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

摘要/Abstract

摘要：

目的通过免疫沉淀联合质谱分析的方法筛选发热伴血小板减少综合征病毒（SFTSV）非结构蛋白（NSs）的宿主互相作用（互作）蛋白，探讨互作蛋白的功能、亚细胞定位及参与的生物途径，为阐明SFTSV的复制和致病机制提供依据。方法将真核表达载体pSFTSV-NSs-Flag（实验组）和Flag-CMV-3（阴性组）分别转染进人胚胎肾293T细胞中，同时设置空白组（不进行任何处理）。收集各组细胞裂解液，采用间接免疫荧光和Western blotting法验证SFTSV NSs在宿主细胞中的表达及定位。蛋白裂解液经protein A/G处理，采用免疫沉淀法富集与NSs结合的宿主蛋白，通过银染和考马斯亮蓝染色初步分析捕获的互作蛋白，观察各组蛋白差异条带。采用液相色谱和串联质谱技术得到蛋白质的序列信息，保留可信蛋白基于UniProt数据库检索，对鉴定到的蛋白进行基因本体论（GO）功能富集分析、蛋白结构域和功能位点数据库（IPR）、真核生物同源蛋白簇（KOGs）功能注释、京都基因与基因组百科全书（KEGG）信号通路富集分析、亚细胞定位和转录因子（TF）功能注释，确定互作蛋白的亚细胞结构、基因功能及参与的生物过程。结果 SFTSV NSs在相对分子质量33 000处表达单一特异性条带，免疫荧光检测其定位于细胞质中，且聚集呈砂粒体包涵体样。银染和考马斯亮蓝染色，实验组和阴性组均存在显著差异条带。质谱筛选出46种潜在互作蛋白；GO功能富集分析、KOGs功能注释和KEGG信号通路富集分析，与病毒翻译、细胞代谢及蛋白质运输相关的生物途径富集到较多数目的蛋白，注释中有8种蛋白具有中间丝蛋白结构域。亚细胞定位所占百分率最高的是细胞质蛋白；与NSs定位场所一致。TF功能注释，有1种蛋白来自NF-Y家族。结论互作蛋白在协助蛋白质正确折叠、参与核糖体翻译和构成细胞骨架等进程中发挥作用，可能参与了抗病毒复制，可作为候选蛋白进一步研究SFTSV的复制机制。

关键词: 发热伴血小板减少综合征病毒, 非结构蛋白, 蛋白-蛋白互相作用, 免疫沉淀, 质谱

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

中图分类号:

R373.3

骆丽可,程子文,程廓,李永刚,王大为,杨宝玲. 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[J]. 吉林大学学报(医学版), 2024, 50(5): 1286-1296.

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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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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