Mechanism Of ABL2 Regulation Of Coxsackie B3 Virus Replication

Background and Objective:Coxsackievirus B3（CVB3）is a standard single positive-stranded RNA virus whose infection causes various diseases,such as myocarditis,pancreatitis and encephalitis.Unfortunately,there is no particularly effective drug or vaccine against CVB3.Tyrosine kinase 2（ABL proto-oncogene 2,ABL2）is a member of the non-receptor tyrosine protein kinase family and is involved in a variety of physiological processes including regulation of cell proliferation,morphology and cell adhesion.Recent studies have reported that ABL2 is involved in the infection process of pathogenic microorganisms,but its role in the replication of CVB3 infection is unknown.Therefore,this study mainly focuses on ABL2 to investigate its effect on CVB3 replication and its regulatory mechanism,aiming to elucidate the molecular mechanism of CVB3 infection replication and provide theoretical basis and drug targets for clinical diagnosis and treatment of CVB3 infection-induced diseases.Methods:1.He La were selected as a classical CVB3 infection cells model,and the difference in ABL2 expression after CVB3 infected cells with a multiplicity of infection（MOI）of 1,5 and 10 was analyzed by Western blot.2.He La cells stably overexpressing ABL2 and silencing ABL2 were constructed separately using lentivirus and validated using Western blot and q RT-PCR.He La cell lines stably overexpressing and silencing ABL2 were infected with CVB3 respectively,and the expression of CVB3 coat protein VP1 was examined by Western blot assay,viral plague assay to analyze CVB3 titer,and CVB3-e GFP fluorescent virus to analyze the effect of ABL2 on CVB3 replication.3.The effect of ABL2 on the replication cycle of CVB3 was analyzed by ABL2inhibitor（Imatinb）and CCK-8 assay.He La cells were treated with different concentrations of Imatinb,infected with CVB3,and cell proteins and supernatants were collected at different times,and the replication of CVB3 was analyzed by Western blot,viral plaque assay and TCID₅₀ method.4.Interacting proteins of ABL2 were analyzed using STING site prediction,combined with immunoprecipitation（Co-IP）,Western blot and immunofluorescence assays.The effects of ABL2 interacting proteins on CVB3 replication and related signaling pathways were further analyzed using Western blot and viral plaque assay methods.5.The regulation of ABL2 overexpression/silencing on reciprocal proteins and related signaling pathways in CVB3 infection was analyzed using Western blot.Both effects on CVB3 replication and related signaling pathways were then analyzed using ABL2 and reciprocal protein co-expression assays.Results:1.CVB3-infected He La cells with different MOIs showed a decrease in ABL2expression;the most significant decrease in ABL2 was observed at MOI=5.2.On the He La cell lines constructed with stable overexpression and silencing of ABL2,overexpression of ABL2 significantly inhibited the expression of CVB3 capsid protein VP1 and reduced the viral titer and fluorescence intensity of the fluorescent virus,while silencing of ABL2 promoted the expression of CVB3 VP1 and increased the viral titer and fluorescence intensity of the fluorescent virus.3.ABL2 inhibitors were added at different times of CVB3 infection,and analysis of Western blot and viral phagocytic spot assays showed that ABL2 inhibited the early stages of CVB3 infection.4.Co-IP and cellular immunofluorescence co-localization experiments revealed that ABL2 and RAC1 interacted,and RAC1 expression was upregulated after CVB3infection of He La cells.Overexpression of RAC1 promoted the expression of VP1,along with increased viral titer and increased the number of CVB3-e GFP fluorescent viruses;and promoted the expression of p-PI3K and p-AKT;silencing RAC1 decreased the expression of VP1,reduced viral titer,decreased the number of fluorescent viruses,and significantly decreased the expression of p-PI3K and p-AKT.5.The expression of RAC1 decreased after overexpression of ABL2,and the detection of p-PI3K and p-AKT signaling pathways also showed a decreasing trend;the expression of RAC1 increased significantly after infection with CVB3 after silencing ABL2,and p-PI3K and p-AKT also showed an increasing trend.co-expression analysis of ABL2 and RAC1 It was further shown that ABL2 interacted with RAC1 to regulate the p-PI3K and p-AKT signaling pathways to inhibit the replication of CVB3 infection.Conclusion:ABL2,as an antiviral gene during CVB3 infection,could inhibits CVB3replication by negatively regulating the expression of its interacting gene RAC1,PI3K/AKT signaling pathway and other pathways,which is expected to provide new ideas for clinical diagnosis and treatment of CVB3 infection.