家蚕核型多角体病毒BmNPV泛素基因缺失显著降低病毒增殖效率

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

摘要:

【目的】泛素是一类进化上高度保守的、由76个氨基酸组成的多肽，是蛋白质泛素化修饰过程中所必需的底物分子，蛋白质泛素化修饰异常会影响宿主的生长和发育。家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus, BmNPV)基因orf26是一个编码病毒泛素的基因，其在病毒增殖中的具体功能尚不清楚。【方法】本研究利用同源重组的方法将氯霉素(chloramphenicol)基因(Cm)表达盒替换家蚕杆状病毒泛素基因序列3¢端的50个碱基对序列，构建携带Cm表达盒的重组BmNPV病毒基因组(Bm-Bacmid^Ub-KO)，利用转座原理将绿色荧光蛋白基因(green fluorescent protein, gfp)表达盒插入Bm-Bacmid^Ub-KO和野生型Bm-Bacmid^WT转座位点，构建重组质粒Bm-Bacmid^Ub-KO-GFP和Bm-Bacmid^WT-GFP，以及异位互补型的质粒Bm-Bacmid^GFP-UbRep。这些重组病毒分别转染家蚕卵巢细胞(Bombyx mori ovarian cell，BmN)后，对转染后的细胞进行绿色荧光观察。【结果】泛素基因缺失后不影响感染性病毒粒子的产生，但与野生型相比，泛素缺失型重组病毒明显降低了感染的细胞中产生的绿色荧光数量。免疫印迹分析表明，泛素缺失后降低重组病毒结构蛋白GP64和VP39在细胞中的表达水平，显著降低病毒增殖效率。生物分析表明，泛素缺失的重组病毒能延缓家蚕半致死时间15 h。【结论】BmNPV泛素基因缺失不影响感染性病毒粒子的产生，但显著降低病毒增殖效率。本研究为阐明泛素在BmNPV增殖中的具体作用提供了实验依据。

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.