Open reading frame Bm21 of Bombyx mori nucleopolyhedrovirus is not essential for virus replication in vitro, but its deletion extends the median survival time of infected larvae

In this report, the open reading frame 21 (Bm21) of Bombyx mori nucleopolyhedrovirus (BmNPV), one of the unique genes of group I NPVs, was characterized. Bm21 is predicted to encode a protein of 55.8 kDa and was found to contain imperfectly conserved leucine-rich repeats. 3' Rapid amplification of cDNA ends (3'RACE) showed that the transcript of Bm21 was first detected from 6 h post-infection and that it also encompassed the complete Bm20. 5'RACE revealed three transcription initiation sites, one of which mapped to the baculovirus early transcription motifs CGTGC and CAGT. Transient-expression and superinfection assays indicated that BM21 localized in the nucleus of infected BmN cells. To study the function of BM21, a Bm21-null virus was constructed using bacmid technology. Viral one-step growth curve analyses showed that the Bm21-null virus had similar budded virus production kinetics to those of the parental virus. Bioassay analyses showed that the median lethal concentration (LC50) of the Bm21-null virus was similar to that of the control virus; however, the median survival time (ST50) of the knockout virus was significantly longer than the control virus. These results indicate that BM21 is not essential for virus replication in vitro, but that deletion of the gene delays the killing of the infected larvae.

在本报告中，对家蚕核型多角体病毒（BmNPV）的开放阅读框21（Bm21）进行了特性分析，Bm21是I类核型多角体病毒的独特基因之一。预测Bm21编码一个55.8 kDa的蛋白质，并且发现其包含不完全保守的富亮氨酸重复序列。3' cDNA末端快速扩增（3'RACE）表明，Bm21的转录本在感染后6小时首次被检测到，并且它还包含完整的Bm20。5'RACE揭示了三个转录起始位点，其中一个定位在杆状病毒早期转录基序CGTGC和CAGT上。瞬时表达和重复感染实验表明，BM21定位于受感染的BmN细胞的细胞核中。为了研究BM21的功能，利用杆粒技术构建了一种Bm21缺失病毒。病毒一步生长曲线分析表明，Bm21缺失病毒与亲本病毒具有相似的出芽病毒产生动力学。生物测定分析表明，Bm21缺失病毒的半数致死浓度（LC50）与对照病毒相似；然而，缺失病毒的半数存活时间（ST50）明显长于对照病毒。这些结果表明，BM21对于病毒的体外复制不是必需的，但该基因的缺失会延迟对受感染幼虫的致死作用。