WO2005071101A1 - Treatment of hepatitis c infection by increasing stat1 methylation - Google Patents

Abstract

The present invention pertains to the signaling involved in suppression of immune function during hepatitis C infection. More specifically, this invention relates to the discovery that PP2A is involved in the inhibition of interferon alpha signaling in hepatitis C infection. Reduced STAT1 methylation and reduced interferon signaling. Restoring methylation by treatment with a methyl group donor increases interferon signaling.

Description

TREATMENT OF HEPATITIS C INFECTION BY INCREASING STAT1 METHYLATION

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims benefit under 35 U.S.C. 119(e) of U.S. Provisional Application No. 60/560,728, filed January 23, 2004, the entire disclosure of which is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention The present invention pertains to the signaling involved in suppression of immune function during hepatitis C infection. More specifically, this invention relates to the discovery that PP2A is involved in the inhibition of interferon alpha signaling in hepatitis C infection. Reduced STAT1 methylation and reduced interferon signaling. Restoring methylation by treatment with a methyl group donor increases interferon signaling.

2. Background Art Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. An important and striking feature of hepatitis C is its tendency towards chronicity. In over 70% of infected individuals, HCV establishes a persistent infection over decades that may lead to cirrhosis and hepatocellular carcinoma. In order to do so, HCV must escape the host immune response. Type I interferons (IFN) are crucial and potent components of the early host response against virus infection¹. Many viruses have evolved strategies to protect themselves against the IFN system². Viruses can interfere with the IFN system by blocking IFN synthesis, by inhibiting IFN signaling or by inhibiting the functions of IFN-induced proteins such as dsRNA-dependent protein kinase (PKR). The most important signal transduction pathway for IFNs is the Jak-STAT pathway³. IFNα and JJFNβ activate STAT1, STAT2 and often STAT3. Signal transducers and activators of transcription (STAT) proteins are activated by members of the Jak kinase family through the phosphorylation of a single tyrosine residue . Activated STATs form dimers, translocate into the nucleus and bind specific DNA elements in the promoters of target genes ' . This activation cycle is terminated by tyrosine dephosphorylation in the nucleus, followed by the decay of dimers and the nuclear export of STATs⁷' ⁸. Negative regulators of this signal transduction pathway have been found at three levels. First, the suppressor of cytoldne signaling (SOCS) family members SOCS1 and SOCS3 prevent phosphorylation and activation of IFN induced STATs by inhibiting the IFN receptor associated Jak kinases⁹. Second, downstream of STAT activation by tyrosine phosphorylation, IFN induced gene transcription can be inhibited by protein inhibitor of activated STAT1 (PIAS1). PIAS1 inhibits binding of STAT1 dimers to the response elements in the promoters of target genes¹⁰. The binding of PIASl to STAT1 is regulated by methylation of STAT1 by protein arginine methyl-transferase PRMT1¹¹. Arginine methylation inhibits binding of PIASl to STAT1, whereas demethylation of STAT1 enhances its association with PIASl. A third step of negative regulation occurs through dephosphorylation and deactivation of STATs in the nucleus. Recently, the 45 lcDa isoform of protein tyrosine phosphatase TC-PTP was identified as the nuclear STAT1 phosphatase ². The inventors (hereinafter sometimes referred to as "we") have shown previously that the expression of HCV proteins in human osteosarcoma cell lines inhibits IFNα induced signaling through the Jak-STAT pathway¹³. The inventors confirmed these findings also in livers of transgenic mice expressing the entire HCV open reading frame under the control of an alphal-antitrypsin gene promoter¹⁴. Both in the cell lines and in the mouse livers, activation of STATs by tyrosine phosphorylation was not inhibited, but binding of activated STAT1, STAT2 and STAT3 to DNA was strongly reduced. The inventors hypothesized that HCV proteins may induce cellular proteins that inhibit STAT DNA binding, for instance PIASl or other yet unknown proteins, or that one or more nuclear phosphatases could be induced, resulting in accelerated dephosphorylation and decay of STAT dimers. Since we could not detect an induction of PIASl by HCV proteins neither in the cell lines nor in the transgenic mouse livers, we screened mouse liver extracts for an overexpressed phosphatase. The inventors have found that the catalytic subunit of protein phosphatase 2 A (PP2Ac) was overexpressed in liver extracts of HCV transgenic mice. Interestingly, expression of an N-terminally modified catalytic subunit of PP2A in the human hepatoma cell line Hulϊ7 resulted in hypomethylation of STAT1 leading to inhibition of STAT1- DNA binding through increased binding to PIASl. The relevance of these findings for the human disease was confirmed in liver biopsies from patients with chronic hepatitis C. Moreover, the inventors have found that treatment with a methyl group donor can restore IFN signaling. S-adenosyl-L-methionine (SAMe, also called AdoMet or S- Adenosylmethionine) is a methyl group donor produced naturally in all living organisms. Under normal circumstances, it is produced in the cells by the transfer of an adenosyl group (derived from ATP) to methionine by the enzyme methionine adenosyl transferase. After enzymatic transfer of its methyl group, SAMe is metabolized to homocysteine, a potential toxic substance. Homocysteine can by recycled to SAMe by transfer of a methyl group from betaine by betaine-homocysteine methyltransferase (BHMT). By using a combination of SAMe and betaine, the inventors found a significant increase in the induction of interferon stimulated genes by IFNα. The inventors have found that treatment of cultured cells with SAMe increases the methylation of STAT 1 and increases IFN signaling. Since methylated STAT1 can not be bound by its inhibitor PIASl, it is a better IFN signal transducer. Currently, patients with chronic hepatitis C are treated with pegylated interferon alpha 2a or 2b in combination with ribavirin. 50-60% of the patients treated with pegylated interferon and ribavirin do not respond to the treatment. For patients with genotype 1, the treatment success rate is 40-45%. The work of the inventors indicates that correction of the methylation state of STAT 1 should increase the responsiveness of patients to therapy for hepatitis C. Although others have suggested treatment of patients with a combination of interferon alpha, ribavirin, and antioxidants including SAMe (see WO 03/024461), they do not appreciate the importance of restoring STAT1 methylation, and they use SAMe as an antioxidant to reduce ribavirin-related hemolysis. Further objects, features and advantages of the present invention will become apparent from the Detailed Description of Preferred Embodiments, which follows, when considered in connection with the attached Drawings.

