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

• Research Article • Previous Articles Next Articles

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

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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

Funding: Innovative Research Group Foundation of the National Natural Science Foundation of China (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].

References

1. Musso D, Gubler DJ.Zika virus. Clin Microbiol Rev 2016; 29(3):487-524. doi: 10.1128/CMR.00072-15.
2. Plourde AR, Bloch EM.A literature review of Zika virus. Emerg Infect Dis 2016; 22(7):1185-92. doi: 10.3201/eid2207.151990.
3. Hasan S, Saeed S, Panigrahi R, et al.Zika virus: a global public health menace: a comprehensive update. J Int Soc Prev Community Dent 2019; 9(4):316-27. doi: 10.4103/jispcd.JISPCD_433_18.
4. Macnamara FN.Zika virus: a report on three cases of human infection during an epidemic of jaundice in Nigeria. Trans R Soc Trop Med Hyg 1954; 48(2):139-45. doi: 10.1016/0035-9203(54)90006-1.
5. Dick GW, Kitchen SF, Haddow AJ.Zika virus. I. Isolations and serological specificity. Trans R Soc Trop Med Hyg 1952; 46(5):509-20. doi: 10.1016/0035-9203(52)90042-4.
6. Barrows NJ, Campos RK, Liao KC, et al.Biochemistry and molecular biology of Flaviviruses. Chem Rev 2018;118(8):4448-82. doi: 10.1021/acs.chemrev.7b00719.
7. Leonhard SE, Mandarakas MR, Gondim FAA, et al.Diagnosis and management of Guillain-Barré syndrome in ten steps. Nat Rev Neurol 2019;15(11):671-83. doi: 10.1038/s41582-019-0250-9.
8. Cao-Lormeau VM, Blake A, Mons S, et al.Guillain-Barré Syndrome outbreak associated with Zika virus infection in French Polynesia: a case-control study. Lancet 2016; 387(10027):1531-9. doi: 10.1016/S0140-6736(16)00562-6.
9. Hancock WT, Marfel M, Bel M.Zika virus, French Polynesia, South Pacific, 2013. Emerg Infect Dis 2014; 20(11):1960. doi: 10.3201/eid2011.141380.
10. Musso D, Nilles EJ, Cao-Lormeau VM.Rapid spread of emerging Zika virus in the Pacific area. Clin Microbiol Infect 2014; 20(10):O595-6. doi: 10.1111/1469-0691.12707.
11. Musso D, Ko AI, Baud D.Zika virus infection—after the pandemic. N Engl J Med 2019; 381(15):1444-57. doi: 10.1056/NEJMra1808246.
12. Song Y, Mugavero J, Stauft CB, et al.Dengue and Zika virus 5' untranslated regions harbor internal ribosomal entry site functions. mBio 2019; 10(2):e00459-19. doi: 10.1128/mBio.00459-19.
13. Shi Y, Gao GF.Structural biology of the Zika virus. Trends Biochem Sci 2017; 42(6):443-56. doi: 10.1016/j.tibs.2017.02.009.
14. Pelletier J, Sonenberg N.Internal initiation of translation of eukaryotic mRNA directed by a sequence derived from poliovirus RNA. Nature 1988; 334(6180):320-5. doi: 10.1038/334320a0.
15. Jang SK, Kräusslich HG, Nicklin MJ, et al.A segment of the 5' nontranslated region of encephalomyocarditis virus RNA directs internal entry of ribosomes during in vitro translation. J Virol 1988; 62(8):2636-43. doi: 10.1128/JVI.62.8.2636-2643.
16. Jaafar ZA, Kieft JS.Viral RNA structure-based strategies to manipulate translation. Nat Rev Microbiol 2019; 17(2):110-23. doi: 10.1038/s41579-018-0117-x.
17. Mailliot J, Martin F.Viral internal ribosomal entry sites: four classes for one goal. Wiley Interdiscip Rev RNA 2018; 9(2):e1458. doi: 10.1002/wrna.1458.
18. Godet AC, David F, Hantelys F, et al.IRES trans-acting factors, key actors of the stress response. Int J Mol Sci 2019; 20(4):924. doi: 10.3390/ijms20040924.
19. López-Ulloa B, Fuentes Y, Pizarro-Ortega MS, et al.RNA-binding proteins as regulators of internal initiation of viral mRNA translation. Viruses 2022; 14(2):188. doi: 10.3390/v14020188.
20. Kafasla P, Morgner N, Robinson CV, et al.Polypyrimidine tract-binding protein stimulates the poliovirus IRES by modulating eIF4G binding. EMBO J 2010; 29(21):3710-22. doi: 10.1038/emboj.2010.231.
21. Asnani M, Kumar P, Hellen CU.Widespread distribution and structural diversity of Type IV IRESs in members of Picornaviridae. Virology 2015; 478:61-74. doi: 10.1016/j.virol.2015.02.016.
22. Kafasla P, Morgner N, Pöyry TA, et al.Polypyrimidine tract binding protein stabilizes the encephalomyocarditis virus IRES structure via binding multiple sites in a unique orientation. Mol Cell 2009; 34(5):556-68. doi: 10.1016/j.molcel.2009.04.015.
23. Xi J, Ye F, Wang G, et al.Polypyrimidine tract-binding protein regulates Enterovirus 71 translation through interaction with the internal ribosomal entry site. Virol Sin 2019; 34(1):66-77. doi: 10.1007/s12250-019-00089-1.
24. Schultz DE, Hardin CC, Lemon SM.Specific interaction of glyceraldehyde 3-phosphate dehydrogenase with the 5'-nontranslated RNA of hepatitis A virus. J Biol Chem 1996; 271(24):14134-42. doi: 10.1074/jbc.271.24.14134.
25. Ali N, Siddiqui A.Interaction of polypyrimidine tract-binding protein with the 5' noncoding region of the hepatitis C virus RNA genome and its functional requirement in internal initiation of translation. J Virol 1995; 69(10):6367-75. doi: 10.1128/JVI.69.10.6367-6375.1995.
26. Hunt SL, Jackson RJ.Polypyrimidine-tract binding protein (PTB) is necessary, but not sufficient, for efficient internal initiation of translation of human rhinovirus-2 RNA. RNA 1999; 5(3):344-59. doi: 10.1017/s1355838299981414.
27. Verma B, Bhattacharyya S, Das S.Polypyrimidine tract-binding protein interacts with coxsackievirus B3 RNA and influences its translation. J Gen Virol 2010; 91(Pt 5):1245-55. doi: 10.1099/vir.0.018507-0.
28. Kafasla P, Mickleburgh I, Llorian M, et al.Defining the roles and interactions of PTB. Biochem Soc Trans 2012; 40(4):815-20. doi: 10.1042/BST20120044.
29. Iioka H, Loiselle D, Haystead TA, et al.Efficient detection of RNA-protein interactions using tethered RNAs. Nucleic Acids Res 2011; 39(8):e53. doi: 10.1093/nar/gkq1316. Epub 2011 Feb 7.
30. De Nova-Ocampo M, Villegas-Sepúlveda N, del Angel RM. Translation elongation factor-1alpha, La, and PTB interact with the 3' untranslated region of dengue 4 virus RNA. Virology 2002; 295(2):337-47. doi: 10.1006/viro.2002.1407.

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