Zhao Cui, Zhang Chen, Chen Bin, Shi Yanghui, Quan Yanping, Nie Zuoming, Zhang Yaozhou, Yu Wei
College of life sciences, Zhejiang Sci-Tech University, Zhejiang Province, Hangzhou 310018, China.
Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine, Zhejiang Province, Hangzhou 310018, China.
PLoS One. 2016 Jul 14;11(7):e0159149. doi: 10.1371/journal.pone.0159149. eCollection 2016.
A DNA-binding protein (DBP) [GenBank accession number: M63416] of Bombyx mori nuclear polyhedrosis virus (BmNPV) has been reported to be a regulatory factor in BmNPV, but its detailed functions remain unknown. In order to study the regulatory mechanism of DBP on viral proliferation, genome replication, and gene transcription, a BmNPV dbp gene knockout virus dbp-ko-Bacmid was generated by the means of Red recombination system. In addition, dbp-repaired virus dbp-re-Bacmid was constructed by the means of the Bac to Bac system. Then, the Bacmids were transfected into BmN cells. The results of this viral titer experiment revealed that the TCID50 of the dbp-ko-Bacmid was 0; however, the dbp-re-Bacmid was similar to the wtBacmid (p>0.05), indicating that the dbp-deficient would lead to failure in the assembly of virus particles. In the next step, Real-Time PCR was used to analyze the transcriptional phases of dbp gene in BmN cells, which had been infected with BmNPV. The results of the latter experiment revealed that the transcript of dbp gene was first detected at 3 h post-infection. Furthermore, the replication level of virus genome and the transcriptional level of virus early, late, and very late genes in BmN cells, which had been transfected with 3 kinds of Bacmids, were analyzed by Real-Time PCR. The demonstrating that the replication level of genome was lower than that of wtBacmid and dbp-re-Bacmid (p<0.01). The transcriptional level of dbp-ko-Bacmid early gene lef-3, ie-1, dnapol, late gene vp39 and very late gene p10 were statistically significantly lower than dbp-re-Bacmid and wtBacmid (p<0.01). The results presented are based on Western blot analysis, which indicated that the lack of dbp gene would lead to low expressions of lef3, vp39, and p10. In conclusion, dbp was not only essential for early viral replication, but also a viral gene that has a significant impact on transcription and expression during all periods of baculovirus life cycle.
据报道，家蚕核型多角体病毒（BmNPV）的一种DNA结合蛋白（DBP）[GenBank登录号：M63416]是BmNPV中的一种调节因子，但其详细功能尚不清楚。为了研究DBP对病毒增殖、基因组复制和基因转录的调控机制，利用Red重组系统构建了BmNPV dbp基因敲除病毒dbp-ko-Bacmid。此外，通过Bac to Bac系统构建了dbp修复病毒dbp-re-Bacmid。然后，将Bacmid转染到BmN细胞中。该病毒滴度实验结果显示，dbp-ko-Bacmid的半数组织培养感染剂量（TCID50）为0；然而，dbp-re-Bacmid与野生型Bacmid相似（p>0.05），表明dbp缺陷会导致病毒粒子组装失败。下一步，采用实时定量聚合酶链反应（Real-Time PCR）分析感染BmNPV的BmN细胞中dbp基因的转录阶段。后一实验结果显示，感染后3小时首次检测到dbp基因的转录本。此外，通过Real-Time PCR分析了用3种Bacmid转染的BmN细胞中病毒基因组的复制水平以及病毒早期、晚期和极晚期基因的转录水平。结果表明，基因组的复制水平低于野生型Bacmid和dbp-re-Bacmid（p<0.01）。dbp-ko-Bacmid早期基因lef-3、ie-1、DNA聚合酶、晚期基因vp39和极晚期基因p10的转录水平在统计学上显著低于dbp-re-Bacmid和野生型Bacmid（p<0.01）。基于蛋白质免疫印迹分析的结果表明，dbp基因的缺失会导致lef3、vp39和p10的低表达。总之，dbp不仅对病毒早期复制至关重要，而且是一种在杆状病毒生命周期的所有阶段对转录和表达都有重大影响的病毒基因。