SUMMARY OF THE INVENTION It is a general object of the invention to increase the level of STAT 1 methylation in vivo. A first preferred embodiment of the invention is a compound increasing methylation of a STAT1 in vivo. A second preferred embodiment is a compound increasing methylation of a STATl in vivo wherein the compound acts by increasing or decreasing an activity of a protein selected from the group consisting of: PP2A2, PRMT1, and PIASl. A further preferred embodiment is a therapeutic composition comprising the compound of second embodiment and a pharmacologically-acceptable carrier. A third preferred embodiment is a compound decreasing activity of a PP2A. Another preferred embodiment is a therapeutic composition comprising a compound of the third embodiment and a pharmacologically-acceptable carrier. A fourth preferred embodiment is a method of selecting a drug to treat hepatitis C, comprising the steps of: providing a plurality of drug candidates, assaying the candidates to ascertain a change in a level of STATl methylation upon application of the candidates to a model system, selecting candidates that increase methylation of STATl, and selecting a candidate from among those selected candidates that is effective in treating hepatitis C. A fifth preferred embodiment is the fourth embodiment, wherein said model system is selected from the group consisting of a protein binding assay, a protein activity assay, a cell culture model, and an animal model. A sixth preferred embodiment is the fifth embodiment wherein the last-named step comprises testing the drug in an animal model of hepatitis C infection. Another preferred embodiment is a drug developed by the method of the sixth embodiment. A seventh preferred embodiment is a method if selecting a drug to treat hepatitis C, comprising the steps of providing a plurality of drug candidates, assaying the candidates to ascertain a change in a level of PP2A activity upon application of the candidates to a model system, selecting candidates that change the level of PP2A activity, and selecting a candidate from among those selected candidates that are effective in treating hepatitis C. Yet another preferred embodiment is the seventh embodiment wherein the model system is selected from the group consisting of a protein binding assay, a protein activity assay, a cell culture model, and an animal model. Still another preferred embodiment is the seventh embodiment wherein the last- named step comprises testing the drug in an animal model of hepatitis C infection. Yet another preferred embodiment is a drug selected by the method of the seventh embodiment. An eighth preferred embodiment is a method of treating hepatitis C infection, comprising enhancing a level of methylation of STAT 1. A ninth preferred embodiment is the method of the eighth embodiment, further comprising increasing or decreasing an activity of a protein selected from the group consisting of: PPA2, PRMT1, and PIASl. Still another preferred embodiment is the method of the ninth embodiment, wherein said increasing or decreasing step is carried out by administering a therapeutic composition. A tenth preferred embodiment of the invention is a method of treating hepatitis C infection, comprising reducing an activity of PP2A. Yet another preferred embodiment of the invention is the method of the tenth embodiment, wherein said reducing is carried out by administering a therapeutic composition. Still another preferred embodiment of the invention is the method of the eighth embodiment further comprising the step of administering a drug selected by the method of the fourth embodiment. Yet another preferred embodiment of the invention is the method of the eighth embodiment further comprising the step of administering a drug selected by the method of the seventh embodiment. Still another preferred embodiment is the use of methyl group donor for the manufacture of a pharmaceutical composition, wherein the pharmaceutical composition comprises a methyl-group donor in an amount effective to increase a level of STATl methylation in a patient. An eleventh another preferred embodiment is the use of a methyl group donor to increase a level of STATl methylation in a patient, comprising (1) selecting a patient, and (2) treating the patient with a methyl group donor in an amount effective to increase the level of STATl methylation in the patient. Yet another preferred embodiment is the use of the eleventh embodiment, further comprising the step of monitoring said level of STATl methylation in said patient. Still another preferred embodiment is a pharmaceutical composition consisting essentially of interferon, ribavarin, a methyl group donor, and a pharmacologically- acceptable carrier. Yet another preferred embodiment is a pharmaceutical composition consisting essentially of interferon, ribavarin, a methyl group donor, and a pharmacologically- acceptable earner. Still another preferred embodiment is any of the previously-named embodiments with a methyl group donor, wherein the methyl group donor is SAMe. Yet another preferred embodiment is any of the previously-named embodiments with a methyl group donor, wherein betaine is used in conjunction with said methyl group donor. A twelfth preferred embodiment is a method of monitoring a patient, comprising the steps of (1) selecting a patient undergoing treatment for viral infection, and (2) testing one or more samples from the patient to ascertain a level of STATl methylation. Still another preferred embodiment is the method of the twelfth embodiment, wherein the viral infection is a hepatitis C infection. Yet another preferred embodiment is a pharmaceutical composition comprising pegylated interferon, ribavarin, SAMe, betaine, and a pharmacologically-acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS Abbreviations as used herein are: DMA, dimethylarginine; EMSA, electrophoretic mobility shift assay; HA-PP2Ac, catalytic subunit of PP2A fused to an influenza hemagglutinin derived nine-amino-acid epitope tag; HCV, hepatitis C virus; hlFNα (hlFNa), human interferon alpha; ISRE, interferon stimulated response element; MTA, 5'-methyl-thioadenosine; OA, Okadaic Acid; PIASl, protein inhibitor of activated STATl; PKR, dsRNA-dependent protein kinase; PP2A, protein phosphatase 2 A; PP2Ac, catalytic subunit of PP2A; PRMT1, protein arginine methyl-transferase 1; SOCS, suppressor of cytokine signaling; SAMe, S- adenosylmethionine; STAT, signal transducer and activator of transcription; TC-PTP, T cell protein tyrosine phosphatase. Figure 1. TC-PTP, STATl phosphorylation, and PP2A expression in HCV transgenic mice. (A) Nuclear tyrosine phosphatase TC-PTP is not upregulated in B6HCV mice. Liver extracts of untreated or IFNα (lOOOU/g of body weight) treated C57BL6 and B6HCV were tested for TC-PTP expression by Western blot. Blots were stripped and reprobed for actin as loading control. No difference in TC-PTP expression was detected.

