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

doi:10.13481/j.1671-587X.20240512

Abstract

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.

Key words: Severe fever with thrombocytopenia syndrome virus, Non-structural protein, Protein- protein interaction, Immunoprecipitation, Mass-spectrometric technique

CLC Number:

R373.3

Like LUO,Ziwen CHENG,Kuo CHENG,Yonggang LI,Dawei WANG,Baoling YANG. Expression and localization of fever with thrombocytopenia syndrome virus nonstructural protein and screening and analysis of host-interacting proteins[J].Journal of Jilin University(Medicine Edition), 2024, 50(5): 1286-1296.

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References 34

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Protein	Gene	PSM	Feature or function
H6VRF8	KRT1	8	Cytoskeletal cross-linking
A0A7I2YQB2	HSPA9	7	Cooperative immunity
A0A7P0TAI0	HSPA5	7	Protein folding companion
A5A3E0	POTEF	3	Promote apoptosis
Q15084	PDIA6	1	Inhibition of misfolded protein aggregation
A0A7S5EUS7	IGHV1-69	1	Activated in response to cellular stress
B4DI39	HSP70-1	3	ATP-dependent protein folding chaperone
Q14257	RCN2	1	Calcium binding
Q4VB24	HIST1H1E	1	Structural components of chromatin
E7EUT5	GAPDH	1	Catalytic activity
B4DJ30	Neutral alpha-glucosidase AB	2	Hydrolase activity
Q6LC01	β-Tubulin	2	Structural components of the cytoskeleton
Q05639	EEF1A2	1	Promoting GTP-dependent binding of aminoacyl tRNAs to the ribosomal A site
V9HWE1	HEL113	1	Binding protein
A6NG45	PRDX4	1	Antioxidant protein
A0A8I5KW59	ERP44	2	Regulation of calcium release
B2R4R0	HIST1H4J	1	Protein transmembrane transport
P36578	RPL4	1	Protein synthesis in cells
H3BT71	RBMX	1	Antiviral activity
A0A024R943	TOR3A	1	Binding protein
Q969H8	MYDGF	1	Secretory protein
J3KRX5	RPL17	1	Structural protein
P69905	HBA1	1	Oxygen transportation
Q6GMX0	Ig-like domains	1	Structural domain protein (SDP)
S6BAN6	IgG L	1	Structural protein

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Expression and localization of fever with thrombocytopenia syndrome virus nonstructural protein and screening and analysis of host-interacting proteins

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