Functional Analysis Of BmNPV ORF40,ORF133/134 And ORF92a

The family Baculoviridae are a large group of insect-specific enveloped viruses with circular,supercoiled,double-stranded DNA genomes.Baculoviruses have been isolated from more than 600 host insect species mainly from the orders Lepidoptera,Hymenoptera and Diptera.Baculoviruses have a typical biphasic infection cycle,and produce two types of progeny virions:budded virus(BV)and occlusion-derived virus(ODV).These two types of virions have identical genetic information,but their virion structures and functions are divergent.Bombyx mori nucleopolyhedrovirus(BmNPV)specifically infects the domestic silkworm(Bombyx mori),and is one of the best-characterized baculoviruses.In this study,we investigated four ODV-associated proteins of BmNPV(ORF40,ORF133/134 and ORF92a)with unknown functions.By use of gene knockout BmNPV mutants,we characterized their different roles in the ODV development process.The main results are summarized as follows.1.Functional analysis of BmNPV ORF40ORF40(bm40)is located at 38,254-39,213 nt in the genome of BmNPV and is a highly conserved gene in sequenced alphabaculovirus genomes.bm40 open reading frame(ORF)contains 960 bp and encodes a putative protein of 319 amino acid residues with a predicted molecular weight of 37.8 kDa.Bioinformatics analysis showed that the Bm40 sequence contains a DnaJ domain at the N terminus and an RNA recognition motif(RRM)at the C terminus.RT-PCR and western blotting results indicated that bm40 is a late gene.To investigate the role of bm40 during virus infection,a bm40-knockout BmNPV bacmid was constructed via ? Red homologous recombination in Escherichia coli.The results showed that the knockout virus exhibited a single-cell infection phenotype,and could not produce infectious BVs,while viral DNA replication was not affected.Electron microscopy results revealed that Bm40 is required for nucleocapsid egress from the nucleus to the cytoplasm,nucleocapsid envelopment to form ODVs and their subsequent embedding into polyhedra.Confocal microscopy analysis showed that Bm40 was predominantly localized in the nuclei from 48 h post-infection(h p.t.)and subsequently condensed on the nuclear membrane and polyhedra at the late phase of infection.The deletion of the RRM motif at the C terminus did not affect BV production and the subcellular localization of Bm40.Taken together,these results demonstrate that Bm40 plays an essential role in BV production and ODV envelopment in the BmNPV infection cycle.2.Functional analysis of BmNPV ORF133/134ORF133(bm133)and ORF134(bm134)are located at 126,858-127,313 nt and 127,342-127,671 nt respectively in the genome of BmNPV,and they are two adjacent genes with opposite transcriptional orientations.The two genes are highly conserved in all sequenced group I NPVs and encode two putative proteins with small molecular weight(Bm133,17.7 kDa;Bm134,12.4 kDa).RT-PCR results indicated that both bm133 and bm134 are late genes.A double bm133-bm134 knockout bacmid was generated to enable the functional study of each gene independently or together.The results showed that the deletion of both bm133 and bm134 did not affect BV production or viral DNA replication in transfected BmN cells.Electron microscopy results revealed that the double-knockout did not affect nucleocapsid assembly,virus-induced intranuclear microvesicle formation or ODV production,however,the number of virions embedded in the polyhedra decreased significantly.Further investigations showed that disruption of either gene was unable to recover the defect of ODV occlusion,suggesting that Bm133 and Bm134 are indispensable to the embedding of ODVs into polyhedra.Confocal microscopy analysis showed that Bm133 and Bm134 distributed throughout the whole cell during viral infection and Bm134 concentrated on the mature polyhedra in lysed cells.These results suggest that although Bm133 and Bm134 are not essential for BV or ODV development,they play vital roles in ODV occlusion and polyhedra morphogenesis.3.Functional analysis of BmNPV ORF92aORF92a(bm92a)is located at 88,983-89,162 nt in the genome of BmNPV and is highly conserved in sequenced baculovirus genomes.bm92a ORF contains 180 bp and encodes a putative protein of 59 amino acid residues with a predicted molecular weight of 7.1 kDa.Bm92a is a homologue of the per os infectivity factor(PIF)AC 110 of Autographa californica multiple nucleopolyhedrovirus(AcMNPV).It contains a highly hydrophobic transmembrane domain(TM)at the N terminus.RT-PCR and western blotting results indicated that bm92a is a late gene.A bm92a-knockout BmNPV bacmid was constructed to investigate the role of Bm92a in the baculovirus life cycle.The results showed that the bm92a-knockout virus had similar growth kinetics to the repair virus.Electron microscopy results indicated that the bm92a deletion did not affect nucleocapsid assembly,ODV envelopment or polyhedra formation.Confocal microscopy analysis revealed that Bm92a was predominantly localized to the intranuclear ring zone and subsequently concentrated on the mature polyhedra during the late phase of infection.Further observations showed that the trafficking and localization patterns of Bm92a were similar to those of PIFland PIF2.Interestingly,when PIF1 was used as bait in yeast two-hybrid(YTH)assays,it was found to interact with Bm92a.Interactions were confirmed by bimolecular fluorescence complementation(BiFC)assays.These results suggest that Bm92a is not required for BV or ODV development,and it may constitute a structural part of the viral PIF complex via interaction with PIF1.