(B) Overexpression of PP2Ac in transgenic mice. Western blot with whole cell extracts from C57BL6 and B6HCV mouse livers show increased amounts of PP2Ac. The membrane was stripped and reprobed with actin antibodies as a loading control. Blots were quantified using NIH Image software and values represent the mean ± SEM of 14 mice in each group. *** Indicates a statistical significant difference (P < 0.001) between B6HCV and C57BL6 groups (ANOVA test).

(C) PP2Ac activity measurements. Serine/threonine phosphatase activity of PP2Ac from whole cell extracts was performed using Serine/Threonine Phosphatase Assay System (Promega) according to the manufacturer's instructions. Shown are the mean values ± SEM of 2 animals in each group. The repetition of the experiment gave the same result (not shown). * indicates a statistical significant difference (P < 0.05) between B6HCV and C57BL6 mice (ANOVA test).

(D) Phospho-serine and phospho-tyrosine status of STATl is not impaired in HCV transgenic mice. C57BL6 and B6HCV mice were left untreated or treated with mlFNα (lOOOU/g of body weight, 20 min). Liver extracts were probed for tyrosine phosphorylation of STATl by Western blot with STATl phosphotyrosine specific antibodies (PY-STAT1) and for serine phosphorylation by a STATl immunoprecipitation followed by Western blot with STATl phosphoserine specific antibodies (PS-STAT1). Blots were stripped and reprobed with STATl antibodies. No difference between C57BL6 and B6HCV was detected. Figure 2. Expression of an epitope-tagged phosphatase 2A catalytic subunit inhibits IFNα induced Jak-STAT signaling.

(A) From eight Huh7 derived clones with stable expression of hemagglutinin (HA) tagged PP2Ac the two clones with the highest expression levels were used for analysis of IFNα induced signal transduction. Shown are the results with one of them (the same results were found with the other clone). Lysates from unstimulated Huh7 and HA-PP2Ac expressing cells were analyzed for the expression of endogenous PP2Ac and transfected HA-PP2Ac by Western blot with PP2Ac specific antibodies. Membranes were then stripped and reprobed with HA antibodies to confirm the presence of the HA-tag and with actin antibodies as a loading control.

(B) Epitope-tagged PP2Ac allows expression of functionally active phosphatase 2A catalytic subunit. Serine/threonine phosphatase activity of PP2Ac from whole cell lysates was measured using the Serine/Threonine Phosphatase Assay System (Promega) according to the manufacturer's instructions. Values are the mean ± SEM of 2 independent experiments.

(C) Expression of HA-PP2Ac impairs Jak-STAT signaling. Huh7 and HA-PP2Ac expressing cells were incubated in the presence or in the absence of hlFNα (lOOOU/ml, 20 min) with or without pretreatment with okadaic acid (lOOnM, 60 min) prior to hlFNα stimulation as indicated. Nuclear extracts were analyzed with EMSAs using the SIE-m67 oligonucleotide probe.

(D) Serine and tyrosine phosphorylation of STATl is not impaired by HA-PP2Ac expression. Whole cell lysates from unstimulated or stimulated cells were probed for tyrosine phosphorylation of STATl (PY-STAT1) or serine phosphorylation (PS-STAT1) with specific antibodies by Western blot or STATl immunoprecipitation / Western blot, respectively. Blots were stripped and reprobed with STATl antibodies. (E) Incubation of purified, active PP2Ac with nuclear extracts from Huh7 cells does not impair Jak-STAT signaling. Nuclear extracts from unstimulated or hlFNα (lOOOU/ml, 20 min) treated cells were incubated with purified PP2A for 10 or 20 min as indicated (samples in duplicates). EMSA was performed with the SIE-m67 oligonucleotide probe. Figure 3. PP2Ac overexpression reduces the arginine methylation of STATl resulting in enhanced binding of PIASl to STATl.

