Analysis of the C-terminal Conserved Region in Baculovirus Ac78

Ac78 of the archetype of the Baculoviridae, Autographa californica multiple nucleopolyhedrovirus( Ac MNPV),is a recently identified baculovirus core gene. It plays an important role in the baculovirus life cycle. An amino acid( aa) alignment of the Ac78 homologs showed that Ac78C-terminal aa 105 ~ 108 are highly conserved in group I NPVs. To investigate the role of Ac78 aa 105 ~108 in the baculovirus life cycle,an Ac78 gene truncated Ac MNPV recombinant( v Ac78: del105-108),in which the Ac78 cassette without residues 105 ~ 108,was constructed successfully with Bac-to-Bac system. To examine the effect on occlusion body morphogenesis and to facilitate examination of virus infection,the Ac MNPV polyhedrin( Polh) gene and the enhanced green fluorescence protein( Egfp;referred to as Gfp in the present study) gene whose expression was driven by its own promoter and theAc MNPV immediate-early gene Ie1 promoter,respectively,were also inserted into the recombinant virus.Fluorescence microscopy and titration assay demonstrated that Sf9 cells transfected with v Ac78: del105-108 or v Ac78: HA showed a normal and steady increase in virus production and similar virus growth kinetics. Electron microscopy showed that the cells transfected with v Ac78: del105-108 exhibited the typical characteristics of baculovirus infection,which were similar to those of v Ac78: HA-transfected cells. Immunofluorescence microscopy showed that,in the nuclei of v Ac78: del105-108-infected Sf9 cells,discrete foci of red fluorescence appeared near the inner nuclear membrane at 24 hour post infection( h p. i.),and they were condensed at 48 h p. i.,by 72 h p. i.,Ac78 was concentrated within the ring zone and formed larger foci. In summary,we provided the evidence demonstrating that removal of the Ac78 aa 105 ~ 108 did not affect the productions of budded virions and M-ODVs and the subcellular localization pattern of Ac78 in Sf9 cells. That is,Ac78 aa 105 ~ 108 is not essential for its role during the baculovirus life cycle.