M'hadheb-Gharbi Manel Ben, El Hiar Raïda, Paulous Sylvie, Jaïdane Hela, Aouni Mahjoub, Kean Katherine M, Gharbi Jawhar
Unité de Pathogenèse et Virulence Virales, Laboratoire des Maladies Dominantes Transmissibles (MDT-01), Faculté de Pharmacie de Monastir, Monastir, Tunisia.
J Mol Microbiol Biotechnol. 2008;14(4):147-56. doi: 10.1159/000107369. Epub 2007 Aug 13.
The lengthy 5' nontranslated region of coxsackievirus B3 (CVB3) forms a highly ordered secondary structure containing an internal ribosome entry segment (IRES), which plays an important role in controlling viral translation and pathogenesis. The stem-loop V (SL-V) of this IRES contains a large lateral bulge loop which encompasses two conserved GNRA motifs. In this study, we analyzed the effects of point mutations within the GNRA motifs of the CVB3 IRES. We characterized in vitro virus production and translation efficiency and we tested in vivo virulence of two CVB3 mutants produced by site-directed mutagenesis. The GNAA1 and GNAA2 RNAs displayed decreased translation initiation efficiency when translated in rabbit reticulocyte lysates. This translation defect was correlated with reduced yields of infectious virus particles in HeLa cells in comparison with the wild type. When inoculated orally into Swiss mice, both mutant viruses were avirulent and caused neither inflammation nor necrosis in hearts. These results highlight the important role of the GNRA motifs within the SL-V of the IRES of CVB3, in directing translation initiation.
柯萨奇病毒B3（CVB3）冗长的5'非翻译区形成了一种高度有序的二级结构，其中包含一个内部核糖体进入片段（IRES），该片段在控制病毒翻译和发病机制中起重要作用。该IRES的茎环V（SL-V）包含一个大的侧向凸起环，其中包含两个保守的GNRA基序。在本研究中，我们分析了CVB3 IRES的GNRA基序内点突变的影响。我们表征了体外病毒产生和翻译效率，并测试了通过定点诱变产生的两个CVB3突变体的体内毒力。当在兔网织红细胞裂解物中翻译时，GNAA1和GNAA2 RNA的翻译起始效率降低。与野生型相比，这种翻译缺陷与HeLa细胞中感染性病毒颗粒产量降低相关。当经口接种到瑞士小鼠体内时，两种突变病毒均无毒，且在心脏中既不引起炎症也不引起坏死。这些结果突出了CVB3 IRES的SL-V内的GNRA基序在指导翻译起始中的重要作用。
