The Mechanism Of SARS-CoV-2 NSP13 In Regulating Inflammatory Immunity Of Lung Epithelial Cells

Since the outbreak of COVID-19)in late 2019,this pandemic has escalated into a global public health issue.The peak of COVID-19 infection is now over,but there are still reports of cases of COVID-19 variant infections nationwide.Sars-Cov-2 consists of four types of structure proteins and sixteen types of non-structure proteins.The structure proteins play an important role in infecting host cells.The non-structure proteins are involved in virus replications and host immune reactions while bringing about multiple physiological changes to the host to provide an appropriate environment for viral existence.Among the non-structure proteins,NSP13 protein is capable of a variety of enzyme activities.NSP13 can participate in viral replication processes such as m RNA capping,the unwinding of double-stranded DNA and double-stranded RNA.Moreover,NSP13 can induce host DNA damage while inhibiting host DNA damage response.NSP13 can also inhibit type I interferon signaling pathway,affecting host immune response.Since NSP13 is highly conservative in gene accounting sequence and protein structure,it has been regarded as an important target for the treatment of COVID-19.This study was intended to propose new lines of thinking in terms of host inflammatory response to confirm that NSP13 can be regarded as a drug target.Part one:the effect of NSP13 on NF-κB signaling pathwayBackgroundAfter SARS-CoV-2 enters cells,virus RNA will be recognized by various pathogen pattern recognition receptors before stimulating diverse immune cells such as monocytes and lymphocytes,causing antiviral response and cytokine production.Subsequently,IFN,NF-κB and JAK/STAT signaling pathways will be activated,indicating that SARS-CoV-2 participate in modulating host cell immune response.According to clinical data,SARS-CoV-2 can promote the production of a wide variety of inflammatory cytokines,leading to severe organ injury and dysfunction,a series of syndromes and even death.IL-6、TNF-α、IL-1 and many other kinds of inflammatory factors,downstream cytokines of NF-κB signal pathway,are detected and used to manifest the inflammation level of patients.It has been confirmed that NSP13 is capable of regulating the type I interferon signaling pathway.In type II lung epithelial cells,which is susceptible to SARS-CoV-2,type I interferon signaling pathway interacts with NF-κB signal pathway.Bioinformatics analysis found that NSP13 interacted with many proteins of NF-κB signal pathway.The relationship between NSP13 and NF-κB signal pathway deserves more extensive investigation.A better knowledge of the effect of NSP13 protein on NF-κB signaling pathway is expected to provide new therapeutic targets for related drugs and to advance our understanding of the interactions between COVID-19 and host inflammation.ObjectiveThis study aimed to explore the effect of NSP13 on secretion of inflammatory cytokines and expressions of important components in the NF-κB signaling pathway.Efforts were made to find out more about the mechanism by which NSP13 regulates the NF-κB signaling pathway in order to illuminate the role of proteins in viral infection and explain how SARS-CoV-2 escapes host immune response in the initial stage of infection.MethodA549 with a high expression of NSP13（A549-NSP13）was constructed via lentivirus infection.RT-q PCR was used to test the m RNA of NSP13.WB was used to test the Flag tag and flow cytometry was adopted to test the transfection efficiency in order to prove that A549-NSP13 was established properly.RT-q PCR and ELISA were used to detect downstream inflammatory factors of NF-κB signal pathway such as chemokines and interleukins.WB was used to test the expression levels of P65,phosphorylated P65（pi-P65）and IκBαso as to explore the mechanism by which NSP13protein regulated NF-κB signal pathway.After cells were treated with the protein synthesis inhibitor CHX（10μg/ml）for 0,15,30,45,90 and 120 minutes,the expression of IκBαwas detected by WB.The gray values of protein bands at different time points were compared to determine the effect of NSP13 on IκBαt_1/2.RT-q PCR and WB were used to test the expression of specific ubiquitin ligase SCF^β-Trcp,which participated in IκBαubiquitination degradation in order to analyze the effect of NSP13 on IκBαdegradation.ResultRT-q PCR showed that the m RNA expression level of NSP13 protein in the A549-NSP13 group was significantly increased.Compared with the control group,WB detected Flag tag in A549-NSP13 group alone while GFP was detected only in the A549-NSP13 group by flow cytometry.The above results showed that A549 cells overexpressing NSP13 protein were constructed.Compared with the A549-CON group,the expression levels of IL-6 and CCL-5 in the A549-NSP13 group decreased significantly,suggesting that NSP13 inhibited inflammatory factors downstream of NF-κB signal pathway.Less pi-P65 and more IκBαwere observed in the