多嘧啶簇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

Q2 Medicine

Chinese Medical Sciences Journal Pub Date : 2024-09-01 DOI:10.24920/004393

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

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引用次数: 0

摘要

目的确定寨卡病毒（ZIKV5'UTR）5'非翻译区 RNA 结合蛋白，并研究结合蛋白对位于 ZIKV5'UTR 的内部核糖体入口位点（IRES）活性和病毒产生的影响。方法使用tRSA标记的ZIKV 5'UTR RNA捕获U251细胞中的相互作用蛋白，并通过SDS-PAGE银染色法观察tRSA-ZIKV 5'UTR RNA结合蛋白。随后，利用液相色谱-串联质谱（LC-MS/MS）、生物信息学分析和 Western 印迹来鉴定与 ZIKV5'UTR 结合的候选蛋白。进行了双链表达试验和斑块形成试验，以分析结合蛋白对 ZIKV IRES 活性和 ZIKV 生成的影响。此外，双荧光素酶报告实验表明，过表达 PTB 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，病毒斑块形成试验表明，上调 PTB 表达可显著提高病毒滴度（t = 6.400，P < 0.01），而降低 PTB 表达水平则会削弱病毒传染性（t = 5.055，P < 0.01）。结论 PTB 与 ZIKV 5'UTR 呈正向相互作用，可增强 IRES 活性和病毒生成。

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

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 chromatographytandem 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 (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).