The Mechanism Of S-phase Arrest Induced By N Protein Of SARS-COV-2 And Drug Screening

Background: The main symptoms of new coronavirus pneumonia(COVID-19)are low fever,mild fatigue,taste and smell disorder;severe symptoms are dyspnea,hypoxemia,even acute respiratory distress syndrome,septic shock,multiple organ failure,etc.Very few patients also have central nervous system involvement and avascular necrosis of the extremities.Now the main pathogen is the novel coronavirus(SARS-CoV-2),which is prevalent in China and other countries.The proteins of SARS-CoV-2 include viral structural proteins and non-structural proteins.The nucleocapsid protein N is structural protein and is relatively stable among all the proteins of the novel coronavirus(SARS-CoV-2),and is involved in the processes of coronavirus genomic RNA synthesis,signal transduction and immune regulation.We investigate whether nucleocapsid protein N regulates host cell cycle because viruses often regulate host cell cycle for own replication;the mechanism of regulating cell cycle;whether nucleocapsid protein N induces DNA damage response because DNA damage response often leads to cell cycle arrest;whether natural compounds protect cells from the damage of nucleocapsid protein N which is very important for revealing the pathogenesis of SARS-CoV-2,and the treatment for SARS-CoV-2.Methods: First,the plasmid of nucleocapsid protein N was constructed and transfected into cells for the expression of nucleocapsid protein N and researching its function.And cell morphology was analyzed for cell colony formation and cell death,flow cytometry was used for cell cycle distribution,no-serum treatment and nocodazole treatment were used to analyze S phase entry and exit.Western blot was used to detect protein expression.Immunoprecipitation was used to comfirm the binding of nucleocapsid protein N with cell cycle-related proteins.Immunofluorescence was confirm the expression of nucleocapsid protein N,and co-localization of nucleocapsid protein N with?-H2 AX.Nuclear staining was used to confirm cell growth.Results And Analysis: 1.Nucleocapsid protein N inhibits cell growth,but does not induce cell death.Cell colony was formed in vector group with the time,while cell colony was not formed well in nucleocapsid protein N transfected cells.Cell counting method found that the cell number in nucleocapsid protein N transfected group was less than the empty vector transfection group.Immunofluorescence technique found that the cells with non-nucleocapsid protein N transfection could proliferate well,while the cells with nucleocapsid protein N transfection could not proliferate well,but with the normal morphology.Therefore,nucleocapsid protein N inhibited cell proliferation,but did not induce cell death.2.Nucleocapsid protein N arrests cell cycle at S phase The results of flow cytometry showed that nucleocapsid protein N arrested cell cycle at S phase in time and dose-dependent manner(P<0.001).Further nucleocapsid protein N promoted S phase entry and inhibited S exit.The expression of cyclin A,cyclin B,cyclin D,CDK1,CDK2,CDK4 and CDK4 was not affected by nucleocapsid protein N,while cyclinE1 was up-regulated in the nucleocapsid protein transfected group(P<0.001),which could explain nucleocapsid protein N promoted S phase entry.Therefore,the results showed that the nucleocapsid protein N could induce cell cycle to inhibit the cell proliferation;and up-regulated cyclinE1 exression to induce S phase arrest.3.The nucleocapsid protein N induces DDR DDR is the up-stream event of cell cycle arrest.Western blot indicated that nucleocapsid protein N up-regulated the level of ?-H2AX(P<0.001),which is the marker of DDR.Meanwhile immunofluorence data showed that ?-H2 AX was localized with nucleocapsid protein N.Therefore nucleocapsid protein N induced DDR.4.Tanshinone II-A and silibinin inhibit the expression of?-H2 AX and protect host cells Rutin,tanshinone II-A and silibinin were reported to have the ability of inhibiting DDR.The western blot results showed that after treatment with tanshinone II-A and silibinin the level of ?-H2 AX protein was decreased in the nucleocapsid protein N transfected group,but rutin did not decreasse the level of ?-H2 AX protein in the nucleocapsid protein N transfected cells.Nuclear staining and cell counting showed that the role of nucleocapsid protein N in up-regualting?-H2 AX was reversed by tanshinone II-A and silibinin treatment,and cell proliferation has been recovered.Therefore,tanshinone II-A and silibinin could reverse the role of nucleocapsid protein N,which might be a candidate for treatment of SARS-CoV-2.Conclusions: 1.Nucleocapsid protein N can inhibit cell growth,but does not induce cell death.2.Nucleocapsid protein N promotes entry into S phase and inhibits exit from S phase;Nucleocapsid protein N up-regualtes the expression of cyclinE1,and Nucleocapsid protein N can induce DDR and increases the level of ?-H2 AX which is the marker of DDR.3.Tanshinone II-A and silibinin could lower the level of?-H2 AX in nucleocapsid protein N expressed cells to promote cell growth.Innovation: 1.Nucleocapsid protein N can inhibit cell growth,but does not induce cell death.2.Nucleocapsid protein N induces cell cycle arrest at S phase throught promoting entry into S phase and inhibiting exit from S phase,which might be important for viral proliferation,and Nucleocapsid protein N up-regualtes the expression of cyclinE1,which might be the reason of nucleocapsid protein N regulating cell cycle,and Nucleocapsid protein N can induce DDR,which is important to reveal the pathogenesis of SARS-CoV-2.3.Tanshinone II-A and silibinin could lower the level of?-H2 AX in nucleocapsid protein N expressed cells to promote cell growth,which might be the candidates for anti-SARS-CoV-2.