Viral protein translation modulation

丙型肝炎病毒是黄病毒科肝炎病毒属的一种单链正链RNA病毒。近年来，从狗、马、蝙蝠和牛等多种动物中分离到与丙型肝炎病毒高度同源的肝炎病毒。因此，对丙型肝炎病毒的基础研究不仅对了解丙型肝炎病毒的生物学至关重要，而且对于了解动物肝炎病毒的生物学也是一个很好的模型病毒。*本研究计划的长期目标是以丙型肝炎病毒为模型研究病毒翻译调控的分子机制。在目前的NSERC发现拨款资助期间(2013-2018)，我们正在研究非结构蛋白-5A(NS5A)在丙型肝炎病毒RNA翻译中的调节作用，以及病毒3‘非翻译区(3’UTR)的功能。我们发现NS5A下调了丙型肝炎病毒的翻译。病毒3‘非编码区中的聚(U/UC)序列是NS5A翻译下调所必需的。详细的定位实验表明，NS5A结构域I的R112、结构域II的K312和结构域III的E446参与了翻译下调。从机制上讲，我们发现NS5A结构域I的R112突变取消了其与聚(U/UC)RNA的结合，表明NS5A与聚(U/UC)RNA的相互作用可能代表了NS5A调控病毒翻译的一种机制。我们还研究了PI3K-Akt通路对丙型肝炎病毒RNA翻译的影响。我们发现，这条途径增加了丙型肝炎病毒的翻译与所有三个Akt亚型的参与。这项研究由三名研究生和一名博士后研究员进行，结果发表了五篇论文。根据我们正在进行的研究，这项建议的短期目标是进一步表征丙型肝炎病毒NS5A调节病毒RNA翻译的分子机制。我们将在五个目标中检验这一假设，即丙型肝炎病毒NS5A蛋白通过多种机制调节丙型肝炎病毒RNA翻译。*1.研究丙型肝炎病毒3‘端非编码区可变区和X区在NS5A调控病毒RNA翻译过程中的作用。*2.表征NS5A二聚化对丙型肝炎病毒RNA翻译的影响。*3.研究NS5A过度磷酸化对丙型肝炎病毒RNA翻译的影响。*4.鉴定丙型肝炎病毒蛋白和细胞蛋白调节NS5A对丙型肝炎病毒RNA翻译的影响的分子机制。*5.研究MAPK信号转导通路在NS5A调控丙型肝炎病毒翻译中的作用。*这项建议将继续我们以丙型肝炎病毒为模型的病毒翻译调制的研究计划。我们的研究将进一步确定丙型肝炎病毒蛋白如何调节病毒翻译。因此，从这一提议中获得的知识也将有助于理解相关动物肝炎病毒的蛋白质翻译调控。这项拟议的研究将由研究生进行。**

Hepatitis C virus (HCV), a positive-sense, single-stranded RNA virus, is a member of the Hepacivirus genus in the Flaviviridae family. Hepaciviruses with close homology to HCV have been isolated in recent years from numerous animal species, such as dog, horse, bat, and cattle. As such, basic research on HCV is not only critical for understanding HCV biology, it also serves as an excellent model virus for understanding the biology of animal hepaciviruses. ***The long-term goal of this research program is to study the molecular mechanisms of viral translation modulation using HCV as a model. During the current NSERC Discovery grant funding period (2013-2018), we are investigating the regulatory role of non-structural protein-5A (NS5A) in HCV RNA translation and the function of the viral 3´untranslated region (3´UTR). We showed that NS5A downregulates HCV translation. The poly(U/UC) sequence in the viral 3´UTR is required for translation downregulation by NS5A. Detailed mapping experiments demonstrated that amino acid R112 in domain I of NS5A, K312 in domain II, and E446 in domain III are involved in translation downregulation. Mechanistically, we showed that mutating R112 in NS5A domain I abrogates its binding to poly(U/UC) RNA, suggesting that NS5A - poly(U/UC) RNA interaction may represent a mechanism of how NS5A modulates viral translation. We also studied the effects of the PI3K-Akt pathway on HCV RNA translation. We found that this pathway increases HCV translation with the involvement of all three Akt isoforms. The research, performed by three graduate students and one post-doctoral fellow, resulted in five publications. Based on our ongoing research, the short-term goal of this proposal is to further characterize the molecular mechanisms by which HCV NS5A modulates viral RNA translation. We will test the hypothesis that HCV NS5A protein regulates HCV RNA translation through multiple mechanisms in five objectives. ******1. Characterize the role of the variable and X regions in the HCV 3´UTR in viral RNA translation modulation by NS5A. ***2. Characterize the effect of NS5A dimerization on HCV RNA translation. ***3. Characterize the effect of NS5A hyper-phosphorylation on HCV RNA translation. ***4. Characterize the molecular mechanisms by which HCV proteins and cellular proteins modulate the effect of NS5A on HCV RNA translation. ***5. To study the role of the MAPK signal transduction pathway in HCV translation modulation by NS5A. ******This proposal will continue our research program on viral translation modulation using HCV as a model. Our research will further determine how an HCV protein regulates viral translation. As such, knowledge gained from this proposal will also help understand protein translation modulation of related animal hepaciviruses. The proposed research will be carried out by graduate students. **