(A) Expression of HA-PP2Ac reduces STATl arginine methylation. Whole cell lysates from Huh7 and HA-PP2Ac were subject to immunoprecipitation with mono/dimethyl arginine antibodies. Resolved proteins were immunoblotted for STATl.

(B) Reduced arginine methylation of STATl in liver extracts of HCV transgenic mice. Liver cell extracts of C57BL6 and B6HCV mice were immunoprecipitated with DMA antibodies. The amount of precipitated STATl was analyzed by Western blot with STATl specific antibodies.

(C) Semiquantitative determination of STATl methylation. Liver extracts of untreated mice were immunoprecipitated with DMA antibodies. The amount of precipitated STATl was analyzed by Western blot with STATl specific antibodies. The signal intensities were quantified with the NIH image computer program. The bar diagram shows the mean values (plus standard errors) of 4 C57BL76 and 4 B6HCV mice.

(D) Increased binding of PIASl to STATl in cells expressing HA-PP2Ac. Whole cell lysates from Huh7 and HA-PP2Ac cells were subject to immunoprecipitation with STATl antibodies and then blotted for PIASl. As a positive control, Huh7 cells were treated with for 1 hour with 0.3 mM MTA.

(E) Increased binding of PIASl to STATl in liver extracts from HCV transgenic mice. Liver extracts from C57BL6 and B6HCV mouse were subject to immunoprecipitation with STATl antibodies and then blotted for PIASl.

(F) Inhibition of PP2Ac with okadaic acid increases arginine methylation of STATl. Huh7 cells and HA-PP2Ac expressing cells were treated with 1000 U/ml hlFNα for 20 minutes after preincubation with or without okadaic acid (lOOnM, 60 min). The reduction of arginine methylation observed in cells expressing HA-PP2Ac in the absence of okadaic acid (lane 2) is partially reversed by pretreatment with lOOnM okadaic acid (lane 3). The lower panel shows the PP2Ac phosphatase activity in the corresponding samples. The activity is increased in untreated HA-PP2Ac expressing cells compared to Huh7 cells, but strongly inhibited by pretreatment with okadaic acid. Serine/threonine phosphatase activity of PP2Ac from whole cell lysates was measured using the Serine/Threonine Phosphatase Assay System (Promega) according to the manufacturer's instructions.

Figure 4. Impaired IFNα induced Jak-STAT signaling in liver biopsies of patients with chronic hepatitis C.

(A) Human liver biopsies can be stimulated in vitro with IFNs. 5-10 mm long tissue cylinders obtained by percutaneous liver biopsy with a 1.4mm thick biopsy needle were incubated immediately after retrieval in phosphate buffered saline (PBS) alone or in PBS with lOOOU/ml human IFNα for the indicated length of time. Cryosections (5 μm) were made and subcellular localization of PY-STAT1 was determined using an antibody against Tyr-701 of STATl. Nuclei were stained with Hoechst stain. The white size bars indicate 20 μm. Activation of STATl and nuclear translocation was uniform throughout the biopsy cylinder. Maximum activation of STATl occurred after 20 min.

(B) Quantification of activated STATl in liver cells nuclei. Shown are values (percent liver cell nuclei with positive staining for PY-STAT1) from 4 patients: Number 53 had a nodular regenerative hyperplasia without liver fibrosis, number 59 had a chronic hepatitis C with a liver cirrhosis,. number 60 had a chronic hepatitis C with mild fibrosis, and number 61 was recovered from hepatitis B and showed no fibrosis on routine histology. For each time point and patient, a minimum of 200 cells was counted.

(C) Semiquantitative assessment of EMS As with nuclear extracts from liver biopsies of patients with chronic hepatitis C or controls. Liver biopsies were incubated in the absence or in the presence of hlFNα (lOOOU/ml) for 20 minutes and nuclear extracts were prepared. EMS As were performed using 10 μl of nuclear extracts with SJE-m67 oligonucleotide probe. Shown are representative samples: Patients number 64, 83 and 104 were controls, patients number 69, 111, 115, 78 and 73 had chronic hepatitis C. An example of signal quantification is shown with patient number 73. The autoradiogram signal intensity was measured using NTH Image software and then normalized to 1 μg of total protein used for EMSA. The response to hlFNα stimulation is calculated by a ratio of the normalized signal intensity after hlFNα treatment divided by the normalized signal intensity of PBS controls (= fold increase).

(D) Impaired IFNα induced signaling in liver biopsies from patients with chronic hepatitis C. Shown are box-plots of the fold increase in EMSA signals after IFNα treatment for 25 patients with chronic hepatitis C and 10 controls. The difference between the groups was analyzed with the Mann- Whitney U test and found to be significant at p = 0.03. Figure 5. Expression, phosphorylation and arginine methylation of STATl, and expression of PP2Ac in human liver cells.

(A) Tyrosine phosphorylation of STATl is not impaired by the presence of the HCV. Biopsies from controls (patients number 64, 83, 89) and from patients with chronic hepatitis C (patients number 69, 119, 132) were incubated with hlFNα (lOOOU/ml, 20 min) and extracts were probed for PY-STAT1 to check the phosphorylation status of STATl. The two bands seen in some samples are the larger STATl α and the shorter STATl β, respectively. Nitrocellulose membranes were then stained with Blot-FastStain to verify the loading amount of total protein. One of the stained proteins (180 kDa in size) is shown as an example.

