Intrahepatic MxA and PKR protein expression in chronic hepatitis C virus infection

The therapeutic effect of interferon-alpha and ribavirin in the treatment of chronic hepatitis C viral infection is limited. To identify patient characteristics that may predict responsiveness to treatment, the intrahepatic protein expression of two directly induced IFN-alpha effector proteins, MxA and PKR, were studied. Forty liver biopsy samples from patients with a variety of chronic liver diseases were stained for MxA and PKR protein using immunohistochemical techniques. In a HCV patient cohort, 30 liver biopsies were stained for MxA and PKR protein prior to treatment with IFN-alpha and ribavirin. PKR protein expression was not upregulated in viral liver disease. In contrast, MxA protein expression was significantly upregulated in viral liver disease (P = 0.005). In chronic HCV liver disease, moderate to strong cytoplasmic expression of MxA protein was observed in hepatocytes and monocytes, indicating endogenous hepatocellular IFN-alpha pathway activation. In the HCV patient cohort treated with combination therapy, strong pre-treatment MxA hepatocyte expression was predictive of a non-response to treatment (odds ratio 9.33; P = 0.01; 95% confidence interval 1.63-53.2). This effect was independent of HCV genotype and viral load. It is concluded that pretreatment hepatocellular MxA expression may become a useful predictor of response to combination treatment with IFN-alpha and ribavirin.     

Interferon-alpha therapy does not modulate hepatic expression of classical type I interferon inducible genes

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease. Treatment with interferon-alpha(2) (IFN-alpha(2)) can induce viral clearance and marked biochemical and histological improvement. IFN-alpha(2) treatment has been shown to stimulate the expression of type I IFN regulated genes in peripheral blood mononuclear cells (PBMCs) of hepatitis C patients; however, whether it affects hepatic expression remains unknown. This study thus aimed at comparing hepatic gene expression with particular emphasis on type I IFN inducible genes in patients with chronic hepatitis C before and during an IFN-alpha(2) monotherapy. Responsiveness to IFN-alpha(2) therapy was monitored by determining serum and hepatic viral load. Differential gene expression analysis was performed by two different techniques, namely suppression subtractive hybridization (SSH) and differential display (DD). Expression of two prototype type I IFN regulated genes was quantified in further PBMC and liver samples. Among different genes found to be up-regulated during an effective, that is, virus clearing, IFN-alpha treatment, only a single one was identified which can be accounted to type I IFN responsive genes. Parallel quantitative real time PCR analyses demonstrated significant induction of the type I IFN regulated genes MxA and PKR in PBMC, but not in the liver. Taken together, while IFN-alpha treatment leads to the induction of type I IFN regulated genes in PBMC, such an induction appears not to occur in the liver of hepatitis C patients. The mechanism by which IFN-alpha treatment causes viral clearance might be independent of hepatic activation of type I IFN regulated genes.     

MxA induction may predict sustained virologic responses of chronic hepatitis B patients with IFN-alpha treatment.

The objective of this study was to find potential biomarkers for predicting sustained virologic responses to interferon-alpha (IFN-alpha) treatment in chronic hepatitis B (CHB) patients. A total of 101 CHB patients were treated with pegylated IFN-alpha2a for 48 weeks and followed up for 24 weeks, including 34 IFN responders (IFN-Rs) and 67 IFN nonresponders (IFN-NRs). After peripheral blood mononuclear cells (PBMCs) and Epstein-Barr virus-transferred B (EBV-B) cell lines were treated with different concentrations of IFN-alpha in vitro, activated IFN-stimulated gene factor3 (ISGF3) and IFN-gamma-activation factor (GAF) were measured by EMSA, and MxA, OAS1, and PKR mRNA were measured by real-time PCR. Polymorphisms in the MxA promoter were genotyped to find the possible association. IFN-alpha-activated ISGF3 and GAF levels were similar between IFN-NRs and IFN-Rs. However, MxA mRNA induction in IFN-Rs was higher than that in IFN-NRs, and such discrepancy increased when highly concentrated IFN was used to stimulate. The OAS1 and PKR mRNA induction have a similar pattern between IFN-Rs and IFN-NRs. In addition, frequency of the MxA-88G/T genotype was significantly different between IFN-Rs and IFN-NRs, and this polymorphism was also functional because MxA mRNA induction in patients with GG genotype was lower than those with GT genotype. Regression analysis showed that MxA mRNA induction after 10,000 IU/mL IFN stimulation could serve as an independent factor for predicting IFN-alpha, with an area under curve (AUC) of 0.838, a positive predictive value of 68% for IFN-Rs, and a negative predictive value of 89% for IFN-NRs. MxA mRNA induced by IFN-alpha might predict sustained virologic responses to IFN-alpha treatment in CHB patients.     

