Toribio René, Ventoso Iván
Departamento de Biología Molecular and Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Cientificas- Universidad Autónoma de Madrid, Universidad Autónoma de Madrid, Cantoblanco 28049 Madrid, Spain.
Proc Natl Acad Sci U S A. 2010 May 25;107(21):9837-42. doi: 10.1073/pnas.1004110107. Epub 2010 May 10.
Infection of cultured cells with lytic animal viruses often results in the selective inhibition of host protein synthesis, whereas viral mRNA is efficiently translated under these circumstances. This phenomenon, known as "shut off," has been well described at the molecular level for some viruses, but there is not yet any direct or indirect evidence supporting the idea that it also should operate in animals infected with viruses. To address this issue, we constructed recombinant Sindbis virus (SV)-expressing reporter mRNA, the translation of which is sensitive or resistant to virus-induced shut off. As found in cultured cells, replication of SV in mouse brain was associated with a strong phosphorylation of eukaryotic initiation factor (eIF2) that prevented translation of reporter mRNA (luciferase and EGFP). Translation of these reporters was restored in vitro, in vivo, and ex vivo when a viral RNA structure, termed downstream hairpin loop, present in viral 26S mRNA, was placed at the 5' end of reporter mRNAs. By comparing the expression of shut off-sensitive and -resistant reporters, we unequivocally concluded that replication of SV in animal tissues is associated with a profound inhibition of nonviral mRNA translation. A strategy as simple as that followed here might be applicable to other viruses to evaluate their interference on host translation in infected animals.
用裂解性动物病毒感染培养细胞通常会导致宿主蛋白合成受到选择性抑制，而在这些情况下病毒mRNA却能有效翻译。这种被称为“关闭”的现象在分子水平上已被一些病毒所熟知，但尚无任何直接或间接证据支持其在感染病毒的动物中也会发生的观点。为了解决这个问题，我们构建了表达报告mRNA的重组辛德毕斯病毒（SV），其翻译对病毒诱导的关闭敏感或有抗性。正如在培养细胞中所发现的，SV在小鼠脑中的复制与真核起始因子（eIF2）的强烈磷酸化有关，这阻止了报告mRNA（荧光素酶和EGFP）的翻译。当病毒26S mRNA中存在的一种称为下游发夹环的病毒RNA结构置于报告mRNA的5'端时，这些报告基因的翻译在体外、体内和离体条件下均得以恢复。通过比较对关闭敏感和有抗性的报告基因的表达，我们明确得出结论，SV在动物组织中的复制与非病毒mRNA翻译的深度抑制有关。这里所采用的这种简单策略可能适用于其他病毒，以评估它们对感染动物中宿主翻译的干扰。
