Complex genetic interactions among non-essential genes of BmNPV revealed by multiple gene knockout analysis

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  • Taka Hitomi
    Laboratory of Applied Molecular Entomology, Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University
  • Ono Chikako
    Laboratory of Applied Molecular Entomology, Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University
  • Sato Masanao
    Okazaki Institute for Integrative Bioscience, National Institute for Basic Biology National Institutes of Natural Sciences
  • Asano Shin-ichiro
    Laboratory of Applied Molecular Entomology, Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University
  • Bando Hisanori
    Laboratory of Applied Molecular Entomology, Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University

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About two thirds of open reading frames (orfs) predicted on Bombyx mori nucleopolyhedrovirus (BmNPV) genome were dispensable for expression of the reporter gene derived from the polyhedrin promoter in BmN cells when knocked out one at a time (Ono et al., 2012). Effects of removal of multiple genes of BmNPV and genetic interactions of the viral genes have been poorly investigated. In this study, we constructed BmNPV lacking multiple non-essential genes at the orf11-12-13-14 gene cluster and analyzed the expression of EGFP under the control of the polyhedrin gene promoter. Viruses lacking more than two genes except for the one lacking orf13 and orf14 showed distinct phenotypes compared to the single gene knockout viruses. Synergistic, compensatory, and additive relationships were observed in the genetic interaction analyses between pairs of adjacent genes in the gene cluster. In addition, the virus lacking both orf12 and orf13 but carrying the orf12 genomic region at downstream of the polyhedrin locus expressed EGFP at higher levels than the control virus BmGFP (Ono et al., 2012) although the transcription of orf12 was not changed by insertion at the different locus. This increased EGFP expression was not observed with the virus in which the defect of orf12 and orf13 was rescued with orf13 at the same position. These observations suggested that inserting orf12 at the position specifically affected expression of the reporter gene at the polyhedrin locus.<br>

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