Molecular Characteristics And Functional Analysis Of Bombvx Mori Nucleopolyhedrovirus F-like Protein

One hallmark of the baculovirus infection cycle is the production of two morphologically and functionally distinct progeny phenotypes:the budded virion(BV)and the occlusion-derived virion(ODV).One of major differences between BV and ODV is the origin of the envelopes.BV appears to have two distinct envelope proteins,F protein and GP64.However,F protein homologs(F-like proteins)in Group I NPVs lack membrane fusion activity and are still retained via other important biological functions during the vial life cycle.BmNPV is a significant pathogen of Bombyx mori,severely impairing economic benefits of sericulture industry.In addition,F-like protein(Bm14)of BmNPV,one of representative Group I NPVs,is still understudied.In this study,a series of functional analyses were conducted to characterize molecular features and biological roles of Bm14.The main conclusions are summarized as follows.1.Characterization of Bm14 and phylogenetic diversity among its homologsBmNPV bml4 gene is located from 13586 nt to 15607 nt of the genome.A typical baculovirus early promoter motif CAGT and two typical late promoter motifs TAAG was found to locate in the upstream of its translation initiation codon ATG,suggesting that bm14 might be an early/late gene.Analysis of functional domains revealed that Bm14 lacks conserved domains associated with membrane fusion and thus can not directly mediate virus-to-cell fusion.Further phylogenetic analysis indicated that Bm14 homologs are ubiquitous in all sequenced alpha-and beta-baculovirus,with significant differences in amino acid similarities and identities among different branches.2.Expression patterns,subcellular and virion localization of Bm14Utilizing the recombinant viruses,bml4 transcript was firstly detected at 6 h p.i.and its specific protein from 24 h p.i.to 96 h p.i.in infected cells.These results indicated that bml4 is an early/late gene which mainly expresses at late and very late stages of viral infection.Immunofluorescence analysis showed that the subcellular localization of Bml4 experienced a dynamic distribution from the nuclear membrane to cytoplasmic membrane throughout the viral infection.Moreover,Bm14 was associated with both BV and ODV and specifically localized to the envelope fractions.3.Effect of bm14 knockout on progeny virus formation,OB morphogenesis and productionDeletion of bm14 resulted in aberrant budding of progeny nucleocapsids,which were trapped in the perinuclear space and could not be released from the outer nuclear membrane into cytoplasm,thus reducing the production rate of infectious BV.In addition,occlusion bodies showed lower levels during the infection with bm14-null mutant.Further analysis revealed that the interaction of Bm14 with polyhedra membrane protein PE regulated the morphogenesis of OBs and its null resulted in rough,pitted a more aberrant OBs with less infectious ODVs.4.Effect of bml4 null on oral infection and efficient spread in host larvaeBm14 exhibited a constant expression in the midgut during BmNPV infection via Western blot analysis,suggesting the role in regulating the primary infection in host midgut.A series of bioassays indicated that bm14 knockout increased survival time and lethal dose of infected larvae,which might result from the weakened binding and fusion of ODV with midgut epithelial cells via the interactions with PIFs.Meanwhile,the disruption of bm14 not only reduced infectious BV production in the hemolymph but also delayed the efficient spread in larval tissues including fat body,trachea and midgut.Further analysis indicated that Bm 14 participated in regulating pH-dependent virus-to-cell fusion via interacting with the Domain I of GP64,thus affecting efficient propagation in BmNPV-infected larvae.5.Identification of viral and host partners interacting with Bm14To identify viral and host partners interacting with Bm14,yeast two-hybrid and coimmunoprecipitation assays were performed.Ten candidate proteins,including Bm-calreticulin,Bm-E3 ubiquitin-protein ligase SINA-like 10,Bm-BAG family molecular chaperone regulator 2,Bm-phosphoribosylaminoimidazole carboxylase,38K,EXONO,VP39,VP80,VP1054 and GP41,were identified using the cytoplasmic tail domain(CTD)of Bm14 as the bait.Further analysis revealed that the 603-673 aa of CTD within Bm14 was a key region mediating the above interactions.