Expression and localization of fever with thrombocytopenia syndrome virus nonstructural protein and screening and analysis of host-interacting proteins
10.13481/j.1671-587X.20240512
- VernacularTitle:发热伴血小板减少综合征病毒非结构蛋白的表达和定位及宿主互作蛋白的筛选和分析
- Author:
Like LUO
1
;
Ziwen CHENG
;
Kuo CHENG
;
Yonggang LI
;
Dawei WANG
;
Baoling YANG
Author Information
1. 锦州医科大学基础医学院病原生物学教研室,辽宁锦州 121000
- Keywords:
Severe fever with thrombocytopenia syndrome virus;
Non-structural protein;
Protein-protein interaction;
Immunoprecipitation;
Mass-spectrometric technique
- From:
Journal of Jilin University(Medicine Edition)
2024;50(5):1286-1296
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To screen the host interaction proteins of the severe fever with thrombocytopenia syndrome virus(SFTSV)nonstructural protein(NSs)by immunoprecipitation combined with mass spectrometry analysis,to discuss the functions,subcellular localization,and biological pathways of these interaction proteins,and to provide the basis for clarifying the replication and pathogenic mechanism of SFTSV.Methods:The eukaryotic expression vectors pSFTSV-NSs-Flag(experimental group)and Flag-CMV-3(negative group)were transfected into the human embryonic kidney 293T cells,and contorl group(no treatment)was set up.The lysates of the cells in various groups were collected,and the expression and localization of SFTSV NSs in the host cells were verified by indirect immunofluorescence and Western blotting methods.The protein lysates were treated with protein A/G and immunoprecipitation was used to enrich host proteins binding to NSs.The captured interaction proteins were initially analyzed by silver staining and Coomassie brilliant blue staining to observe the differential protein bands in various groups;liquid chromatography-tandem mass spectrometry was used to obtain the information of protein sequences;the reliable proteins were retained and searched by UniProt database;Gene Ontology(GO)functional enrichment analysis,IPR,eukaryotic orthologous groups(KOGs)functional annotation,Kyoto Encyclopedia of Genes and Genomes(KEGG)signaling pathway enrichment analysis,subcellular localization,and transcription factor(TF)functional annotation were used to determine the subcellular structure,gene functions,and biological processes of the interaction proteins.Results:The immunofluorescence results showed that the SFTSV NSs expressed a single specific band at relative molecular mass 33 000 and was localized in the cytoplasm in a granular inclusion body-like manner.The silver staining and Coomassie brilliant blue staining results showed there were significant differential protein bands between experimental group and negative group.The mass spectrometry results identified 46 potential interaction proteins.The GO functional enrichment analysis,KOGs functional annotation,and KEGG signaling pathway enrichment analysis results showed that the biological pathways related to viral translation,cellular metabolism,and protein transport were enriched with a considerable number of proteins.Eight annotated proteins had intermediate filament domains.The highest percentage of subcellular localization was cytoplasmic proteins,consistent with the NSs localization site.The TF functional annotation analysis results showed one protein from the NF-Y family.Conclusion:The interaction proteins play roles in assisting the proper protein folding,participating in the cribosome translation,and forming the cytoskeleton,which may be involved in antiviral replication.These proteins can be used as candidate proteins for further study on the replication mechanism of SFTSV.
