Viral protein translation modulation

丙型肝炎病毒（HCV）是一种正义单链RNA病毒，属于黄病毒科肝炎病毒属。近年来，已经从许多动物物种，如狗、马、蝙蝠和牛中分离出与HCV具有密切同源性的肝炎病毒。因此，对HCV的基础研究不仅对理解HCV生物学至关重要，而且也是理解动物肝炎病毒生物学的极好模型病毒。* 本研究计划的长期目标是以HCV为模型研究病毒翻译调节的分子机制。在当前的NSERC发现资助期间（2013-2018），我们正在研究非结构蛋白-5A（NS 5A）在HCV RNA翻译中的调节作用和病毒3UTR的功能。我们发现NS 5A下调HCV翻译。病毒3UTR中的poly（U/UC）序列是NS 5A翻译下调所需的。详细的定位实验表明，NS 5A结构域I中的氨基酸R112、结构域II中的氨基酸K312和结构域III中的氨基酸E446参与翻译下调。从机制上讲，我们发现NS 5A结构域I中的突变R112消除了其与poly（U/UC）RNA的结合，表明NS 5A- poly（U/UC）RNA相互作用可能代表NS 5A如何调节病毒翻译的机制。我们还研究了PI 3 K-Akt通路对HCV RNA翻译的影响。我们发现，这一途径增加了HCV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，发表了五篇论文。基于我们正在进行的研究，该提案的短期目标是进一步表征HCV NS 5A调节病毒RNA翻译的分子机制。我们将在五个目标中检验HCV NS 5A蛋白通过多种机制调节HCV RNA翻译的假设。*1.描述HCV 3UTR可变区和X区在NS 5A调节病毒RNA翻译中的作用。*2。表征NS 5A二聚化对HCV RNA翻译的影响。*3。描述NS 5A过度磷酸化对HCV RNA翻译的影响。*4。描述HCV蛋白和细胞蛋白调节NS 5A对HCV RNA翻译影响的分子机制。*5。目的探讨丝裂原活化蛋白激酶（MAPK）信号转导通路在NS 5A对丙型肝炎病毒（HCV）翻译调控中的作用。** 本提案将继续我们以HCV为模型的病毒翻译调节研究计划。我们的研究将进一步确定HCV蛋白如何调节病毒翻译。因此，从该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节。这项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. **