The absence of ubiquitin gene reduces the proliferation efficiency of Bombyx mori nucleopolyhedrovirus (BmNPV)

Abstract:

[Objective] Ubiquitins, a class of highly conserved 76-amino acid peptides, are essential substrate molecules in the ubiquitination of proteins. The abnormal ubiquitination of proteins is directly associated with the individual growth and development. The role of orf26, a gene encoding ubiquitin of silkworm baculovirus, in virus proliferation remains unclear. [Methods] The chloramphenicol gene (Cm) expression cassette was used to replace a 50 bp fragment at the 3′ end of the ubiquitin gene sequence by homologous recombination, and the recombinant Bombyx mori nucleopolyhedrovirus (BmNPV) carrying Cm expression cassette (Bm-Bacmid^Ub-KO) was constructed. A green fluorescent protein expression cassette was inserted into Bm-Bacmid^Ub-KO and Bm-Bacmid^WT via transposition for the construction of recombinant Bm-Bacmid^Ub-KO-GFP and Bm-Bacmid^WT-GFP, respectively. Meanwhile, the repair type of Bm-Bacmid^GFP-UbRep was constructed in the study. [Results] The deletion of ubiquitin gene did not affect the generation of infectious virions in BmN cells, while it decreased the production of progeny virions. Western blotting showcased that the deletion of ubiquitin gene down-regulated the expression levels of viral structural proteins GP64 and VP39, which were directly involved in the production of progeny virions. Furthermore, the bioassay results showed that the deletion of ubiquitin gene caused a 15-h delay in the median lethal time of silkworm. [Conclusion] The deletion of ubiquitin gene in BmNPV did not affect the production of infectious virions while significantly reducing the efficiency of viral proliferation. This study lays a foundation for clarifying the specific role of ubiquitin in BmNPV proliferation.