Evidence That Hepatitis C Virus Resistance to Interferon Is Mediated through Repression of the PKR Protein Kinase by the Nonstructural 5A Protein

Hepatitis C virus (HCV) is the major cause of non-A non-B hepatitis and a leading cause of liver dysfunction worldwide. While the current therapy for chronic HCV infection is parenteral administration of type 1 interferon (IFN), only a fraction of HCV-infected individuals completely respond to treatment. Previous studies have correlated the IFN sensitivity of strain HCV-1b with mutations within a discrete region of the viral nonstructural 5A protein (NS5A), termed the interferon sensitivity determining region (ISDR), suggesting that NS5A may contribute to the IFN-resistant phenotype of HCV. To determine the importance of HCV NS5A and the NS5A ISDR in mediating HCV IFN resistance, we tested whether the NS5A protein could regulate the IFN-induced protein kinase, PKR, a mediator of IFN-induced antiviral resistance and a target of viral and cellular inhibitors. Using multiple approaches, including biochemical, transfection, and yeast genetics analyses, we can now report that NS5A represses PKR through a direct interaction with the protein kinase catalytic domain and that both PKR repression and interaction requires the ISDR. Thus, inactivation of PKR may be one mechanism by which HCV avoids the antiviral effects of IFN. Finally, the inhibition of the PKR protein kinase by NS5A is the first described function for this HCV protein.     

Polymorphisms in interferon-induced genes and the outcome of hepatitis C virus infection: roles of MxA,OAS-1 and PKR

Interferon stimulates the expression of a number of genes encoding enzymes with antiviral activities, including myxovirus resistance-1 (MxA), 2-5-oligoadenylate synthetase 1 (OAS-1) and double-stranded RNA-dependent protein kinase (PKR). We examined whether polymorphisms in these genes influenced the outcome of hepatitis C virus (HCV) infection. We observed a lower frequency of the GG genotype at position -88 in the MxA gene promoter in self-limiting HCV infection (OR=0.56; 95% CI: 0.35-0.8; P=0.010) and in nonresponders to therapy (OR=0.49; 95% CI: 0.25-0.95; P=0.020). This genotype predominantly influenced the outcome of treatment in patients with viral genotype 1 (OR=0.22 95% CI: 0.07-0.67; P=0.002). A polymorphism in the 3'-untranslated region of the OAS-1 gene was associated with outcome of infection (GG genotype less frequent in self-limiting infection: OR=0.43; 95% CI: 0.21-0.86; P=0.010). A polymorphism at position -168 in the promoter region of the PKR gene was associated with self-limiting infection (CT genotype: OR=2.75; 95% CI: 1.45-5.24; P=0.002). Further associations were found with a CGG trinucleotide repeat in the 5'UTR region of the PKR gene. Polymorphisms in the interferon-induced genes, MxA, OAS-1 and PKR appear thus associated with HCV outcome.     

