National Veterinary Institute, Technical University of Denmark, Lindholm, Kalvehave, Denmark.
J Virol. 2012 Aug;86(16):8681-92. doi: 10.1128/JVI.00346-12. Epub 2012 Jun 6.
Modulation of translation initiation efficiency on classical swine fever virus (CSFV) RNA can be achieved by targeted mutations within the internal ribosome entry site (IRES). In this study, cDNAs corresponding to the wild-type (wt) or mutant forms of the IRES of CSFV strain Paderborn were amplified and inserted into dicistronic reporter plasmids encoding Fluc and Rluc under the control of a T7 promoter. The mutations were within domains II, IIId(1), and IIIf of the IRES. The plasmids were transfected into baby hamster kidney (BHK) cells infected with recombinant vaccinia virus vTF7-3, which expresses the T7 RNA polymerase. IRES mutants with different levels of IRES activity were identified and then introduced by homologous recombination into bacterial artificial chromosomes (BACs) containing CSFV Paderborn cDNA downstream of a T7 promoter. From the wt and mutant BACs, full-length CSFV RNA transcripts were produced in vitro and electroporated into porcine PK15 cells. Rescued mutant viruses were obtained from RNAs that contained mutations within domain IIIf which retained more than 75% of the wt translation efficiency. Sequencing of cDNA generated from these rescued viruses verified the maintenance of the introduced changes within the IRES. The growth characteristics of each rescued mutant virus were compared to those of the wt virus. It was shown that viable mutant viruses with reduced translation initiation efficiency can be designed and generated and that viruses containing mutations within domain IIIf of the IRES have reduced growth in cell culture compared to the wt virus.
通过在内部核糖体进入位点（IRES）内进行靶向突变，可以调节经典猪瘟病毒（CSFV）RNA 的翻译起始效率。在这项研究中，扩增了与 CSFV 株 Paderborn 的野生型（wt）或 IRES 突变体对应的 cDNA，并将其插入双顺反子报告质粒中，该质粒在 T7 启动子的控制下编码 Fluc 和 Rluc。突变位于 IRES 的结构域 II、IIId（1）和 IIIf 内。将质粒转染到感染重组痘苗病毒 vTF7-3 的仓鼠肾细胞（BHK）中，该病毒表达 T7 RNA 聚合酶。鉴定出具有不同 IRES 活性水平的 IRES 突变体，然后通过同源重组将其引入含有 CSFV Paderborn cDNA 的细菌人工染色体（BAC）中，该 cDNA 位于 T7 启动子下游。从 wt 和突变 BAC 中，体外产生全长 CSFV RNA 转录本，并用电穿孔将其转入猪 PK15 细胞中。从含有突变的 RNA 中获得了突变病毒，这些突变位于保留了超过 75%wt 翻译效率的 IIIf 结构域内。从这些拯救的病毒中获得的 cDNA 的测序证实了 IRES 内引入的变化得以维持。比较了每个拯救的突变病毒的生长特性与 wt 病毒的生长特性。结果表明，可以设计和生成具有降低翻译起始效率的存活突变病毒，并且与 wt 病毒相比，含有 IRES 中 IIIf 结构域突变的病毒在细胞培养中的生长速度较慢。
