Abstract：Baculoviruses are a family of enveloped, rod-shaped, double-stranded DNA viruses which are specifically pathogenic to arthropods, mainly belonging to the orders Lepidoptera, Hymenoptera, and Diptera. ac78 of the archetype of the Baculoviridae, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) was one of the 38 core genes of baculoviruses. Previous studies showed that Ac78 played an important role in the baculovirus life cycle, and Ac78 conserved amino acid (aa) 2~25 and 64~88 were essential for Ac78 functions during baculovirus life cycle, but the mechanism by which Ac78 employs that affected the budded virion (BV) production and multiple nucleocapsid-enveloped occlusion-derived virion (M-ODV) formation was still unknown. An aa alignment of the Ac78 homologs showed that Ac78 N-terminal aa 9 (R9) and 22 (K22) were highly conserved in baculoviruses, suggesting that R9 and K22 might be important in the functions of Ac78. In the present study, the ac78 with a HA tag prior to the stop codon, in which the codon of R9 or K22 was mutated into the codon of alanine (A), together with the enhanced green fluorescence protein gene (egfp; referred to as gfp in the present study) and the AcMNPV polyhedrin (polh) gene, was inserted into the polh locus of the ac78-knockout AcMNPV bacmid (bAc78KO) to construct the recombinant viruses, vAc78R9A:HA or vAc78K22A:HA. All recombinant viruses were confirmed by PCR analysis and further DNA sequencing. The ac78-knockout virus (vAc78KO), ac78-repaired virus (vAc78:HA), vAc78R9A:HA, or vAc78K22A:HA bacmid DNA was transfected into Sf9 (Spodoptera frugiperda IPLB-Sf21-AE clonal isolate 9) cells, viral replication and infection were monitored by a fluorescence microscope. At 24 h post transfection (h p.t.), no obvious differences in the amounts of GFP-positive cells were observed among all 4 groups, indicating comparable transfection efficiencies. At 72 h p.t., almost all Sf9 cells transfected with vAc78R9A:HA or vAc78:HA bacmid DNA showed GFP fluorescence, the number of fluorescent cells increased slightly onwards in the vAc78KO- or vAc78K22A: HA-transfected cells, but the amount of GFP-positive cells in Sf9 cells transfected with vAc78K22A:HA was a little more than that in Sf9 cells transfected with vAc78KO. Using virus supernatants harvested from vAc78KO-, vAc78:HA-, vAc78R9A:HA-, or vAc78K22A:HA-transfected Sf9 cells, viral growth curve analysis was performed by fifty percent tissue culture infective dose endpoint dilution assay to further evaluate the ability of vAc78R9A:HA and vAc78K22A:HA to produce infectious BVs. There was no significant difference between vAc78R9A:HA and vAc78:HA in capabilities to generate infectious BVs, while the titer of vAc78K22A:HA was approximately 100-fold lower than that of vAc78:HA at 120 h p.t. The results showed that the R9 mutation had no effect on infectious BV production, but the K22 mutation significantly reduced the production of the infectious BVs. Thin sections of vAc78KO-, vAc78:HA-, vAc78R9A:HA-, or vAc78K22A:HA-transfected Sf9 cells at 72 h p.t. were observed using an electron microscope. In vAc78:HA- or vAc78R9A:HA-transfected Sf9 cells, numerous M-ODVs and occlusion bodies (OBs) embedding the numerous M-ODVs emerged in the ring zone, but in the vAc78KO- or vAc78K22A:HA-transfected cells, the M-ODVs in the ring zone and OBs were significantly fewer than those in the vAc78:HA-transfected Sf9 cells, showing that the R9 mutation did not affect the M-ODV formation, or the embedding of M-ODVs into the OBs, while the K22 was important in the formation of M-ODVs and the embedding of M-ODVs into the OBs. Taken together, the results demonstrated that Ac78 R9 was not essential for the infectious BV production and M-ODV formation, but K22 played an important role in the Ac78 functions. This study enriches the information of Ac78, and would lay a foundation for uncovering the mechanism employed by Ac78.
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