Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India.
J Gen Virol. 2010 May;91(Pt 5):1245-55. doi: 10.1099/vir.0.018507-0. Epub 2010 Jan 13.
We have investigated the possible role of trans-acting factors interacting with the untranslated regions (UTRs) of coxsackievirus B3 (CVB3) RNA. We show here that polypyrimidine tract-binding protein (PTB) binds specifically to both 5' and 3' UTRs, but with different affinity. We have demonstrated that PTB is a bona fide internal ribosome entry site (IRES) trans-acting factor (ITAF) for CVB3 RNA by characterizing the effect of partial silencing of PTB ex vivo in HeLa cells. Furthermore, IRES activity in BSC-1 cells, which are reported to have a very low level of endogenous PTB, was found to be significantly lower than that in HeLa cells. Additionally, we have mapped the putative contact points of PTB on the 5' and 3' UTRs by an RNA toe-printing assay. We have shown that the 3' UTR is able to stimulate CVB3 IRES-mediated translation. Interestingly, a deletion of 15 nt at the 5' end or 14 nt at the 3' end of the CVB3 3' UTR reduced the 3' UTR-mediated enhancement of IRES activity ex vivo significantly, and a reduced interaction was shown with PTB. It appears that the PTB protein might help in circularization of the CVB3 RNA by bridging the ends necessary for efficient translation of the viral RNA.
我们研究了与柯萨奇病毒 B3（CVB3）RNA 的非翻译区（UTR）相互作用的反式作用因子的可能作用。我们在此表明，多嘧啶 tract 结合蛋白（PTB）特异性结合 5'和 3'UTR，但亲和力不同。我们通过在 HeLa 细胞中体外部分沉默 PTB 来表征其对 CVB3 RNA 的影响，证明了 PTB 是 CVB3 RNA 的真正内部核糖体进入位点（IRES）反式作用因子（ITAF）。此外，据报道，BSC-1 细胞中内源性 PTB 水平非常低，其 IRES 活性明显低于 HeLa 细胞。此外，我们通过 RNA 足迹印迹测定法对 PTB 在 5'和 3'UTR 上的假定接触点进行了映射。我们表明，3'UTR 能够刺激 CVB3 IRES 介导的翻译。有趣的是，CVB3 3'UTR 的 5'端缺失 15 个核苷酸或 3'端缺失 14 个核苷酸会显著降低 3'UTR 对 IRES 活性的体外增强作用，并且与 PTB 的相互作用减少。PTB 蛋白可能通过桥接病毒 RNA 有效翻译所需的末端，有助于 CVB3 RNA 的环化。
