The Role Of USP18 In Interferon Resistance Of Dengue Virus And Its Underlying Mechanism

Background:Previous studies demonstrated that type I IFNs inhibited dengue virus(DENV)replication if the cells were pre-treated with IFNs prior to infection,while cells infected with DENV first developed IFN resistance.Ubiquitin-specific protease 18(USP18)is a negative regulator of the type-I interferon(IFN)signaling and its expression level is significantly increased following DENV infection both in cell lines and in the peripheral blood mononuclear cells(PBMCs)isolated from patients' blood.However,the role of USP18 in DENV replication and in IFN-a resistance of DENV is elusive.Aims:We sought to explore the role of USP18 in DENV replication and in IFN-?resistance of DENV-2.Methods:Hela cells,2fTGH cells and USA cells were infected with DENV-2 to determine the expression of USP18.USP18 was overexpressed or knocked down by USP18 palsmid or by siRNA,respectively.The transfection efficiency was confirmed by Western blot.The cells transfeted with USP18 siRNA or USP18 plasmid were treated with IFNa following DENV-2 infection.The expression level of USP18,type I Interferons(IFN?/?)and DENV-2 RNA were quantified by Real-Time PCR.The Jak/STAT signaling pathway was investigated by testing the p-STAT1 levels(western blot),Interferon-sensitive response element(ISRE)activity(dual-luciferase reporter assay),and ISG expression(real-time PCR).DENV proteins including Capsid,PrM,E,NS1,NS2A,NS2B,NS3,NS4A and NS4B were constructed and expressed in 293T cells,Co-immuneprecipitation was performed to identify which DENV-2 protein can interact with USP18.Results:DENV-2 infection led to the increased expression of USP18 in Hela cells,2fTGH cells and U5A cells.IFNa treatment further increased USP18 expression in 2fTGH cells but not in IFNAR-dificient USA cells.Overexpression of USP18 stimulated DENV-2 RNA replication while silencing of USP18 inhibited DENV-2 RNA replication.Decreased USP18 expression by siRNA made the DENV-2 more sensitive to IFN treatment in DENV-2 infected Hela cells and 2fTGH cells.However,we did not observe further decrease of DENV-2 replication after treatment with IFNa in USP18 knockdown U5A cells compared with USP18 knockdown alone.Silencing of USP18 activated the IFNa-mediated Jak/STAT signaling pathway as shown by the increased expression of p-STAT1,enhanced ISRE activity,and elevated expression of ISGs.In addition,USP18 could interact with envelope,NS1 and NS3 proteins of DENV-2.Conclusion:USP18 plays an important role in DENV-2 replication and interferon resistance.DENV-2 infection induces USP 18 expression independent of IFN-I-mediated Jak/STAT signaling pathway.USP 18 overexpression promotes DENV-2 replication and blocks the anti-DENV-2 activity of IFNa.Silencing of USP 18 rescues the IFN resistance of DENV-2 through enhanced activation of the IFN-a-induced Jak/STAT signaling pathway.In addition,USP 18 can interact with envelope,NS1 and NS3 of viral proteins to facilitate viral replication although that detailed mechanism needs to be further investigated.