(B) STATl is overexpressed in liver cells of HCV patients. Biopsy extracts from healthy (numbers 83, 143, 64) and HCV patients (numbers 81,82,87) were probed for STATl to determine the expression level. Nitrocellulose membranes were then stained with Blot- FastStain to ensure equal protein loading. One of the proteins (180 kDa in size) is shown.

(C) Semiquantitative assessment of STATl expression. The STATl signal intensity in Western blots was quantified with NIH Image software. The results (arbitrary units) are shown in a box plot diagram. The difference between controls and HCV patients is statistically significant (P < 0.0001, Mann- Whitney U test).

(D) Overexpression of PP2Ac in liver cells of HCV patients. Liver biopsy extracts from controls (numbers 83, 143, 64) and HCV patients (numbers 81, 82, 87) were analyzed with Western blot using a PP2Ac specific antibody. The same 180 kDa protein as above is shown as a loading control.

(E) Semiquantitative assessment of PP2Ac expression. The PP2Ac signal intensity in Western blots was quantified with NIH Image software. The results (arbitrary units) are shown in a box plot diagram. The difference is significant at p = 0.04 (Mann- Whitney U test).

(F) STATl is hypomethylated in HCV patients. Whole cell lysates from unstimulated healthy (numbers 153, 154) and HCV patients (numbers 132, 138) were subject to immunoprecipitation with DMA antibodies and then blotted for STATl. The two bands seen in some samples are STATlα and STATlβ, respectively. (G) Quantification of STATl methylation. The STATl specific signal intensities after immunoprecipitation with DMA antibodies (upper panel of figure 5F) were divided through the STATl specific signal intensities in a Western blot (not shown) for two controls (numbers 153, 154) and two patients with chronic hepatitis C (numbers 132, 138). STATl is hypomethylated in extracts from liver biopsies of patients with chronic hepatitis C as compared to controls.

(H) Increased binding of PIASl to STATl in HCV patients. Whole cell extracts from unstimulated control patients (number 148, 154) and HCV patients (numbers 132, 138) were subject to immunoprecipitation with STATl antibodies and resolved proteins were blotted for PIASl.

(I) Nuclear tyrosine phosphatase TC-PTP is equally expressed in human liver cells of healthy and HCV patients. Whole cell lysates from unstimulated liver biopsies were probed for TC-PTP to determine the expression level. Nitrocellulose membranes were then stained with Blot-FastStain to verify the loading amount of total protein. Figure 6. STATl expression, PP2Ac expression and EMSA signal intensity of STATl are independent of inflammation and fibrosis of liver biopsies. Pathological grading and staging of liver biopsies was according to Hytiroglou et al.¹⁶: Grade of inflammation: 0 = no inflammation, 1 = mild, 2 = moderate, 3= severe; Stage of fibrosis: 0 = no fibrosis, 1 = mild, 2 = moderate, 3 = severe, 4 = cirrhosis. Biopsies were grouped in 4 categories of inflammation (left column) or 5 categories of fibrosis (right column). Shown are box plot diagrams of protein expression levels of STATl (first row) and PP2Ac (second row) and of STATl gel shift signal intensities (third row). None of the differences between groups were statistically significant when tested with the parametric one-factor ANOVA test or the non-parametric Kruskal-Wallis test by ranks. Figure 7. Treatment to restore STATl methylation and interferon signaling. (A) EMSA with m67 oligonucleotide showing that treatment with AdoMet (SAMe) restores IFN signaling in cells expressing HA-PP2Ac. (B) Immunoprecipitation analysis shows that AdoMet (SAMe) restores STATl methylation in cells expressing HA-PP2Ac and reduces association of STATl with the inhibitor Piasl. Figure 8. Treatment of Huh7 and HA-PP2A cells with Adomet and Betaine increases the induction of the IP- 10 gene by IFNα. The cells were treated overnight with AdoMet and/or Betaine using the concentrations indicated on the graph. They were ten treated again with AdoMet, Betaine, and IFNα for 6 hours. After extraction of RNA, real time RT-PCR was performed to quantify the amount of IP- 10 mRNA.

