Viral protein translation modulation

丙型肝炎病毒（HCV）是一种阳性意义的单链RNA病毒，是黄病毒科肝炎病毒属的成员。近年来，从狗、马、蝙蝠和牛等许多动物物种中分离出与丙型肝炎病毒具有密切同源性的肝炎病毒。因此，HCV的基础研究不仅是了解HCV生物学的关键，也是了解动物肝炎病毒生物学的一个很好的模型病毒。本研究计划的长期目标是研究以HCV为模型的病毒转译调节的分子机制。在目前的NSERC发现资助期内（2013-2018），我们正在研究非结构蛋白5a （NS5A）在HCV RNA翻译中的调节作用和病毒3untranslational region （3UTR）的功能。我们发现NS5A下调HCV翻译。病毒3UTR中的poly（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氨基酸参与了翻译下调。在机制上，我们发现突变NS5A结构域I中的R112消除了它与poly(U/UC) RNA的结合，这表明NS5A - poly(U/UC) RNA相互作用可能是NS5A调节病毒翻译的一种机制。我们还研究了PI3K-Akt通路对HCV RNA翻译的影响。我们发现这条通路在所有三种Akt亚型的参与下增加了HCV的翻译。这项研究由三名研究生和一名博士后完成，发表了五篇论文。基于我们正在进行的研究，本提案的短期目标是进一步表征HCV NS5A调节病毒RNA翻译的分子机制。我们将通过五个目标来验证HCV NS5A蛋白通过多种机制调节HCV RNA翻译的假设。1. 研究HCV 3UTR中可变区和X区在NS5A介导的病毒RNA翻译调节中的作用。表征NS5A二聚化对HCV RNA翻译的影响。3. 表征NS5A超磷酸化对HCV RNA翻译的影响。4. 研究HCV蛋白和细胞蛋白调控NS5A对HCV RNA翻译作用的分子机制。5. 研究MAPK信号转导通路在NS5A介导HCV翻译中的作用。本研究将继续以HCV为模型进行病毒转译调节的研究。我们的研究将进一步确定HCV蛋白如何调节病毒转译。因此，从这一建议中获得的知识也将有助于了解相关动物肝炎病毒的蛋白质翻译调节。建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 3untranslated region (3UTR). We showed that NS5A downregulates HCV translation. The poly(U/UC) sequence in the viral 3UTR 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 3UTR 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.