NS5AISDR‐V3 region genetic variability of Tunisian HCV‐1b strains: Correlation with the response to the combined interferon/Ribavirin therapy

In the non‐structural protein 5A (NS5A) of hepatitis C virus (HCV), mutations within the interferon sensitivity‐determining region (ISDR), the PKR‐binding domain (PKR‐BD), the variable region 3 (V3), and the interferon/ribavirin resistance‐determining region (IRRDR) have been correlated with the IFN‐based therapy response. In Tunisia, where a high prevalence of HCV‐1b has been found, no data regarding the implication of NS5A in treatment response were available. The current study examined the relationship between the pre‐treatment mutation number within ISDR, PKR‐BD, V3, IRRDR, as well as in the entire ISDR‐V3 region of NS5A (aa 2209–2379) and the response to the 48‐week course of combined IFN plus ribavirin therapy in 15 HCV‐1b‐infected Tunisian patients. Referring to HCV‐J sequence, a significant high genetic variability was observed within PKR‐BD in the sustained virological responder patients compared to non‐responders (P = 0.040). More importantly, when considering the entire region from ISDR to V3, referred to as NS5A_ISDR‐V3, a clear difference in the mutation number was observed between sustained virological responders (19.6 ± 3.16) and non‐responders (15.0 ± 1.41) (P = 0.002). Additionally, a more detailed analysis of NS5A_ISDR‐V3 region revealed an elevated degree of mutation rate within the region located between amino acids 2282 and 2308 (P = 0.0006). Interestingly, an analysis of specific amino acid variations defined proline and serine at position 2300 as signature patterns for sensitive and resistant strains, respectively. The genetic variability within the NS5A region of HCV‐1b strains was associated with the response to the combined IFN plus ribavirin therapy in our Tunisian cohort. J. Med. Virol. 81:2021–2028, 2009. © 2009 Wiley‐Liss, Inc.     

The NS5a gene of hepatitis C virus in patients treated with interferon-alpha

Patients infected with hepatitis C virus (HCV) genotype 3 have a better response to interferon-alpha (IFN-alpha) therapy than those infected with genotype 1. There are extensive sequence differences between genotypes in the 3' half of the NS5a gene. An association between IFN-alpha response and the interferon sensitivity-determining region (ISDR) (amino acids 2209-2248) of HCV genotype 1b has been described [Enomoto et al. (1996) New England Journal of Medicine 334:771-776]. A prospective study was conducted to determine whether the derived NS5A amino acid sequence or quasi-species diversity could predict response to IFN-alpha therapy. Serum samples were obtained before, during, and after treatment from 35 IFN-alpha-treated patients chronically infected with HCV (eight with type1b,13 with type1a, and 14 with type3a). Nucleotide sequences were determined, and amino acid sequences corresponding to residues 2178-2390 of the polyprotein were derived. Quasi-species complexity was analysed by amplification of the ISDR region (2270-2403), followed by single-stranded conformation polymorphism (SSCP). No amino acid sequence that could be used to predict response to treatment was found, and there was no selection of specific amino acid residues during treatment. A striking lack of variability was seen in HCV genotype 3a, but the small degree of variation could suggest an effect on response. SSCP showed that variation in the predominant NS5a sequence occurred in the presence and absence of therapeutically administered IFN-alpha. HCV quasi-species diversity pretreatment did not predict IFN-alpha treatment outcome. The conclusion of the study is that the amino acid sequence of NS5a cannot be used to predict the efficacy of treatment with IFN-alpha in HCV-infected patients in Scotland. No evidence was found to support the selection of IFN-alpha-resistant strains in the NS5a gene.     

