Characterization of AcMNPV with a deletion of ac69 gene

Jianhao Ke,Riqiang Deng,Lin Lin,Liao Yaoping,Xunzhang Wang,Jinwen Wang,Bei Jinlong

doi:10.4081/mr.2011.e4

Abstract

ORF69 (Ac69) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is conserved in some baculovirus genomes. Although it has been shown that Ac69 has cap 0-dependent methyltransferase activity and is not required for budded virus production in Spodoptera frugiperda Sf-9 cells, its role in occlusion-derived virus synthesis and virus oral infectivity is not known. This paper describes generation of an ac69 knockout AcMNPV bacmid mutant and analyses of the influence of ac69 deletion on the viral infectivity in Sf-9 cells and Trichoplusia ni larvae so as to investigate the role of ac69 in the viral life cycle. Results indicated that ac69 deletion has little effect on the production rates and morphogenesis of budded virus and occlusion-derived virus in Sf-9 cells. In addition, animal experiment revealed that the deletion mutant did not affect AcMNPV infectivity for Trichoplusia ni larvae in LD50 and LT50 bioassay when administered orally. These results suggest that ac69 may be dispensable for viral infectivity both in vitro and in vivo.

Highlights

Materials and MethodsThe insect Spodoptera frugiperda cells line Sf9 cells were cultured at 27°C in Grace’s medium (Invitrogen Life Technologies) supplemented
Introduction expression assay system10 Wu and coworkers with 10% fetal bovine serum, penicillin (100 showed that Ac69 which was synthesized dur- μg/mL) and streptomycin (30 μg/mL)
Bacmid ing the late phase of infection had cap 0- bMON14272 (Invitrogen Life Technologies), Autographa californica multiple nucleopoly- dependent methyltransferase activity.[11] containing the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) genome, can propagate hedrovirus (AcMNPV) is the most intensively studied member of the Baculovirade family, which encompasses a group of arthropod-specific DNA viruses with a circular covalently

Summary

Materials and Methods

The insect Spodoptera frugiperda cells line Sf9 cells were cultured at 27°C in Grace’s medium (Invitrogen Life Technologies) supplemented. The 58841-59399) homologous to the upstream ment CAGT motif.[4,5,6] By the virus-encoded RNA results described demonstrate that sequence of ac[69] was PCR amplified from polymerase, the late and very late gene tran- ac[69] deletion mutant had no striking pheno- AcMNPV genome. Z. With primers ac69D1 and ac69D2, a 727-bp later by GFP expression and occlusion body for- titers were determined by TCID50 end point fragment The resulting product was cell, and colonies were screened for sensitivity points the titers were determined by a TCID50 digested with BamHI/XbaI and cloned into to tetracycline to ensure that the isolated end-point dilution assay in Sf-9 cells. BamHI/XbaI digested vector pET28a-US-Z to bacmid was free of the helper plasmid

Electron microscopy analysis

Findings

HindIII BamHI NotI HindIII BamHI XbaI XbaI XhoI