Preliminary Study On The Effects Of SARS-CoV-2 Nsp1 Protein On NF-κB Signal Pathway

Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)infection causes an acute respiratory disease with high incidence rate and strong infectivity,namely novel coronavirus disease 2019(COVID-19).The SARS CoV-2 genome is a single stranded positive strand RNA,which can encode 29 viral protein.Nsp1 protein is the first non structural protein encoded by SARS-CoV-2 virus and is a determining factor affecting virus virulence.Previous studies have found that SARS-CoV-2 Nsp1 protein can inhibit the production of inflammatory factors(IL-6and IL-8),but the mechanism is still unclear.In order to further investigate the function of SARS-CoV-2 Nsp1 protein,this study first constructed eukaryotic recombinant vectors p CMV-Myc-Nsp1 and p EGFP-N1-Nsp1 using seamless cloning technology.Western blot technology was used to test the successful expression of SARS-CoV-2 Nsp1 recombinant plasmid in cells.Subsequently,the prokaryotic expression plasmid p ET-28a-SARS-CoV-2-Nsp1 was purchased,and the prokaryotic recombinant plasmid was induced by IPTG to express and purify the protein.The SARS-CoV-2 Nsp1 protein was purified by Coomassie Brilliant Blue staining,Western blot and affinity chromatography.When the NF-κB signaling pathway is activated,it can mediate the release of downstream inflammatory factors.To investigate the effect of SARS-CoV-2 Nsp1 protein on the production of inflammatory factors,this study transfected p CMV-Myc-Nsp1 and p EGFP-N1-Nsp1 into cells,respectively.Under TNF-α and LPS stimulation or overexpression of My D88 and p65 conditions,the transcription of inflammatory factors IL-6,IL-8,and TNF-α mRNA was detected using RT-PCR technology.The results showed that SARS-CoV-2 Nsp1 protein can inhibit the transcription of inflammatory factors IL-6,IL-8,and TNF-α m RNA mediated by TNF-α,LPS,My D88,and p65.These results indicate that SARS-CoV-2 Nsp1 protein can inhibit the production of inflammatory factors.The p65,p50,and IκBα Proteins play a crucial role in the NF-KB signaling pathway.To further investigate the effects of SARS-CoV-2Nsp1 protein on p65,p50,and IκBα Does the protein have an impact.In this study,recombinant plasmids were transfected into cells and Western blot and RT-PCR techniques were used to detect p65,p50,and IκBα The protein expression and m RNA transcription levels of.The results showed that SARS-CoV-2Nsp1 protein could inhibit the transcription level of p65 mRNA.These results indicate that the SARS-CoV-2Nsp1 protein inhibits the production of inflammatory factors by inhibiting p65 m RNA transcription.The mRNA modification database shows that p65 mRNA has m6 A modification.To investigate whether the inhibition of p65 m RNA transcription by SARS-CoV-2 Nsp1 protein is related to m RNA degradation mediated by m6 A modification,this study transfected the p EGFP-N1-Nsp1 plasmid into cells and detected the expression of the m6 A reading protein YTHDF2 protein using Western blot.The results showed that SARS-CoV-2 Nsp1 protein upregulated the expression of YTHDF2,and this study further found that overexpression of YTHDF2 protein could inhibit p65 m RNA transcription.These results indicate that SARS-CoV-2 Nsp1 protein can inhibit p65 m RNA transcription by upregulating YTHDF2 protein,thereby reducing p65 protein expression.To investigate the key structural domains of SARS-CoV-2 Nsp1 protein inhibiting NF-κB signaling pathway activation,three SARS-CoV-2 Nsp1 gene truncations(p EGFP-N1-S1,p EGFP-N1-S2,and p EGFP-N1-S3)were constructed and transfected into cells.Western blot and RT-PCR techniques were used to detect p65,p50,and IκBα Expression of YTHDF2 protein and IL-6,IL-8,TNF-α Transcription of p65 m RNA.The results showed that pEGFP-N1-S3 could inhibit IL-6,IL-8,and TNF-α Transcription of p65 m RNA and upregulation of YTHDF2 protein expression.These results indicate that the 126-180 amino acid domain of SARS-CoV-2 Nsp1 protein is a key structural domain for Nsp1 protein to inhibit NF-κB signaling pathway activation.In summary,this study found that the 126-180 amino acid segment of SARS-CoV-2Nsp1 protein can promote the degradation of p65 m RNA by upregulating the expression of YTHDF2 protein,thereby inhibiting the production of inflammatory factors and blocking the activation of NF-B signaling pathway.This study preliminarily analyzed the new function of SARS-CoV-2Nsp1 protein,providing new ideas for understanding the pathogenicity of SARS-CoV-2 virus and treating COVID-19 disease.