MUTATIONAL ANALYSIS OF THE J-K STEM-LOOP REGION OF THE ENCEPHALOMYOCARDITIS VIRUS IRES
MUTATIONAL ANALYSIS OF THE J-K STEM-LOOP REGION OF THE ENCEPHALOMYOCARDITIS VIRUS IRES
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DOI：
10.1128/jvi.69.7.4399-4406.1995
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发表时间：
1995-07-01
影响因子：
5.4
通讯作者：
PALMENBERG, AC
中科院分区：
文献类型：
--
作者：
HOFFMAN, MA;PALMENBERG, AC
脑心肌炎病毒（EMCV）RNA的非帽依赖性翻译由5'非翻译区的一段称为内部核糖体进入位点（IRES）的片段控制。IRES包含一系列茎环结构元件。J和K茎（EMCV碱基682至795），靠近IRES的中心，在所有心脏病毒、口疮病毒和肝病毒中非常保守。我们通过构建EMCV J-K序列内的突变并测试突变在翻译、翻译竞争、UV交联和病毒感染性测定中的活性来研究这些元件的生物学作用。J和K的螺旋连接附近的突变被证明对下游顺反子的细胞翻译和无细胞翻译都是严重有害的。相同的突变降低了IRES在竞争测定中竞争细胞因子的能力，并降低了携带这些病变的病毒基因组的感染性。在J的末端环中的突变给出了类似的结果。相反，K末端环内的突变对体外翻译活性和IRES竞争能力的影响最小，然而，K环突变的体内分析揭示了细胞翻译过程中的缺陷，并进一步显示了HeLa细胞中感染性的显著降低。UV交联实验确定了一个49-kDa的蛋白质与J-K区域强烈相互作用，但这种蛋白质的身份及其对IRES活性的贡献尚不清楚。
Cap-independent translation of encephalomyocarditis virus (EMCV) RNA is controlled by a segment of the 5' untranslated region termed the internal ribosomal entry site, or IRES. The IRES contains a series of stem-loop structural elements. The J and K stems (EMCV bases 682 to 795), near the center of the IRES, are well conserved among all cardio-, aphtho-, and hepatoviruses. We have examined the biological roles of these elements by constructing mutations within the J-K sequences of EMCV and testing the mutations for activity in translation, translation competition, UV cross-linking, and viral infectivity assays. Mutations near the helical junction of J and K proved severely detrimental to both cellular translation and cell-free translation of downstream cistrons. The same mutations reduced the ability of the IRES to compete for cellular factors in competition assays and reduced the infectivity of viral genomes carrying these lesions. A mutation in the terminal loop of J gave similar results. In contrast, mutations within the terminal loop of K had minimal impact on in vitro translation activity and IRES competitive ability, However, in vivo analysis of the K-loop mutations revealed deficiencies during cellular translation and further showed markedly reduced infectivity in HeLa cells. UV cross-linking experiments identified a 49-kDa protein which interacts strongly with the J-K region, but the identity of this protein and its contribution to IRES activity are unclear.