Verena Christen

Interferon signaling and treatment outcome in chronic hepatitis C.

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. The current standard therapy for chronic hepatitis C (CHC) consists of a combination of pegylated IFN alpha (pegIFNα) and ribavirin. It achieves a sustained viral clearance in only 50–60% of patients. To learn more about molecular mechanisms underlying treatment failure, we investigated IFN-induced signaling in paired liver biopsies collected from CHC patients before and after administration of pegIFNα. In patients with a rapid virological response to treatment, pegIFNα induced a strong up-regulation of IFN-stimulated genes (ISGs). As shown previously, nonresponders had high expression levels of ISGs before therapy. Analysis of posttreatment biopsies of these patients revealed that pegIFNα did not induce expression of ISGs above the pretreatment levels. In accordance with ISG expression data, phosphorylation, DNA binding, and nuclear localization of STAT1 indicated that the IFN signaling pathway in nonresponsive patients is preactivated and refractory to further stimulation. Some features characteristic of nonresponders were more accentuated in patients infected with HCV genotypes 1 and 4 compared with genotypes 2 and 3, providing a possible explanation for the poor response of the former group to therapy. Taken together with previous findings, our data support the concept that activation of the endogenous IFN system in CHC not only is ineffective in clearing the infection but also may impede the response to therapy, most likely by inducing a refractory state of the IFN signaling pathway.

Activation of endoplasmic reticulum stress response by hepatitis viruses up‐regulates protein phosphatase 2A

The up‐regulation of protein phosphatase 2 A (PP2A) is an important factor leading to an inhibition of IFNα signaling caused by viral protein expression. Here, we describe the molecular mechanism involved in PP2Ac up‐regulation by HCV and HBV. HCV and HBV protein expression in cells induces an ER stress response leading to calcium release from the ER. HCV protein expression induces CREB activation, probably through calcium/calmodulin‐dependent protein kinase. CREB binds to a CRE element in the promoter of PP2Ac and induces its transcriptional up‐regulation. Because PP2Ac is involved in many important cellular processes including cell‐cycle regulation, apoptosis, cell morphology, development, signal transduction and translation, its up‐regulation during ER stress has potentially important implications. (HEPATOLOGY 2007.)

Inhibition of Alpha Interferon Signaling by Hepatitis B Virus

ABSTRACT Alpha interferon (IFN-α) and pegylated IFN-α (pegIFN-α) are used for the treatment of chronic hepatitis B (CHB). Unfortunately, only a minority of patients can be cured. The mechanisms responsible for hepatitis B virus (HBV) resistance to pegIFN-α treatment are not known. pegIFN-α is also used to treat patients with chronic hepatitis C (CHC). As with chronic hepatitis B, many patients with chronic hepatitis C cannot be cured. In CHC, IFN-α signaling has been found to be inhibited by an upregulation of protein phosphatase 2A (PP2A). PP2A inhibits protein arginine methyltransferase 1 (PRMT1), the enzyme that catalyzes the methylation of the important IFN-α signal transducer STAT1. Hypomethylated STAT1 is less active because it is bound by its inhibitor, PIAS1. In the present work, we investigated whether similar molecular mechanisms are also responsible for the IFN-α resistance found in many patients with chronic hepatitis B. We analyzed the expression of PP2A, the enzymatic activity of PRMT1 (methylation assays), the phosphorylation and methylation of STAT1, the association of STAT1 with PIAS1 (via coimmunoprecipitation assays), the binding of activated STAT1 to interferon-stimulated response elements (via electrophoretic mobility shift assays), and the induction of interferon target genes (via real-time RT-PCR) in human hepatoma cells expressing HBV proteins as well as in liver biopsies from patients with chronic hepatitis B and from controls. We found an increased expression of PP2A and an inhibition of IFN-α signaling in cells expressing HBV proteins and in liver biopsies of patients with CHB. The molecular mechanisms involved are similar to those found in chronic hepatitis C.