Morphine withdrawal enhances hepatitis C virus replicon expression

We previously demonstrated that morphine enhances hepatitis C virus (HCV) replication in human hepatic cells. Here we describe the impact of morphine withdrawal (MW), a recurrent event during the course of opioid abuse, on HCV replicon expression in human hepatic cells. MW enhanced both viral RNA and protein expression in HCV replicon cells. Blocking opioid receptors by treatment with naloxone after morphine cessation (precipitated withdrawal, PW) induced greater HCV replicon expression than MW. Investigation of the mechanism responsible for MW- or PW-mediated HCV enhancement showed that both MW and PW inhibited the expression of endogenous interferon-alpha (IFN-alpha) in the hepatic cells. This down-regulation of intracellular IFN-alpha expression was due to the negative impact of MW or PW on IFN-alpha promoter activation and on the expression of IFN regulatory factor 7 (IRF-7), a strong transactivator of the IFN-alpha promoter. In addition, both MW and PW inhibited the anti-HCV ability of recombinant IFN-alpha in the hepatic cells. These in vitro observations support the concept that opioid abuse favors HCV persistence in hepatic cells by suppressing IFN-alpha-mediated intracellular innate immunity and contributes to the development of chronic HCV infection.     

Ratio of serum gamma-GT/ALT rather than ISDR variability is predictive for initial virological response to IFN-alpha in chronic HCV infection.

Chronic hepatitis C virus (HCV) infection in humans is treated at present with interferon (IFN)-alpha. Because the proportion of patients responding to therapy with sustained or even just with transient elimination of viral RNA is low, several potential prognostic parameters have been evaluated to predict the outcome of the therapy. The present study aimed to prove the validity of a predictive parameter described previously for initial virological response, namely the ratio of serum gamma-glutamyltransferase/alanine transaminase (gamma-GT/ALT) activity in connection with virus genotypes 1a, 1b, and 3a, prospectively and to compare the predictive value of these combined parameters with amino acid variability within the interferon sensitivity determining region (ISDR). The prospective analysis confirmed previous data on the predictive value of the serum gamma-GT/ALT ratio. Concerning ISDR variability, the majority of ISDR sequences obtained from the mostly nonresponding type 1b-infected individuals (23/28) resembled nonmutant types (27/ 28). Isolates from type 3a-infected patients responding to therapy in the majority of cases (13/ 20) exclusively resembled nonmutant types when compared with databank type 3a sequences, but were mutant when compared with the prototype sequence HCV-J. However, the initial virological responsiveness among both type 1b- and type 3a-infected patients did not correlate to ISDR variability. In contrast, virological responsiveness was closely related to serum gamma-GT/ALT ratio. The data are not necessarily contrary to the concept that the number of amino acid exchanges within the ISDR compared with the prototype HCV-J sequence is related to some extent to IFN-alpha sensitivity. The ratio of serum gamma-GT/ALT in combination with HCV genotype, however, was found to be a more reliable and stringent predictive parameter.     

Rapid and simple detection of IFN-neutralizing antibodies in chronic hepatitis C non-responsive to IFN-alpha

Different mechanisms have been proposed for the failure of interferon (IFN) therapy in patients with chronic hepatitis C and multiple sclerosis, for example, the presence of IFN-neutralizing antibodies. In this study, a novel assay system based on the IFN-inducible Mx-promoter was used to detect IFN-neutralizing antibodies in sera of patients with chronic hepatitis C. To monitor IFN bioactivity in IFN-treated patients, a real-time RT-PCR for MxA gene expression in PBMCs was established. Using these two methods, patients with chronic hepatitis C virus (HCV) infection receiving IFN therapy and patients with treatment induced HCV clearance were monitored. Importantly, neutralizing anti-IFN antibodies were detected in the sera of 3 of 38 chronically HCV-infected patients who failed to respond to therapy but none in sera of patients who cleared HCV after IFN therapy. Interestingly, the presence of these antibodies correlated with the lack of MxA induction in PBMCs after initiation of IFN-alpha therapy. Retrospective analysis of one patient's sera revealed that the anti-IFN-alpha antibodies had already developed after the first of four unsuccessful IFN therapies, suggesting that neutralizing antibodies may have contributed to the failure of previous IFN treatments. In summary, a novel screening assay was established that may be helpful for testing IFN non-responders for the presence of clinically relevant anti-IFN-alpha antibodies and for selecting alternative IFN preparations not neutralized by these antibodies.     

