Viral protein translation modulation

病毒蛋白翻译调节

• 批准号: RGPIN-2018-04138
• 负责人: Liu, Qiang
• 金额: $ 2.33万
• 依托单位: University of Saskatchewan
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=712753
• 关键词: Viral; protein; translation; modulation

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.

丙型肝炎病毒（HCV）是一种正义单链RNA病毒，是黄病毒科肝炎病毒属的成员。近年来，已从许多动物物种（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，HCV的基础研究不仅对于了解HCV生物学至关重要，而且还可以作为了解动物肝炎病毒生物学的优秀模型病毒。 该研究项目的长期目标是以HCV为模型研究病毒翻译调节的分子机制。在当前的 NSERC Discovery 资助期间（2013-2018），我们正在研究非结构蛋白 5A (NS5A) 在 HCV RNA 翻译中的调节作用以及病毒 3 非翻译区 (3UTR) 的功能。我们发现 NS5A 下调 HCV 翻译。病毒 3UTR 中的聚 (U/UC) 序列是 NS5A 翻译下调所必需的。详细的作图实验表明，NS5A 结构域 I 中的氨基酸 R112、结构域 II 中的 K312 和结构域 III 中的 E446 参与翻译下调。从机制上讲，我们发现 NS5A 结构域 I 中的 R112 突变会消除其与聚（U/UC）RNA 的结合，这表明 NS5A - 聚（U/UC）RNA 相互作用可能代表了 NS5A 调节病毒翻译的机制。我们还研究了 PI3K-Akt 通路对 HCV RNA 翻译的影响。我们发现该途径在所有三种 Akt 亚型的参与下增加了 HCV 翻译。这项研究由三名研究生和一名博士后研究员进行，发表了五篇出版物。根据我们正在进行的研究，该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。我们将测试 HCV NS5A 蛋白通过五个目标的多种机制调节 HCV RNA 翻译的假设。 1. 描述 HCV 3UTR 中的可变区和 X 区在 NS5A 病毒 RNA 翻译调节中的作用。 2. 表征 NS5A 二聚化对 HCV RNA 翻译的影响。 3. 表征 NS5A 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 NS5A 对 HCV 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HCV翻译中的作用。 该提案将继续我们以 HCV 作为模型进行病毒翻译调节的研究计划。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，从该提案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。拟议的研究将由研究生进行。