S‐adenosylmethionine and betaine correct hepatitis C virus induced inhibition of interferon signaling in vitro

Hepatitis C virus (HCV) infection is an important cause of chronic liver disease. Standard therapy, pegylated interferon α (pegIFNα) combined with ribavirin, results in a sustained response rate in approximately half of patients. The cause of treatment failure in the other half of the patients is unknown, but viral interference with IFNα signal transduction through the Jak‐STAT pathway might be an important factor. We have shown previously that the expression of HCV proteins leads to an impairment of Jak‐STAT signaling because of an inhibition of STAT1 methylation. Unmethylated STAT1 is less active because it can be bound and inactivated by its inhibitor, protein inhibitor of activated STAT1 (PIAS1). We show that treating cells with S‐adenosyl‐L‐methionine (AdoMet) and betaine could restore STAT1 methylation and improve IFNα signaling. Furthermore, the antiviral effect of IFNα in cell culture could be significantly enhanced by the addition of AdoMet and betaine. In conclusion, we propose that the addition of these drugs to the standard therapy of patients with chronic hepatitis C could overcome treatment resistance. (HEPATOLOGY 2006;43:796–806.)

Upregulation of Protein Phosphatase 2Ac by Hepatitis C Virus Modulates NS3 Helicase Activity through Inhibition of Protein Arginine Methyltransferase 1

ABSTRACT Hepatitis C virus (HCV) is a major cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma worldwide. HCV has a positive-strand RNA genome of about 9.4 kb in size, which serves as a template for replication and for translation of a polyprotein of about 3,000 amino acids. The polyprotein is cleaved co- and posttranslationally by cellular and viral proteases into at least 10 different mature proteins. One of these proteins, nonstructural protein 3 (NS3), has serine protease and NTPase/RNA helicase activity. Arginine 467 in the helicase domain of NS3 (arginine 1493 in the polyprotein) can be methylated by protein arginine methyltransferase 1 (PRMT1). Here we report that the methylation of NS3 inhibits the enzymatic activity of the helicase. Furthermore, we found that PRMT1 activity itself is regulated by protein phosphatase 2A (PP2A). PP2A inhibits PRMT1 enzymatic activity and therefore increases the helicase activity of NS3. This is important, because we found an increased expression of PP2A in cell lines with inducible HCV protein expression, in transgenic mice expressing HCV proteins in hepatocytes, and in liver biopsy samples from patients with chronic hepatitis C. Interestingly, up-regulation of PP2A not only modulates the enzymatic activity of an important viral protein, NS3 helicase, but also interferes with the cellular defense against viruses by inhibiting interferon-induced signaling through signal transducer and activator of transcription 1 (STAT1). We conclude that up-regulation of PP2A might be crucial for the efficient replication of HCV and propose PP2A as a potential target for anti-HCV treatment strategies.

Hepatitis C virus–induced up‐regulation of protein phosphatase 2A inhibits histone modification and DNA damage repair

The molecular mechanisms underlying hepatocarcinogenesis in chronic viral hepatitis are poorly understood. A potential tumorigenic pathway could involve protein phosphatase 2A (PP2A) and protein arginine methyltransferase 1 (PRMT1), because both enzymes are dysregulated in chronic hepatitis C, and both enzymes have been involved in chromatin remodeling and DNA damage repair. We used cell lines that allow the inducible expression of hepatitis C virus proteins (UHCV57.3) and of the catalytic subunit of PP2A (UPP2A‐C8) as well as Huh7.5 cells infected with recombinant cell culture–derived hepatitis C virus (HCVcc) to study epigenetic histone modifications and DNA damage repair. The induction of viral proteins, the overexpression of PP2Ac, or the infection of Huh7.5 cells with HCVcc resulted in an inhibition of histone H4 methylation/acetylation and histone H2AX phosphorylation, in a significantly changed expression of genes important for hepatocarcinogenesis, and inhibited DNA damage repair. Overexpression of PP2Ac in NIH‐3T3 cells increased anchorage‐independent growth. These changes were partially reversed by the treatment of cells with the methyl‐group donor S‐adenosyl‐L‐methionine (SAMe). Conclusion: Hepatitis C virus–induced overexpression of PP2Ac contributes to hepatocarcinogenesis through dysregulation of epigenetic histone modifications. The correction of defective histone modifications by S‐adenosyl‐L‐methionine makes this drug a candidate for chemopreventive therapies in patients with chronic hepatitis C who are at risk for developing hepatocellular carcinoma. (HEPATOLOGY 2010.)