Interferon-alpha (IFN-alpha) enhances cytotoxicity in healthy volunteers and chronic hepatitis C infection mainly by the perforin pathway.

Cell-mediated cytotoxicity is exerted via perforin and Fas ligand (FasL). We have recently shown that IFN-alpha up-regulates FasL expression in T cells isolated from healthy volunteers and augments activation-induced T cell death. Since the Fas/FasL system is implicated in the pathogenesis of hepatic failure and both molecules have been shown to be up-regulated in hepatitis C virus (HCV) infection, we studied whether cytotoxicity via the FasL system is enhanced by IFN-alpha and therefore could contribute to hepatic injury. We investigated FasL and perforin expression in peripheral blood mononuclear cells (PBMC) derived from HCV+ donors by Northern analysis and soluble FasL synthesis by ELISA. Natural killer (NK) cell and cytotoxic T lymphocyte (CTL) cytotoxicity was studied by 51Cr-release assays. IFN-alpha up-regulates FasL mRNA and protein synthesis in mitogen-activated PBMC of HCV+ individuals, as previously found in healthy subjects. Stimulation with IFN-alpha increases perforin mRNA levels in PBMC. In NK cytotoxicity assays, the enhancement of cytotoxicity by IFN-alpha is mainly due to the perforin pathway, while the FasL pathway plays only a minor role. In CTL cytotoxicity experiments neither the FasL nor the perforin pathway is further enhanced by IFN-alpha. Our data suggest that up-regulation of perforin by IFN-alpha results in elevated cytotoxicity, suggesting that IFN-alpha might support elimination of virally infected cells via this pathway. In contrast, the major effect of IFN-alpha on the Fas/FasL system might be the enhanced elimination of activated T cells as a means of finally limiting a T cell response.     

Factors predictive of response to interferon-alpha therapy in hepatitis C virus type 1b infection

Interferon-alpha (IFN-alpha) has been used to treat hepatitis C Virus (HCV)-induced infection but has been effective in only about half of all patients. It is suggested that the different responses to IFN-alpha treatment in HCV infection may be influenced by HCV genotypes, HCV RNA titer at the beginning of IFN-alpha therapy, and the sequences of the interferon sensitivity determining region (ISDR). However, there have also been reports showing that these have no relation to an IFN-alpha effect. In a previous study, it was found that the nucleotide sequence variation in the hypervariable region (HVR) 1 of the HCV could predict the effect of IFN-alpha. In the present investigation, an attempt was made to determine the predictive factors of IFN-alpha therapy. Twenty-six patients with HCV infection were treated with IFN-alpha. Among these, 13 patients recovered after 3 to 6 months of IFN-alpha treatment, although the other 13 patients showed no response after 6 months of treatment with IFN-alpha. In order to determine the predictive factors of IFN-alpha therapy, the ALT levels, HCV genotypes, HCV serum titer, and the quasispecies of HVR 1 were compared between responders and non-responders. It is suggested that the variation in the HVR 1 and HCV serum titer can be used to predict the effect of IFN-alpha.     

Upregulation of endogenous intrahepatic interferon stimulated genes during chronic hepatitis C virus infection

The success of interferon-alpha and ribavirin combination therapy for the treatment of chronic hepatitis C viral infection differs between patients. In an attempt to identify predictors of host response to therapy, the levels of mRNA for interferon (IFN) stimulated genes: MxA, PKR, 2'5' OAS, ISG15, and interleukin 8 (IL-8), were examined in liver by real-time RT-PCR prior to commencement of therapy. The levels of intrahepatic classical IFN stimulated genes, but not IL-8, in chronic HCV disease (n = 44) were found to be significantly upregulated (P < 0.001) compared to the control cohort (n = 12). The genotype of the infecting HCV strain did not influence IFN stimulated gene expression. These results suggest that the endogenous type 1 IFN antiviral effector pathway is broadly activated during chronic HCV disease, although the levels of mRNA for any of the IFN-stimulated genes tested did not predict the outcome of combination therapy.     

