SARS-CoV-2 B.1.1.7核衣壳蛋白突变与宿主天然免疫应答及COVID-19临床状态的相关性

doi:10.11958/20251997

天津医药 ›› 2025, Vol. 53 ›› Issue (12): 1240-1245.doi: 10.11958/20251997

SARS-CoV-2 B.1.1.7核衣壳蛋白突变与宿主天然免疫应答及COVID-19临床状态的相关性

何先珍¹(), 付亚男², 尤婉玲³, 耿澳华³, 孙晓光³, 曾凤³, 刘龙¹^,³^,^△()

¹ 十堰市人民医院（湖北医药学院附属人民医院）儿童医疗中心（邮编442000）
² 玉溪市疾病预防控制中心
³ 湖北医药学院基础医学院病原生物学教研室

收稿日期:2025-05-16 修回日期:2025-08-04 出版日期:2025-12-15 发布日期:2025-12-08
通讯作者: ^△E-mail：liulong2015@outlook.com
作者简介:何先珍（1987），女，主治医师，主要从事感染性疾病诊疗研究。E-mail：xianzhenhappy@sina.com
基金资助:
国家自然科学基金资助项目(82002149);湖北省医学领军人才培养工程(2024-2027);十堰市“科技领军人才扶持计划”(2023)

The correlation between SARS-CoV-2 B.1.1.7 nucleocapsid protein mutation with host innate immune response and clinical manifestation of COVID-19

HE Xianzhen¹(), FU Ya'nan², YOU Wanling³, GENG Aohua³, SUN Xiaoguang³, ZENG Feng³, LIU Long¹^,³^,^△()

¹ Children's Medical Center, Shiyan People's Hospital (Affiliated People's Hospital of Hubei University of Medicine), Shiyan 442000, China
² Center for Disease Control and Prevention of Yuxi City
³ Department of Pathogenic Biology, School of Basic Medical Sciences, Hubei University of Medicine

Received:2025-05-16 Revised:2025-08-04 Published:2025-12-15 Online:2025-12-08
Contact: ^△E-mail：liulong2015@outlook.com

何先珍, 付亚男, 尤婉玲, 耿澳华, 孙晓光, 曾凤, 刘龙. SARS-CoV-2 B.1.1.7核衣壳蛋白突变与宿主天然免疫应答及COVID-19临床状态的相关性[J]. 天津医药, 2025, 53(12): 1240-1245.

HE Xianzhen, FU Ya'nan, YOU Wanling, GENG Aohua, SUN Xiaoguang, ZENG Feng, LIU Long. The correlation between SARS-CoV-2 B.1.1.7 nucleocapsid protein mutation with host innate immune response and clinical manifestation of COVID-19[J]. Tianjin Medical Journal, 2025, 53(12): 1240-1245.

摘要/Abstract

摘要：

目的阐明新型冠状病毒（SARS-CoV-2） B.1.1.7变异株核衣壳蛋白（N蛋白）特异性突变体（N_D3L、N_M234I和N_{R203K-G204R-T205I}）与新型冠状病毒感染（COVID-19）患者临床状态的相关性，揭示其对N蛋白液-液相分离（LLPS）现象和宿主天然免疫应答的影响。方法基于全球共享流感数据倡议组织（GISAID）数据库的SARS-CoV-2 B.1.1.7谱系全基因组测序数据，筛选与轻/重症临床表型显著相关的非同义突变位点。针对高频N蛋白突变体，采用双荧光素酶报告系统定量检测干扰素（IFN）-β启动子转录活性；qPCR检测IFN-β、白细胞介素（IL）-6、肿瘤坏死因子（TNF）-α mRNA表达水平；通过激光共聚焦显微观察LLPS特征。蛋白印迹检测宿主线粒体抗病毒信号蛋白（MAVS）的泛素化修饰状态。结果共鉴定出17 640个非同义突变位点，其中突变频率>1%且有65个与轻症相关，20个与重症相关。与重症相关的N蛋白突变位点为D3L、M234I、R203K-G204R-T205I。N蛋白及N_M234I、N_{R203K-G204R-T205I}突变体均可抑制IFN-β的启动子活性（P<0.05）。相较于野生型N，N_{R203K-G204R-T205I}突变体可显著降低IFN-β、IL-6和TNF-α mRNA水平（P<0.05），同时可通过分散LLPS凝聚点的形成而改变相分离状态。但N突变体不影响宿主MAVS的泛素化修饰。结论 SARS-CoV-2 B.1.1.7变异株N蛋白突变体可通过改变LLPS状态抑制天然免疫应答，进而影响COVID-19患者的临床预后。

