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

doi:10.11958/20251997

Abstract

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

CLC Number:

R373.1

Figures/Tables 6

References 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^＊＊