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Fundación para el Estudio de las Hepatitis Virales, Madrid, Spain
Background.
Hepatitis C virus (HCV) proteins interfere with the interferon (IFN)induced Jak/signal transducer and activator of transcription (STAT) pathway. Which protein is responsible for this effect and whether this interference results in down-regulation of IFN-induced genes remain controversial. We analyzed the effect of HCV core (HCV-Co) protein on expression of IFN-induced antiviral genes.
Methods.
HepG2 cells were transfected with the plasmid pHCV-Co, and, after treatment with IFN-, levels of MxA, protein kinase R (PKR), and 25 oligoadenylate synthetase (25OAS) mRNA were determined. Chloramphenycol acethyl transferase (CAT) analysis was performed on cells cotransfected with pHCV-Co and pMx4CAT (containing the MxA gene promoter) and treated with IFN. Electrophoretic mobility shift assays were used, and Western-blot analysis of STAT 1 and 2 was performed.
Results.
Levels of MxA mRNA in pHCV-Cotransfected cells decreased in a dose-dependent manner, by down-regulation of the MxA gene promoter. HCV-Co protein inhibits binding of IFN-stimulated gene factor 3 (ISGF3) to the IFN-stimulated response element (ISRE). Intracellular distribution of STAT 1 and 2 was not modified after treatment with IFN. Expression of HCV-Co protein also results in down-regulation of expression of PKR and 25OAS genes.
Conclusion.
HCV-Co protein inhibits IFN-induced transcription of antiviral genes by decreasing binding of ISGF3 to the ISRE.
Chronic hepatitis C virus (HCV) infection is a progressive disease that may end in liver cirrhosis and, eventually, in hepatocellular carcinoma [1]. Current treatment for chronic HCV infection consists of administration of interferon (IFN) alone or in combination with ribavirin [24]. However, resolution of infection occurs in only 40%60% of cases [5], indicating that HCV may possess molecular mechanisms of resistance to the action of IFN-.
IFNs are pleiotropic cytokines with antiproliferative, immunomodulatory, and antiviral activities [6]. Binding of IFNs to their membrane receptors promotes activation of 2 receptor-associated tyrosine kinases, Jak1 and Tyk2. These kinases recruit and activate a set of proteins termed signal transducer and activator of transcription (STAT) 1, 2, and 3, which form homo- and heterodimers [7]. IFN- promotes formation of heterodimers between STAT 1 and 2, which further binds to a third protein, p48, to form the IFN-stimulated gene factor 3 (ISGF3) complex, which is translocated to the cell nucleus, where it binds to the IFN-stimulated response element (ISRE) sequence in IFN-stimulated gene promoters. The antiviral activity of type I IFNs (/) is mediated by effector proteins, such as double-stranded RNA-activated protein kinase (PKR), 25oligoadenylate synthetase (25OAS), and MxA [8]. Different viruses, such as hepatitis B virus, cytomegalovirus, and mumps virus, interfere with Jak/STAT signaling, as a persistence mechanism [911], and there is molecular evidence indicating that HCV may also have evolved mechanisms to counteract the antiviral action of IFNs. In this sense, it has been reported that the structural protein E2 and the nonstructural protein 5A repress the activity of PKR [1214]. Furthermore, it has also been reported that expression of HCV proteins in cell lines and in transgenic animals interferes with the IFN-induced Jak/STAT signaling pathway [15, 16]. With regard to the question of which protein is responsible for this effect, HCV core (HCV-Co) protein is the best candidate, because it has been shown that this protein, apart from its ability to interact with genomic HCV-RNA to form nucleocapsids [17, 18], is capable of modulating several cellular signaling pathways [1922]. In fact, it has been reported that HCV-Co protein modulates the Jak/STAT signaling pathway, although it does not affect activation of IFN-responsive genes [23]. In contrast, other authors have reported HCV-Co proteinmediated activation of 25OAS gene transcription [24]. So whether this viral structural protein interferes with the Jak/STAT signaling pathway and whether this interference modifies expression of antiviral IFNinduced genes remain to be demonstrated. For these reasons, in the present study, we analyzed the effect of HCV-Co protein on the IFN-induced Jak/STAT signaling pathway and on the downstream antiviral genes.
MATERIALS AND METHODS
Plasmids.
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