多嘧啶束结合蛋白通过与内部核糖体进入位点的5'UTR结合增强寨卡病毒的翻译能力

doi:10.24920/004393

Chinese Medical Sciences Journaldoi: 10.24920/004393

多嘧啶束结合蛋白通过与内部核糖体进入位点的5'UTR结合增强寨卡病毒的翻译能力

茉丽都尔·哈米提¹, 张心湉², 朱瑞敏^3,4, 刘云鹏^3,4, 阴彬¹, 舒鹏程¹, 彭小忠^1,3,4

¹中国医学科学院基础医学研究所北京协和医学院基础学院,重大疾病共性机制研究全国重点实验室,国家医学分子生物重点实验室,医学灵长类动物研究中心,神经科学中心,北京 100005;
²中国医学科学院北京协和医学院,医学生物学研究所,昆明 650031,中国;
³中国医学科学院北京协和医学院,呼吸和共病全国重点实验室,北京 100021;
⁴中国医学科学院北京协和医学院,医学实验动物研究所,北京 100021

收稿日期:2024-06-28 接受日期:2024-08-13 发布日期:2024-09-25

Polypyrimidine Tract-Binding Protein Enhances Zika Virus Translation by Binding to the 5' UTR of Internal Ribosomal Entry Site

Moliduer Hamiti¹, Xin-Tian Zhang², Rui-Min Zhu^3,4, Yun-Peng Liu^3,4, Bin Yin¹, Peng-Cheng Shu¹, Xiao-Zhong Peng^1,3,4

¹State Key Laboratory of Common Mechanism Research for Major Diseases, Department of Molecular Biology and Biochemistry, Medical Primate Research Center, Neuroscience Center, Institute of Basic Medical Sciences Chinese Academy of Medical Sciences & School of Basic Medicine Peking Union Medical College, Beijing 100005, China;
²Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Kunming 650031, China;
³State Key Laboratory of Respiratory Health and Multimorbidity, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100021, China;
⁴Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100021, China

Received:2024-06-28 Accepted:2024-08-13 Online:2024-09-25
Contact: ^* E-mail: 15299037756@163.com

1. 4393附带文件.pdf(483KB)

摘要/Abstract

摘要： 目的识别寨卡病毒（Zika virus,ZIKV）5'非翻译区（5' untranslated region,5'UTR）的RNA结合蛋白,并探讨结合蛋白对位于ZIKV5'UTR的内部核糖体进入位点（internal ribosomal entry site,IRES）活性及病毒产生的影响。
方法使用tRSA标记的ZIKV 5'UTR RNA捕获U251细胞中与之相互作用的蛋白,采用SDS-PAGE银染法获得tRSA-ZIKV 5'UTR RNA候选结合蛋白的条带。随后,利用液相色谱-串联质谱法、生物信息学分析和Western blot分析研究与ZIKV 5'UTR结合的候选蛋白。采用双顺反子表达实验和蚀斑形成实验分析结合蛋白对ZIKV IRES活性和ZIKV产生的影响。
结果 tRSA RNA pull-down分析、液相色谱-串联质谱法和Western blot分析显示PTB为与ZIKV 5'UTR结合的蛋白。双荧光素酶报告基因检测结果显示：PTB过表达能显著增强ZIKV的IRES活性（t = 10.220,P < 0.001）,而敲除PTB则效果相反（t = 4.897,P < 0.01）。此外,病毒噬斑形成实验表明：上调PTB表达能显著提高病毒滴度（t = 6.400,P < 0.01）,而降低PTB表达水平则会削弱病毒的感染能力（t = 5.055,P < 0.01）。
结论 PTB对ZIKV 5'UTR发挥正向调节作用,能增强IRES活性和提高病毒产量。

关键词: 内部核糖体进入位点, 多嘧啶束结合蛋白, 寨卡病毒, tRSA RNA pull-down, 双荧光素酶报告基因检测

Abstract: Objectives To identify the 5' untranslated region of Zika virus (ZIKV5'UTR) RNA-binding proteins and to investigate the impact of the binding protein on the activity of internal ribosomal entry site (IRES) located in ZIKV5'UTR and virus production.
Methods Interacting proteins in U251 cells were captured using tRSA-tagged ZIKV 5'UTR RNA and tRSA-ZIKV 5'UTR RNA-binding proteins were visualized by SDS-PAGE silver staining. Subsequently, liquid chromatography-tandem mass spectrometry (LC-MS/MS), bioinformatics analysis, and western blot were used to identify the candidate proteins binding to ZIKV5'UTR. Dicistronic expression assay and plaque forming assay were performed to analyze the effect of the binding protein on ZIKV IRES activity and ZIKV production.
Results tRSA RNA pull-down assay, LC-MS/MS, and western blot analysis showed that polypyrimidine tract-binding protein (PTB) bound to the ZIKV 5'UTR Furthermore, dual luciferase reporter assay revealed that overexpression of PTB significantly enhanced the IRES activity of ZIKV (t = 10.220, P < 0.001), while PTB knockdown had the opposite effect (t = 4.897, P < 0.01). Additionally, virus plaque forming assay demonstrated that up-regulation of PTB expression significantly enhanced viral titer (t = 6.400, P < 0.01), whereas reducing PTB expression level weakened virus infectivity (t = 5.055, P < 0.01).
Conclusion PTB positively interacts with the ZIKV 5'UTR and enhances IRES activity and virus production.

Key words: internal ribosomal entry site, polypyrimidine tract-binding protein, Zika virus, tRSA RNA pull-down, dual-luciferase reporter assay

基金资助: 国家自然科学基金创新研究群体项目（82221004）

Moliduer Hamiti, Xin-Tian Zhang, Rui-Min Zhu, Yun-Peng Liu, Bin Yin, Peng-Cheng Shu, Xiao-Zhong Peng. Polypyrimidine Tract-Binding Protein Enhances Zika Virus Translation by Binding to the 5' UTR of Internal Ribosomal Entry Site[J/OL].Chinese Medical Sciences Journal, .DOI:10.24920/004393.[Epub ahead of print].

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