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

Moliduer Hamiti; Xin-Tian Zhang; Rui-Min Zhu; Yun-Peng Liu; Bin Yin; Peng-Cheng Shu; Xiao-Zhong Peng

doi:10.24920/004393

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

Chinese Medical Sciences Journal Vol. 39, Issue 3, Pages: 162-170(2024)

Research Articles | Updated：2025-01-08

- Polypyrimidine Tract-Binding Protein Enhances Zika Virus Translation by Binding to the 5'UTR of Internal Ribosomal Entry Site
- Affiliations：
  
  1.State 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
  2.Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Kunming 650031, China
  3.State Key Laboratory of Respiratory Health and Multimorbidity, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100021, China
  4.Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100021, China
- Author bio：
  
  E-mail: pengxiaozhong@pumc.edu.cn
- Funds：
- DOI：10.24920/004393
  CLC：
- Published：30 September 2024，
  
  Published Online：25 September 2024，
  
  Received：28 June 2024，
  
  Accepted：2024-08-13
- Accepted：
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茉丽都尔·哈米提 ,张心湉,朱瑞敏等.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[J]. 中国医学科学杂志（英文）,2024,39(03):162-170.

Hamiti Moliduer,Zhang Xin-Tian,Zhu Rui-Min,et al.Polypyrimidine Tract-Binding Protein Enhances Zika Virus Translation by Binding to the 5'UTR of Internal Ribosomal Entry Site[J].Chinese Medical Sciences Journal,2024,39(03):162-170.
茉丽都尔·哈米提 ,张心湉,朱瑞敏等.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[J]. 中国医学科学杂志（英文）,2024,39(03):162-170. DOI： 10.24920/004393.

Hamiti Moliduer,Zhang Xin-Tian,Zhu Rui-Min,et al.Polypyrimidine Tract-Binding Protein Enhances Zika Virus Translation by Binding to the 5'UTR of Internal Ribosomal Entry Site[J].Chinese Medical Sciences Journal,2024,39(03):162-170. DOI： 10.24920/004393.

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ZIKV 5’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分析显示多嘧啶束结合蛋白（polypyrimidine tract-binding protein，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（

= 10.220，

0.001），而降低PTB表达则效果相反（

= 4.897，

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（

= 6.400，

0.01），而降低PTB表达水平则会削弱病毒的感染能力（

= 5.055，

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。

Abstract

Objectives

To identify the 5' untranslated region of Zika virus (ZIKV 5'UTR) RNA-binding proteins and to investigate the impact of the binding protein on the activity of internal ribosomal entry site (IRES) located in ZIKV 5'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 ZIKV 5'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

respecitvely.

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 (

= 10.220

0.001)

while PTB knockdown had the opposite effect (

= 4.897

0.01). Additionally

virus plaque forming assay demonstrated that up-regulation of PTB expression significantly enhanced viral titer (

= 6.400

0.01)

whereas reducing PTB expression level weakened virus infectivity (

= 5.055

0.01).

Conclusion

PTB positively interacts with the ZIKV 5'UTR and enhances IRES activity and virus production.

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

Keywords

internal ribosomal entry sitepolypyrimidine tract-binding proteinZika virustRSA RNA pull-downdual-luciferase reporter assay

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