DETAILED DESCRIPTION OF THE INVENTION Signaling through the Jak-STAT pathway is crucial for most of the known effects of both type I and type II interferons. In cell lines and in transgenic mice the inventors have previously found inhibition of IFNα induced binding of activated STATs to their cognate response elements by HCV proteins. Interestingly, in none of these cases any of the well- known Jak-STAT inhibitors SOCS1, SOCS3 or PIASl were induced. The inventors have now found that hypomethylation of STATl is the basis for the observed inhibition of Jak- STAT signaling. Methylation of STATl has recently been recognized as a third important posttranslational modification besides tyrosine and serine phosphorylation that regulates STAT mediated transcriptional activation of target genes¹ . Methylation of of STATl is catalyzed by PRMT1, and inhibits the binding of PIASl to STATl ⁿ. Binding of PRMT1 to the IFNα receptor has been reported before . The same authors observed impairment of the antiviral and antiproliferative effects of IFNβ in HeLa cells when the expression of PRMT1 was reduced by antisense cDNA²⁷. Interestingly, methyl-thioadenosine (MTA) is an inhibitor of PRMT1 that accumulates in many transformed cells. Through inhibition of STATl methylation, MTA might contribute to the relative unresponsiveness of many cancer cells towards the antiproliferative effects of IFNs¹¹. The inventors have found for the first time that a virus, HCV, can induce IFNα hyporesponsiveness by blocking methylation of STATl. The expression of HCV proteins in liver cells of transgenic mice reduced STATl methylation and consequently increased association of STATl with PIASl. Importantly, reduced methylation and increased PIASl -STATl binding was also observed in liver biopsies from patients with chronic hepatitis C. HCV proteins may directly inhibit PRMT1 or stimulate demethylation by an as yet unknown enzyme. However, the inventors have found that PP2A is involved in mediating this effect: First, PP2A was found to be overexpressed in HCV transgenic mice and in liver biopsies from patients with chronic hepatitis C. Second, Huh7 cell stably expressing the catalytically active HA-PP2Ac had hypomethylated STATl in the absence of any HCV proteins. Third, inhibition of PP2A with okadaic acid partially restored methylation of STATl. Alternatively, PP2A might influence DNA binding of STATl directly by changing the phosphorylation status of STATl, without having any influence on the methylation status of STATl. For example, PP2A has been reported to inhibit DNA binding of STATl by dephosphorylation of serine 727 without affecting tyrosine phosphorylation in an in vitro assay with extracts from U937 cells²⁶. In human T cells or lymphoma cells, inhibition of PP2A was found to induce serine phosphorylation of STAT3, inhibit tyrosine phosphorylation of STAT3, and to induce relocation of STAT3 from the nucleus to the cytoplasm²³. Furthermore, PP2A inhibition by okadaic acid was found to strongly enhance LL-3 induced tyrosine phosphorylation of STAT5 in 32Dcl3 myeloid progenitor cells²⁴. While these various experimental systems yield different results with regard to the influence of PP2A on Jak-STAT signal transduction, they nevertheless raise the possibility that PP2Ac upregulation in HCV transgenic mice and in liver cells of patients with chronic hepatitis C may influence STAT function by altering tyrosine or serine phosphorylation of STATl. The inventors therefore looked carefully for changes of serine or tyrosine phosphorylation caused by overexpression of PP2A, but did not detect significant differences in extracts from HA-PP2Ac expressing cells compared with Huh7 cells nor in liver extracts from HCV transgenic mice compared with control mice. The inventors conclude that in our experimental systems, the influence of PP2A is indirect through the inhibition of methylation or the stimulation of demethylation of STATl. So far, we found no good evidence for a direct interaction between PP2A and PRMT1, and we can not exclude that the effect of PP2A is mediated through other proteins. However, the fact that expression of HA-PP2Ac in Huh7 cells caused a hypomethylation of STATl and inhibitors of PP2A restored methylation indicates that PP2Ac is upstream of PRMT1. Interestingly, the PP2A holoenzyme assembly itself is regulated by reversible methylation of the catalytic subunit PP2Ac. However, the methyltransferase involved is PPMT and not PRMT1^{28, 29}. PP2A is involved in a wide range of cellular processes including cell cycle regulation, cell morphology, development, signal transduction, translation, apoptosis and stress response²¹. A number of viral proteins have been found to interact with subunits of PP2A, amongst them polyoma small t and middle T, SV40 small t, the HIV protein vpr and the adenovirus E4orf4 protein. These associations alter the function of PP2A without affecting its expression level. The expression of PP2Ac is controlled by an autoregulatory translational mechanism²². However, downregulation of PP2Ac has been found, for instance during all-trα«-f-retinoic acid-induced differentiation of HL-60 cells³⁰ or during peroxisome proliferator-activated receptor-γ induced adipocyte differentiation³¹. Likewise, upregulation of PP2Ac was found in macrophages in response to colony-stimulating factor 1 . Here we report for the first time that expression of viral proteins can upregulate PP2Ac. Given the central role of PP2A in the regulation of cellular homeostasis, it is likely that this upregulation has many additional effects on cells infected with HCV. However, IFN signaling was found to be an important target for viral interference with host defense, and inhibition of STATl might well be a crucial effect of PP2Ac upregulation by HCV. What cells in the liver are crucial for the observed interference of HCV with IFNα signaling? In human liver biopsies (Figure 4A), not only hepatocytes, but also non- parenchymal resident liver cells and infiltrating lymphocytes have nuclear localized phospho-STATl, and all of them could be the site of PP2Ac upregulation and inhibition of STATl signaling. However, the inventors have found that the HCV transgenic mice have no inflammatory infiltrates¹⁴, but still show increased PP2Ac expression levels and impaired STATl signaling. Furthermore, the inventors have found no correlation was found between the degree of inflammation in liver biopsies and the amount of STATl, PP2Ac or the strength of STATl DNA binding (Figure 6). Therefore, the most likely site of HCV interference with IFNα signaling is the hepatocyte. HCV has a striking capability to establish a chronic infection, and attenuation of IFNα signaling is an important advantage of the virus early in the course of the infection. However, HCV can not block IFNα signaling completely, as demonstrated by the success of therapies based on the application of pharmacological doses of recombinant IFNα in some but not all patients with chronic hepatitis C. It is not well understood, which host factors determine the response to IFNα therapies. PP2Ac expression levels and the strength of IFNα induced signaling through the Jak-STAT pathway might represent such host factors. The mean PP2Ac expression level of the 8 patients that were not cured by standard therapies with pegylated IFN-α and ribavirin is 5812 (arbitrary units), compared to 590 for the patients with a sustained response. Likewise, the mean fold increase in EMSA signals after ex vivo FFNα treatment of biopsies is 3.3 in therapy responders, and 2.3 in non responders (table 1). However, the low sample size in this study does not allow a statistical analysis, and we conclude that we do not know if patients with high expression levels of PP2Ac are less responsive to IFNα treatments. Presently, we can only speculate that interference of HCV with IFNα signaling might also impair the success of IFNα therapies. Should further studies find that increased PP2Ac levels impair the response to IFNα based treatments, then treatment strategies aimed at reversing the inhibitory effect of PP2Ac on IFNα signaling might enhance the response rates to current standard therapies with IFNα and ribavirin.