关键词: 新型冠状病毒, 冠状病毒核壳蛋白质类, 干扰素β, 临床状态, B.1.1.7谱系, 液-液相分离

Abstract:

Objective To elucidate the correlation between specific nucleocapsid (N) protein mutant of the SARS-CoV-2 B.1.1.7 variant and clinical stratification in COVID-19 patients, revealing their impact on N protein liquid-liquid phase separation (LLPS) and host innate immune response. Methods Based on whole-genome sequencing data of the SARS-CoV-2 B.1.1.7 lineage from the GISAID database, non-synonymous mutation sites significantly associated with mild/severe clinical phenotypes were screened. For high-frequency N protein mutant, IFN-β promoter transcriptional activity was quantitatively measured using a dual-luciferase reporter system. qPCR was used to detect the mRNA expression levels of interferon (IFN)-β, interleukin (IL)-6 and tumor necrosis factor (TNF)-α. LLPS characteristics were observed by confocal microscopy. The ubiquitination status of host MAVS was detected by Western blot assay. Results A total of 17 640 non-synonymous mutation sites were identified, among which 65 were associated with mild cases and 20 were related to severe cases, with a mutation frequency >1%. The N protein mutation sites associated with severe cases were D3L, M234I and R203K-G204R-T205I. N protein and the mutants N_M234I, N_{R203K-G204R-T205I}inhibited the promoter activity of IFN-β (P<0.05). Compared to the wild type N protein, N_{R203K-G204R-T205I} mutation significantly reduced the mRNA levels of IFN-β, IL-6 and TNF-α (P<0.05), and altered the phase separation state by dispersing the formation of LLPS condensates. However, N mutant did not affect the ubiquitination modification of host MAVS. Conclusion N protein mutants of the SARS-CoV-2 B.1.1.7 variant can influence the clinical prognosis of COVID-19 patients by altering LLPS status and suppressing the innate immune responses. These finding provides a theoretical basis for the design of antiviral drugs targeting the N protein.

Key words: SARS-CoV-2, coronavirus nucleocapsid proteins, interferon-beta, clinical manifestations, B.1.1.7 lineage, liquid liquid phasese separation

中图分类号:

R373.1

图/表 6

参考文献 24

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基因名称	引物序列（5'→3'）	产物大小/bp
IFN-β	上游：ATTGCCTCAAGGACAGGAG	167
	下游：GGCCTTCAGGTAATGCAGAA	167
IL-6	上游：CCTGACCCAACCACA	142
	下游：CTACATTTGCCGAAGAG	142
TNF-α	上游：CAAGCCCTGGTATGAGC	179
	下游：GCAATGATCCCAAAGTAGA	179
GAPDH	上游：GAGTCAACGGATTTGGTCGT	183
	下游：TGGGATTTCCATTGATGACA	183

基因名称	引物序列（5'→3'）	产物大小/bp
IFN-β	上游：ATTGCCTCAAGGACAGGAG	167
	下游：GGCCTTCAGGTAATGCAGAA	167
IL-6	上游：CCTGACCCAACCACA	142
	下游：CTACATTTGCCGAAGAG	142
TNF-α	上游：CAAGCCCTGGTATGAGC	179
	下游：GCAATGATCCCAAAGTAGA	179
GAPDH	上游：GAGTCAACGGATTTGGTCGT	183
	下游：TGGGATTTCCATTGATGACA	183

