRT-III/Vps4,and viral proteins Ac76 and Ac103 might form an “budding complex” to mediate efficient budding and release of progeny nucleocapsids of BV virions and ODV assembly.Vps4... |
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