We describe here that HCV interferes with IFNα signaling via upregulation of PP2Ac. As a consequence, STATl methylation is reduced. Because hypomethylated STATl is bound by its inhibitor PIASl, the transcriptional activation of IFN target genes is impaired. The inhibition of the IFNα induced antiviral response might allow HCV to evade this important first line of defense of the host and to establish a chronic infection. Restoring STATl methylation in cells in culture restores IFN signaling, thus treatment of patients receiving therapy for HCV infection with an agent to restore STATl methylation should improve the efficacy of therapy. One example of such an agent is SAMe. Betaine used in conjunction with SAMe potentiates the effects of SAMe on STATl methylation and interferon signaling. After transfer of the methylgroup, SAMe is metabolized to homocysteine, a potential toxic metabolite. Homocysteine can be recycled to SAMe using betaine as a methyl group donor by the enzyme betaine-homocysteine- methyltransferase (BHMT). The combined use of SAMe and betaine significantly improves the methylation of STATl and the induction of interferon stimulated genes by IFNα. Another way to achieve the therapeutic goal of increased STATl methylation would be to decrease activity of PP2A.

Methods as used in the examples

Patients, Biopsies From February, 2001, to April, 2002, all patients with chronic hepatitis C referred to the outpatient liver clinic of the University Hospital Basel were asked for their permission to use part of the liver biopsy for this study. The protocol was approved by the ethical commission of Basel. Written informed consent was obtained from all patients that agreed to participate in the study. After removal of a 20 to 25mm long biopsy specimen for routine histopathological workup for grading and staging of the liver disease according to Hytiroglou et al.¹⁶, the remaining 5 to 20 mm long biopsy cylinders were used for the preparation of cytoplasmic and nuclear extracts and in some cases for immuno fluorescence studies. For EMSA and Western blots, all samples that yielded at least 0.5 micrograms per microliter were used. The other samples were discarded (about 30-40% of all biopsies obtained). For non-HCV controls, patients that underwent ultrasound-guided liver biopsies of focal lesions (mostly metastasis of carcinomas) were asked for their permission to obtain a biopsy from the normal liver tissue outside the focal lesion. Written informed consent was obtained. Again, a part of the biopsy was used for routine histopathological diagnosis, and the remaining tissue for the preparation of cell extracts as described below. Only samples with confirmed absence of liver disease in the routine histopathological workup were used as controls in the present study.

Cell culture, Transfection Cells were cultured in 10% calf seram Minimum Essential Medium (Invitrogen) HA-PP2Ac cell lines were obtained by stably transfecting Huh7 cells in 6 cm tissue culture dishes using Effectene Transfection Reagent kit (Qiagen) according to the manufacturer's instructions. After 48 hours, the cells were rinsed with PBS and incubated with fresh medium (10% calf serum/Minimum Essential Medium) supplemented with penicillin, streptomycin and 800 μg/ml G418. Individual colonies were picked 7 to 10 days later and propagated. In eight clones out of 20 that were analyzed for HA-PP2Ac expression the transgene was detected by anti-HA Western blot. The two clones with the highest expression level were used for the analysis of IFNα induced intracellular signaling.

Preparation of extracts from cells Cells were lysed on the plates in 200 μl of lysis buffer containing 100 mM NaCl, 50 mM TRIS pH 7.5, 1 mM EDTA, 0.1% Triton X-100, 10 mM NaF, 1 mM PMSF, and 1 mM vanadate. Lysates were centrifuged at 14,000 rpm for 5 min and protein concentration was determined by Lowry (BioRad Protein Assay). For mouse and human whole cell extracts, samples were dounce homogenized in 500 μl or 120 μl of lysis buffer respectively. For cell nuclear extracts, cells were rinsed with cold PBS, scraped from plate and lysed in Low Salt Buffer containing 200 mM Hepes pH 7.6, 10 mM KC1, 1 mM EDTA, 1 mM EGTA, 0.2% NP-40, 10% glycerol and 0.1 mM vanadate. Then they were centrifuged at 15,000 rpm for 5 min and the pellet was resuspended in High Salt Buffer containing Low Salt Buffer supplemented with 420 mM NaCl. After centrifugation, aliquots were made for EMSA.

