College of Animal Sciences, Zhejiang University, Hangzhou 310058, China.
Appl Microbiol Biotechnol. 2012 Aug;95(3):697-705. doi: 10.1007/s00253-012-3996-1. Epub 2012 Apr 4.
The 5' untranslated region plays an important role in positive-sense single-stranded RNA virus translation initiation, as it contains an internal ribosome entry site (IRES) that mediates cap-independent translation and is applied to simultaneously express several proteins. Infectious flacherie virus (IFV) is a positive-sense single-stranded RNA virus; however, the IRES function is still not proved. To investigate whether the sequences of IFV contain IRES activity, a series of bicistronic reporter (DsRed and enhanced green fluorescent protein) recombinant baculoviruses were constructed to infect the insect cells and silkworm using the Bombyx mori baculovirus expression system. Results showed that the upstream 311, 323, 383, 551, and 599 nt have IRES activity except for the 155-nt region in BmN cells. More importantly, the tetraloop structure containing region between 551 and 599 nt appeared to be responsible for the enhanced IRES activity in different insect cell lines and silkworm. These results indicated that the IRES activity is not species specific and tissue specific. Therefore, our findings may provide the basis for the simultaneous expression of two or various different genes under the same promoter in baculovirus expression system.
5' 非翻译区在正链单链 RNA 病毒翻译起始中发挥重要作用，因为它包含内部核糖体进入位点（IRES），介导无帽依赖性翻译，并应用于同时表达几种蛋白质。传染性 Flacherie 病毒（IFV）是一种正链单链 RNA 病毒；然而，IRES 的功能仍未得到证实。为了研究 IFV 的序列是否含有 IRES 活性，构建了一系列双顺反子报告基因（DsRed 和增强型绿色荧光蛋白）重组杆状病毒，并用家蚕杆状病毒表达系统感染昆虫细胞和家蚕。结果表明，上游 311、323、383、551 和 599nt 除了在 BmN 细胞中的 155nt 区域外，均具有 IRES 活性。更重要的是，551 和 599nt 之间包含四环结构的区域似乎负责不同昆虫细胞系和家蚕中增强的 IRES 活性。这些结果表明，IRES 活性不是种属特异性和组织特异性的。因此，我们的发现可能为杆状病毒表达系统中同一启动子下同时表达两个或多个不同基因提供基础。
