Roles Of Host NSF In Entry And Egress Of Budded Virions Of Autographa Californica Multiple Nucleopolyhedrovirus

In eukaryotic cells,the soluble N-ethylmaleimide-sensitive factor attachment protein receptor(SNARE)proteins consist of the minimal machinery that triggers the fusion of transport vesicles with the target membranes.Compared genome analysis revealed that the components of SNARE system are highly conserved in yeast,insects,and human.Recently,Transcriptomics showed most of the SNARE proteins were up-regulated after AcMNPV infection.We found that cellular NSF(N-ethylmaleimide-sensitive factor),the key ATPase for disassembly and recycling of the SNARE complex,is associated with budded virions(BVs)of AcMNPV.To determine whether NSF is required for the replication of AcMNPV,the cDNA of full-length NSF from the host insect,Spodoptera frugiperda,was cloned as wild-type(WT)and dominant negative(DN)forms.We found that overexpression of DN forms of NSF or knock-down of the expression of NSF,significantly affected the infectious AcMNPV production.In cells expressing DN NSF,entering virions were trapped in the cytoplasm or transported to the nucleus with low efficiency.Next,recombinant bacmids(viral genomes)expressing WT or DN NSF proteins were used to examine virus egress.We found that the production of infectious AcMNPV BV was inhibited after the expression of DN NSF.Even though the presence of DN NSF substantially reduced the cell surface levels of the major viral envelope glycoprotein GP64,we found that the reduced GP64 levels did not account for the lack of infectious virus production in DN NSF expressing cells.Transmission electron microscopy(TEM)analysis revealed that the progeny nucleocapsids were retained in the perinuclear space and could not be released from nuclear membrane in host cells expressing DN NSF proteins.Several baculovirus core proteins,including Ac76,Ac78,GP41,Ac93,and Ac103,which required for infectious budded virions production,were detected to interact with NSF,and NSF was found to be assembled into budded virions.Together,our findings demonstrate that cellular SNARE system is involved in AcMNPV infection and NSF is required for efficient entry and nuclear egress of budded virions of AcMNPV.