突变蛋白	轻症	重症
ORF3	G172V、G196V、Q57H、S171L、S74F、W131C	P42L
ORF6	无	I33T
ORF7a	无	L116F
ORF8	Q27stop、R52I、Y73C、L84S、C61F、S24L、K68stop、R24C	T11I
N	D3L、P67S、S194L、S197L、P199L、S202N、R203K、G204R、T205I、S235F、T362I、A376T	D3L、M234I、R203K-G204R-T205I
Spike	A243del、A570D、A701V、D1118H、D1259H、D215G、D80A、E1258D、E484K、H69del、K417N、L18F、L242del、L244del、N501Y、P681H、S477N、S982A、S98F、T716I、T732A、V1176F、V143del、V70del、Y144del	D950N、E156G、F157del、G142D、R158del、T19R
NSP2	L550F、R27C、T85I、V381A	P129L
NSP4	F17L、F308Y、M324I、T492I	A446V、L438P
NSP6	F108del、G107del、I49V、S106del	V149A
NSP7	无	L71F、S25L
NSP14	N129D、P451S	P46L

突变蛋白	轻症	重症
ORF3	G172V、G196V、Q57H、S171L、S74F、W131C	P42L
ORF6	无	I33T
ORF7a	无	L116F
ORF8	Q27stop、R52I、Y73C、L84S、C61F、S24L、K68stop、R24C	T11I
N	D3L、P67S、S194L、S197L、P199L、S202N、R203K、G204R、T205I、S235F、T362I、A376T	D3L、M234I、R203K-G204R-T205I
Spike	A243del、A570D、A701V、D1118H、D1259H、D215G、D80A、E1258D、E484K、H69del、K417N、L18F、L242del、L244del、N501Y、P681H、S477N、S982A、S98F、T716I、T732A、V1176F、V143del、V70del、Y144del	D950N、E156G、F157del、G142D、R158del、T19R
NSP2	L550F、R27C、T85I、V381A	P129L
NSP4	F17L、F308Y、M324I、T492I	A446V、L438P
NSP6	F108del、G107del、I49V、S106del	V149A
NSP7	无	L71F、S25L
NSP14	N129D、P451S	P46L

组别		IL-6 mRNA	TNF-α mRNA
对照组		1.00±0.03	1.00±0.23
RIG-I组		3.51±0.48	4.79±0.79
N组		1.52±0.27^a	1.65±0.49^a
N_D3L组		6.29±0.95^b	6.95±0.94^b
N_M234I组		2.07±0.39^ab	2.39±0.51^ab
N_{R203K-G204R-T205I}组		0.75±0.04^ab	0.54±0.05^ab
F		11.370^＊＊	19.430^＊＊
组别	IFN-β-Luc活性			IFN-β mRNA
对照组	1.00±0.06			1.00±0.42
RIG-I组	13.81±0.81			15.83±1.30
N组	6.79±0.48^a			3.48±0.48^a
N_D3L组	14.58±0.96^b			19.30±1.50^b
N_M234I组	9.08±0.73^ab			9.12±1.94^ab
N_{R203K-G204R-T205I}组	6.66±0.43^a			2.73±0.70^ab
F	76.120^＊＊			31.060^＊＊

SARS-CoV-2 B.1.1.7核衣壳蛋白突变与宿主天然免疫应答及COVID-19临床状态的相关性

The correlation between SARS-CoV-2 B.1.1.7 nucleocapsid protein mutation with host innate immune response and clinical manifestation of COVID-19

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参考文献 24

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组别	GFP	FLAG	Ub-K63
对照组	0.03±0.01	0.04±0.01	0.67±0.17
MAVS-Flag组	0.07±0.02	0.72±0.10^a	9.76±0.12^a
N-EGFP组	0.95±0.12^a	0.56±0.08^a	9.46±0.23^a
N_D3L-EGFP组	1.26±0.12^a	0.73±0.08^a	8.72±0.38^a
N_M234I-EGFP组	1.07±0.11^a	0.75±0.07^a	8.13±0.40^a
N_{R203K-G204R-T205I}-EGFP组	1.30±0.15^a	0.71±0.10^a	9.69±0.34^a
F	32.430^＊＊	12.680^＊＊	144.300^＊＊

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