Screening and molecular docking for host proteins interacting with human rotavirus nonstructural protein 2
10.3760/cma.j.cn112309-20230712-00207
- VernacularTitle:人轮状病毒非结构蛋白2与宿主互作蛋白的筛选及分子对接研究
- Author:
Jiafeng LIN
1
;
Siman HU
;
Zhuojing JIANG
;
Wei ZHAO
;
Yonggang LI
Author Information
1. 绍兴市疾病预防控制中心微生物检验科，绍兴 312071
- Keywords:
Rotavirus;
NSP2;
Interacting protein;
Molecular docking
- From:
Chinese Journal of Microbiology and Immunology
2023;43(11):886-892
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To screen host cell proteins interacting with rotavirus nonstructural protein 2(NSP2) and provide a theoretical basis for the discovery of antiviral targets.Methods:E. coli BL21(DE3) was transformed with a recombinant plasmid NSP2-pGEX-6P-1 and induced by IPTG to express NSP2-GST. GST affinity chromatography was used to purify NSP2-GST and Western blot was performed for verification. NSP2-GST and GST proteins were used as targets to capture interacting proteins in MA104 cells via GST pull-down. After silver staining, differential proteins were screened by in-gel enzymatic hydrolysis and mass spectrometry. Protein pilot platform was used to filter peptides, and the names and the biological functions of the proteins were obtained by Paragon algorithm. The potential connection between the interacting proteins was demonstrated by protein interaction network diagrams and GO functional annotation analysis. Moreover, the molecular docking of the top three proteins with NPS2 was predicted using HDOCK server and verified by docking and confidence scores, so as to investigate the visual docking model between interacting proteins. Results:SDS-PAGE and Western blot showed the successful purification of the recombinant protein NSP2. Ten host proteins including PKM2, which might interact with NSP2, were identified by GST pull-down and protein profiling. GO analysis and interaction diagrams revealed that RPS4X, EZR, SUPT16H and EIF2S3 mediated molecular expression; PKM2, LDHA and ATP5A1 participated in energy metabolism; HSP90, ACTB and ANXA2 were involved in biological movement. Besides, there were functional connections and interaction networks among them. Molecular docking further verified PKM2, HSP90 and RPS4X did interact with NSP2, and the interaction force were strong enough to form a stable structure.Conclusions:This study successfully discovered several host proteins including PKM2, HSP90 and RPS4X that could interact with NSP2, providing reference for investigating the process of rotavirus infection and making related prevention and control strategies.
