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 (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.