| Background: Tick borne encephalitis virus(TBEV)is a highly toxic neurophilic virus that can cause permanent neurological complications or death,with a mortality rate of up to 20%,posing a great threat to life and health.A large number of studies have shown that viral infection can promote self-replication by regulating cell cycle progression and triggering host cell damage response.However,the effect of TBEV on the cell cycle and its regulation of cell cycle-related factors remain unknown.In this paper,the effects of TBEV infection on host cell cycle progression and viral infection on the expression of cell cycle factors were deeply studied,and the effect of host cell cycle arrest on TBEV replication and proliferation was discussed.This study provides a theoretical and experimental basis for clinical treatment and drug development,and provides a feasible direction for the study of flavivirus virus.Method: The main research methods of this topic include virus-infected cells,flow cytometry,real-time PCR,Western Blot,etc.First,the effect of TBEV on cell cycle was detected by flow cytometry in TBEV-susceptible cells(including Vero,A549 and Caco-2);Then,the expression of intracellular and extracellular viral RNA and proteins at different time points after viral infection was analyzed by real-time PCR,Western Blot,and immunofluorescence staining experiments to study the effect of cell cycle arrest on viral replication;Western Blot was used to detect the expression of cell cycle-related factors at different time points after TBEV infection;The effect of TBEV protein on cell cycle was further detected by flow cytometry;Finally,real-time PCR and other methods were used to study the effect of drugs on TBEV replication.Research result: Through flow cytometry experiments,we found that TBEV infection caused cell cycle arrest at G0/G1 phase in Vero,A549 and Caco-2 cells,and further verified that TBEV infection can cause the cell cycle arrest at G0/G1 phase through the synchronous treatment;Moreover,it was also found that G0/G1 phase arrest could promote TBEV adsorption and replication,while G2/M phase arrest could inhibit the replication of TBEV;The results of flow cytometry showed that cells were suppressed from G0/G1 to S phase in the early stage of viral infection,and cells were promoted from G2/M phase to G0/G1 phase in the late stage of infection;Proteomics results showed that the protein levels of CDK2/4/6 were significantly decreased,and the protein levels of CDK1 and P53 were significantly increased after TBEV infection;Western blot results showed that the level of key cytokine protein in G0/G1 phase changed with the increase of infection time,The expression of CDK2 and CDK6 decreased at 12 h and 24 h after infection,the expression of CDK4 decreased at 36 h and 48 h after infection,and the expression of P21 and P53 increased in the infected group,while the expression of key cytokines in the G2/M phase including CDK1 and cyclin B1 increased significantly after 36 h of virus infection.Flow cytometry and Western Blot results showed that TBEV NS5 protein caused G0/G1 phase cell cycle arrest;The results of investigating the effect of drugs on the virus showed that chrysin could significantly inhibit the replication of TBEV and reverse the cycle arrest caused by TBEV.Conclusion: TBEV causes arrest of Vero,A549 and Caco-2 cells and cycle-synchronized Vero cells in the G0/G1 phase.1.Early TBEV infection suppressor cells enter the S phase from the G0/G1 stage,and the late infection promotes the transition from the G2/M phase to the G0/G1 phase.2.The G0/G1 phase significantly promoted the adsorption and replication of TBEV,while G2/M phase arrest inhibited the replication of TBEV.3.TBEV infection can down-regulate the expression of CDK2/4/6 and up-regulate the expression of P21、P53,CDK1 and Cyclin B1.4.TBEV proteins NS2 B,NS3,NS4 B,NS5,M can cause G0/G1 phase arrest.5.Chrysin,an agonist of CDK4,inhibited viral replication by inhibiting TBEV-induced G0/G1 phase arrest. |
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