Characterization of AcMNPV with a deletion of me53 gene |
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Authors: | Qianyun Xi Jinwen Wang Riqiang Deng Xunzhang Wang |
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Institution: | (1) State Key Laboratory for Biocontrol, School of Life Sciences, Sun Yat-sen University, 510275 Guangzhou, People’s Republic of China;(2) College of Animal Science, South China Agricultural University, 510642 Guangzhou, People’s Republic of China |
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Abstract: | The Autographa Californica multiple nucleopolyhedrovirus (AcMNPV) me53 gene, which was previously reported as one of the major early-transcribed genes, was deleted through homologous recombination
from an AcMNPV genome propagated as a bacmid DNA in E. coli, generating a me53 gene knockout bacmid. Green fluorescent protein (GFP) expression analysis and supernatant passage assay revealed that the
me53 knockout bacmid was unable to replicate in cell culture, while me53 repair bacmid, which was generated by reinsertion of the me53 gene into the mutant at a different locus (the gentamicin locus) with ET-recombination technique, resumed viral replication
ability at wild-type levels, indicating that the defective phenotype of the me53 knockout virus was directly due to the deletion of the me53-ORF. Subsequent electron microscopy revealed that the me53 knockout bacmid failed to form nucleocapsid in the nuclei of the transfected cells, though viral infection seemed to be
initiated. Meanwhile, real-time PCR analysis based on SYBR Green fluorescence indicated abolishment of the viral DNA replication
by me53 gene inactivation. Thus, it is demonstrated for the first time that me53 knockout blocked viral DNA replication, nucleocapsid formation, and consequent BV and ODV production. |
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Keywords: | AcMNPV me53 gene Viral production Viral DNA replication |
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