A549-NSP13group than in the A549-CON group,indicating that NSP13 could block P65phosphorylation by increasing IκBαprotein content.After CHX treatment,a longer t_1/2of IκBαwas detected in the A549-NSP13 group than in the A549-CON group,which was a sign that NSP13 inhibited IκBαprotein degradation.The effect of NSP13 protein on IκBαwas also studied.It was found that NSP13 inhibited not onlyβ-Trcp of specific ubiquitin ligase E3 SCF^β-Trcp,but also the overall ubiquitination level of host cells,suggesting that NSP13 inhibited IκBαubiquitin-dependent pathway viaβ-Trcp protein.ConclusionNSP13 inhibited IκBαubiquitin-dependent pathway viaβ-Trcp protein blocking the phosphorylation of P65 and inhibiting NF-κB signal pathway,which resulted in the reduction of downstream inflammatory factors like IL-6 and CCL-5.Part two:Effect of NSP13 on expressions of host receptorsBackgroundThe susceptibility of host cells to SARS-CoV-2 is also an important factor determining the degree of cellular inflammation.The tissues and organs sensitive to SARS-CoV-2 have stronger inflammatory immunity.Therefore,it is also of great significance to explore the effect of NSP13 protein on host cells in inflammatory immunity.The spike protein of SARS-CoV-2 binds to cellular angiotensin converting enzyme 2（ACE-2）receptor.Finally,virus RNA is released into cells and infects host cells.Several amino acid variations were observed in the middle of the binding domain of SARS-CoV-2,which provided an increased affinity to bind to ACE2 more effectively.ACE-2,as a functional host cell receptor,has been identified in multiple cells and organs.Besides,ACE-2 has been proved to be helpful in reducing acute injury and inhibiting fibrogenesis of the lungs and protecting the cardiovascular system.In various cells,ACE-2 receptor inhibitors can reduce the infection rate of COVID-19 and the level of inflammatory factors in cells,which is conducive to the survival of host cells.As such,ACE-2 is now receiving more attention as a potential target for anti-viral therapeutics.As an interferon stimulator,ACE-2 expression is regulated by the type I interferon signal pathway.And NSP13 protein has been confirmed to participate in the regulation of the host type I interferon signal pathway.In our study,it was asserted that NSP13 protein could regulate the expression of ACE-2 receptor.In addition,due to the homology between SARS-CoV-2 and other coronaviruses,some coronavirus receptors also play an important role in SARS-CoV-2 infection,which is of significance for research.So this research also focused on how NSP13 protein affected the expression of other coronavirus receptors in host cells.ObjectiveThis research was intended to analyze the GSE152075 data set of nasopharyngeal swabs from COVID-19 patients and construct the overexpression NSP13 cell lines to explore the effect of NSP13 protein on expressions of ACE-2 and other coronavirus receptors.The interactions between SARS-CoV-2 and host were also studied in order to gain keen insights into the impact of SARS-CoV-2 on virus infection efficiencyMethodThe GSE152075 data set of nasopharyngeal swabs from COVID-19 patients was used to analyze the expressions of host receptors,such as ACE-2.HEK293T-NSP13were constructed via plasmid transfection.RT-q PCR was used to detect the expression of NSP13 at the m RNA level and WB was used to detect the NSP13 expression to find out whether HEK293T-NSP13 for the subsequent experiments were properly constructed.RT-q PCR was used to detect the m RNA expression of coronavirus cell receptors and WB was adopted to detect its protein expression so as to analyze the effect of NSP13 on expressions of coronavirus receptors in host cells.WB was used to detect the expressions of SATA1,STAT2 and other important proteins of the type I interferon signal pathway to explore the effect of NSP13 on the type I interferon signal pathway and whether NSP13 regulated ACE-2 receptors through the type I interferon signal pathway.ResultBy analyzing the nasopharyngeal swab data set of COVID-19 patients,we found that compared with the negative,levels of DPP4,ACE-2 and CEACAM-1 in SARS-CoV-2 positive patients increased,but that of TMPRSS2 decreased.Compared with the A549-CON group,ACE-2 in the A549-NSP13 group was increased based on RT-q PCR,but NSP13 had no effect on the other cell surface receptors.The same was observed in HEK293T cells.STAT1,transcription enhancer CREB and the ligand My D88 of type I interferon pathway were down-regulated in A549 and HEK293T cells,indicating that NSP13 inhibited the type I interferon signal pathway,and that NSP13protein might regulate the expression of ACE-2 receptor through other signal pathways.ConclusionNSP13 protein can upregulate the expression of ACE-2 receptor m RNA in host cells,but has no significant effect on its protein expression.NSP13 protein can inhibit the type I interferon signal pathway,but NSP13 can upregulate the expression of host ACE-2 receptor through other signal pathways.