Identification of functional domains of the interferon-induced enzyme PKR in cells lacking endogenous PKR.

The interferon (IFN)-induced, double-stranded RNA (dsRNA)-activated human protein kinase (PKR) has been shown to exert antiviral and antiproliferative effects. Activation of the enzyme in mammalian cells results in protein synthesis inhibition and cell death by apoptosis. Previous studies on the structure-function relationship of PKR have been based on vectors expressing the enzyme in mammalian cells containing endogenous PKR. As exogenously expressed PKR can form heterodimers with endogenous PKR, the results obtained on the functional characterization of mutant forms of PKR have been taken with caution. To address the natural consequences of heterodimer formation between endogenous and exogenous PKR, we have analyzed the structure-function relationship of PKR ectopically expressed from vaccinia virus (VV) recombinants in cells lacking the endogenous enzyme. We demonstrate that PKR-mediated inhibition of protein synthesis and induction of apoptosis is not dependent on the presence of endogenous PKR. Further, PKR activity is independent of the presence of dsRNA binding motifs (dsRBM). Moreover, single-point mutations of the third basic domain decreased PKR activation. Our findings demonstrate that PKR can be activated in the absence of its N-terminal domain (amino acids 1-232) and that the third basic domain is important for its biologic function.     

MxA and PKR expression in chronic hepatitis C.

The effectiveness of therapy for chronic hepatitis C (CHC) patients has greatly improved in the last few years, and the gold standard is currently held to be pegylated interferon (IFN) in combination with ribavirin. Overall, however, the percentage of patients achieving a sustained virologic response (SVR) is only around 50%,and it is not possible to predict those patients who will benefit from therapy. The molecular mechanisms underlying lack of therapeutic response remain unknown. In this study, we investigated the tissue expression of MxA and RNA-dependent protein kinase (PKR), two antiviral proteins modulated by IFN, in biopsy samples from hepatitis C patients before the beginning of therapy. Our results show that expression of MxA, but not of PKR, is significantly lower in responders compared with nonresponders. No differences were observed regarding the hepatitis C virus (HCV) genotype and the viral load. These results suggest that expression of the MxA protein could play a role among the mechanisms underlying responsiveness to therapy.     

Intrahepatic MxA expression is correlated with interferon-alpha expression in chronic and fulminant hepatitis

Interferon-alpha (IFN-alpha) has potent pro-inflammatory and anti-viral functions. It exerts its effects by inducing intracellular proteins such as MxA. To analyse the role of intrahepatic interferon activation, IFN-alpha and MxA expression was studied by immunohistochemistry in explant livers of 20 patients with fulminant hepatic failure (FHF), 41 patients with chronic liver disease (CLD), and ten normal controls (NCs). In NCs only small numbers of Kupffer cells, but no hepatocytes, showed IFN-alpha and MxA expression. In contrast, significantly enhanced numbers of IFN-alpha- and MxA-positive Kupffer cells, along with small numbers of MxA-positive and larger numbers of IFN-alpha-positive lymphocytes, were found in CLD and in FHF. MxA protein was also expressed on hepatocytes and bile ducts in the vicinity of IFN-alpha-positive inflammatory infiltrates (hepatocytes: NCs: 0%, CLD: 8%, FHF: 68%; bile ducts: NCs: 19%, CLD: 46%, FHF: 83%). A significant correlation was found between the numbers of IFN-alpha- and MxA-positive cells (r=0.67, p<0.001). Thus, large amounts of IFN-alpha are released in the livers of patients with FHF, which is likely to contribute to immune-mediated liver cell damage. Intrahepatic MxA expression corresponds to IFN-alpha produced particularly by infiltrating inflammatory cells, rather than by hepatocytes themselves.