The regulation of hepatitis C virus (HCV) internal ribosome-entry site-mediated translation by HCV replicons and nonstructural proteins
The regulation of hepatitis C virus (HCV) internal ribosome-entry site-mediated translation by HCV replicons and nonstructural proteins
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DOI：
10.1099/vir.0.18658-0
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发表时间：
2003-03-01
影响因子：
3.8
通讯作者：
Katze, MG
中科院分区：
文献类型：
--
作者：
He, YP;Yan, W;Katze, MG
丙型肝炎病毒（HCV）是全球慢性肝病的主要病因，具有编码大的多蛋白的正义ssRNA基因组。HCV多聚蛋白翻译是由位于病毒基因组5 '端的内部核糖体进入位点（IRES）以帽非依赖性方式启动的，但该过程的调控机制仍知之甚少。在这项研究中，我们的特点是在HCV复制子细胞和瞬时转染的人肝细胞表达HCV非结构蛋白的HCV IRES定向翻译的HCV非结构蛋白的影响。使用携带HCV或其他病毒IRES序列的双顺反子报告基因构建体，我们发现HCV IRES介导的翻译在HCV复制子细胞中特异性上调。复制子细胞对HCV IRES介导的翻译的增强作用可被干扰HCV基因组复制的药物I型干扰素或利巴韦林抑制，这表明增强作用可能是由于HCV编码的蛋白功能所致。在复制子细胞中观察到eIF2alpha和eIF4E的磷酸化水平降低，这与我们先前的发现一致，并表明NS5A非结构蛋白可能参与调控机制。事实上，NS5A或NS4B在人肝细胞中的瞬时表达刺激了HCV IRES活性。有趣的是，NS5A的ISDR突变扰乱了HCV IRES活性的刺激。所有这些结果表明，第一次，HCV非结构蛋白优先刺激病毒帽独立，IRES介导的翻译。
Hepatitis C virus (HCV), the global leading cause of chronic liver disease, has a positive-sense, ssRNA genome that encodes a large polyprotein. HCV polyprotein translation is initiated by an internal ribosome-entry site (IRES) located at the 5' end of the viral genome, in a cap-independent manner, but the regulatory mechanism of this process remains poorly understood. In this study, we characterized the effect of HCV nonstructural proteins on HCV IRES-directed translation in both HCV replicon cells and transiently transfected human liver cells expressing HCV nonstructural proteins. Using bicistronic reporter gene constructs carrying either HCV or other viral IRES sequences, we found that the HCV IRES-mediated translation was specifically upregulated in HCV replicon cells. This enhancement of HCV IRES-mediated translation by the replicon cells was inhibited by treatment with either type I interferon or ribavirin, drugs that perturb HCV genome replication, suggesting that the enhancement is probably due to HCV-encoded protein function(s). Reduced phosphorylation levels of both eIF2alpha and eIF4E were observed in the replicon cells, which is consistent with our previous findings and indicates that the NS5A nonstructural protein may be involved in the regulatory mechanism(s). Indeed, transient expression of NS5A or NS4B in human liver cells stimulated HCV IRES activity. Interestingly, mutation in the ISDR of NS5A perturbed this stimulation of HCV IRES activity. All these results suggest, for the first time, that HCV nonstructural proteins preferentially stimulate the viral cap-independent, IRES-mediated translation.