| Hepatitis B virus(HBV)can cause chronic hepatitis B,which may lead to cirrhosis and liver cancer.Despite severe side effects,type I interferon(IFN)treatment remains an antiviral option for treating chronic hepatitis B.Type I IFNs(IFN-?,IFN-?,IFN-? and others)have been recognized as the major antiviral cytokine in vertebrates,constituting one of the most important innate immune responses to viral infection including hepatitis B virus and hepatitis C virus infection.Type I interferon is an approved drug for the treatment of chronic hepatitis B.However,the fundamental mechanisms of antiviral action by type I IFN and the downstream signaling pathway are unclear.Due to co-evolution,many viruses have acquired the ability to counter the antiviral response induced by type I IFN.A thorough understanding of the interactions between host cells and HBV will improve antiviral treatment.Trim25 is a member of the tripartite motif family of E3 ubiquitin ligases.It plays major roles in innate immunity and defence against viral infection,control of cell proliferation and migration of cancer cells.It can catalyse the addition of K48 and K63-linked polyubiquitin chains and has roles including the targeting of the scaffold protein 14-3-3? for degradation and as an effector of downstream signalling in the innate immune response to the presence of viral RNA.TRIM25 is an IFN-stimulated gene(ISG)that has an important role in RIG-I ubiquitination and activation.Whether TRIM25 is induced in liver cells by type I IFN to mediate anti-HBV function remains unclear.Our results are as follows(1)TRIM25 was upregulated by IFN treatment in both 293 T and Hep G2 cells.TRIM25 m RNA was significantly induced in 293 T cells;TRIM25 protein waselevated after IFN? treatment;IFN-induced protein expression was important in TRIM25 m RNA induction.;supernatant may contain the secreted IFN?-induced cytokines that facilitated TRIM25 expression;IL-27 was critical for TRIM25induction;TRIM25 induction was blocked at both the m RNA and protein levels by IFN IL-27-neutralizing antibody;TRIM25 induction was blocked in both 293 T and Hep G2 IL27 R KO cells;type I IFN-induced TRIM25 was also significantly inhibited in BMMs IL27 R KO cells;both TRIM25 m RNA and protein level were upregulated by recombinant IL27 protein stimulation.(2)Both STAT1 and STAT3 were required for TRIM25 induction by IFN.Both TRIM25 basal and induction level was significantly reduced in STAT1 or STAT3 KO 293 T cells;IL-27 protein promoted both IFN-induced STAT1 and STAT3 activation in Hep G2 cells;The activation of STAT1 and STAT3 was inhibited by IL-27-neutralizing antibody upon IFN or IL-27 recombinant protein retreatment;STAT3 was activated in IFN-treated Hep G2 cells at different time points;STAT3activation was completely blocked by the STAT3 specific inhibitor Stattic;type I IFN-induced TRIM25 expression was significantly inhibited by Stattic;STAT1 KO blocked the induction of TRIM25 at the m RNA and protein levels;STAT3 KO also inhibited TRIM25 m RNA and protein production;small intefering RNA-mediated STAT3 knockdown in Hep G2 cells also inhibited IFN?-induced TRIM25 production;The basal levels of TRIM25 in both 293 T and Hep G2 cells were reduced after STAT3 KO or knockdown.(3)TRIM25 KO in Hep G2 cells facilitated HBV replication.The overexpression of TRIM25 with the HA tag inhibited HBe Ag secretion and HBV DNA replication in Hep G2 cells;TRIM25 was completely knocked out in Hep G2 cells through the CRISPR/Cas9 systems;HBe Ag and HBV DNA levels were significantly increased in TRIM25 KO Hep G2 cells compared with WT cells;HBe Ag and HBs Ag were significantly reduced in the supernatant of the overexpressed TRIM25 cells;HBe Ag in the supernatant of p HBV1.3-transfected TRIM25 KO Hep G2 cells was increased after IFN? treatment,and HBV DNA in the cells was alsoinduced by IFN?.(4)HBV replication increased in STAT3 KO cells.HBV DNA,HBe Ag and HBs Ag were significantly increased after STAT3 KO in Hep G2 cells,and that HBV replication was enhanced by IFN? treatment.(5)TRIM25 promoted type I IFN production in Hep G2 cells.Type I IFN was increased in TRIM25-overexpressed cells,and TRIM25 KO reduced the production of type I IFN in Hep G2 cells;transfection with polyd Ad T,poly IC or p HBV1.3 plasmids,an IFN? or ISRE-Luc reporter assay indicated that luciferase activity induction was significantly inhibited in TRIM25 KO Hep G2 cells;the induction of ISGs by p HBV1.3 transfection was also significantly inhibited.(6)HBV infection inhibited TRIM25 expression.TRIM25 expression in PBMCs isolated from HBV patients was significantly decreased compared with healthy controls;the addition of HBV serum could remarkably inhibit TRIM25 expression in Hep G2 cells;the expression of TRIM25 in Hep G2.2.15 cells in which HBV keeps replicating was significantly inhibited compared with Hep G2 cells;The induction of TRIM25 by IFN? stimulation was inhibited after the Hep G2 cells were co-cultured with Hep G2.2.15 supernatants HBV appears to resist IFN treatment by inhibiting TRIM25 expression.Here we report that interleukin-27(IL-27)has a critical role in IFN-induced TRIM25 upregulation.TRIM25 induction requires both STAT1 and STAT3.In TRIM25 knockout Hep G2 cells,type I IFN production was consistently attenuated and HBV replication was increased,whereas overexpression of TRIM25 in Hep G2 cells resulted in elevated IFN production and reduced HBV replication.More interestingly,we found that TRIM25 expression was downregulated in HBV patients and the addition of serum samples from HBV patients could inhibit TRIM25 expression in Hep G2 cells,suggesting that HBV might have involved a mechanism to inhibit antiviral ISG expression and induce IFN resistance.Collectively,our results demonstrate that type I IFN-induced TRIM25 is an important factor in inhibiting HBV replication,and the IFN-IL-27-TRIM25 axis may represent a new target fortreating HBV infection. |
PDF Full Text Request