Immunofluorescence with human liver biopsies For liver biopsy immunostaining, biopsies were performed with a Menghini liver biopsy set (Sterylab, Rho, Italy). Parts of the biopsy were incubated in PBS or PBS with 1000 U/ml IFNα at 37°C for different periods of time. Cryosections (5 μm) were made onto glass slides. Slices were fixed in 4% paraformaldehyde for 30 min at room temperature, permeabilized in 0.1% Triton X-100 for 2 hours and blocked in 3% BSA- 0.1% Triton X-100 for 1 hour. Then they were washed with TBST prior to incubation with anti-PY-STATl (Cell Signalling) overnight at 37°C. After three washes with TBST, they were incubated with Cy3 conjugated secondary antibody (Amersham) for lh30 hour at room temperature. Nuclear staining was performed with Hoechst (Amersham) for 5 min at room temperature. After washing, a coverslip was mounted in FluorSave Reagent (Calbiochem). For quantification of nuclear phospho-STATl staining, a minimum of 100 cells was counted under fluorescent microscope for each sample.

Preparation of extracts from human liver biopsies For human biopsy cytoplasmic/nuclear extracts, samples were homogenized in 120 μl of Buffer A containing 20 mM Hepes pH 7.6, 10 mM NaCl, 3 mM MgC12, 0.1% NP-40, 10% glycerol, 0.2 mM EDTA and 1 mM DTT. After centrifugation at 2000 rpm for 5 min, supernatant (cytoplasmic extracts) was removed and the pellet was washed with 200 μl of Buffer B containing 20 mM Hepes pH 7.6, 0.2 mM EDTA, 20% glycerol and 1 mM DTT, and then centrifuged at 3000 rpm for 5 min. The pellet was then resuspended in 60 μl of Buffer C containing 20 mM Hepes pH 7.6, 400 mM NaCl, 0.2% EDTA, 20% glycerol and ImM DTT. After centrifugation, the supernatant (nuclear extracts) was taken and 10 μl aliquots were made for EMSA.

Immunoprecipitation and immunoblotting Cell, mouse liver and human liver lysates were incubated with anti-PP2A (Upstate), anti-STATl (Transduction Laboratories), anti-TC-PTP, anti-Actin (Santa Cruz) or monoclonal mono/dimethyl-arginine (Abeam) antibodies for overnight at 4°C. Then they were added to protein A-Sepharose (Sigma) for 3 hours at 4°C. After SDS-PAGE and transfer onto nitrocellulose membrane (Schleicher & Schuell), proteins were detected with anti-STATl or anti-PIASl antibodies. Blots were analyzed by densitometry using NIH image software. For loading amount control of human liver biopsy samples, nitrocellulose membrane was stained using Blot-FastStain (GenoTech) according to the manufacturer's instruction.

Electrophoretic mobility shift assays EMS As were performed using 2 μl nuclear extract aliquots for cells and 10 μl aliquots for human biopsies, and end-labeled oligonucleotide SIE-m67 corresponding to STAT response element sequences¹³.

PP2A phosphatase activity assay PP2A activity was performed with whole cell extracts or nuclear extracts using Serine/Threonine Phosphatase Assay System (Promega) according to the manufacturer's instructions, in PPTase-2A buffer containing 250 mM imidazole pH 7.2, 1 mM EGTA, 0.P/o β-mercaptoethanol and 0.5 mg/ml BSA.

Example 1 : PP2Ac overexpression in HCV transgenic mice Previously, we reported inhibition of IFNα induced signaling through the Jak- STAT pathway by HCV proteins in cell lines and in livers of transgenic mice¹³' ¹⁴. In both experimental systems, interference with IFNα signaling occurred at the level of STAT- DNA interactions, and could be detected by electrophoretic mobility shift assays (EMSA) with nuclear extracts and radiolabeled DNA oligonucleotide containing the interferon stimulated response element (ISRE) or a mutated serum inducible element (SJE) designated SIE-m67. The activation of STAT proteins through tyrosine phosphorylation at the receptor kinase complex was not inhibited. Furthermore, we could not detect a block in the nuclear import of activated STATs. We therefore concluded that the interference of HCV proteins with the Jak-STAT signaling occurs in the nucleus, either through inhibition of STAT-DNA binding, or through an increased rate of STAT dephosphorylation by one or more phosphatases. Recently, TC-PTP was identified as the elusive nuclear STATl protein tyrosine 1 phosphatase . TC-PTP might be upregulated by viral proteins and could increase the dephosphorylation rate of phospho-STATl in the nucleus. However, the amount of TC- PTP in liver cell extracts of transgenic mice was the same as in control mice (Figure 1A). A review of the literature drew our attention to protein phosphatase 2 A (PP2A). PP2A is a heterotrimeric protein phosphatase consisting of a 36 kDa catalytic C subunit (PP2Ac), a 65 kDa structural A subunit and a variable regulatory B subunit. PP2A is primarily a serine/threonine phosphatase, but under certain conditions it has an independent phosphotyrosyl phosphatase activity¹⁷. The B subunits determine the substrate specificity and the cellular localization of the enzyme¹⁸. Most PP2A holoenzymes are in the cytoplasm, but some B subunits target PP2A to the nucleus¹⁹. We speculated that HCV proteins might upregulate a nuclear PP2A holoenzyme with phosphotyrosyl phosphatase activity. When we tested liver cell extracts from hepatitis C transgenic mice (B6HCV) for the expression of the catalytic subunit of PP2A, we observed consistently about 30%) higher protein expression compared to C57BL/6 control liver extracts (Figure IB). Not only the expression level of PP2Ac was increased, but also the phosphatase activity, as determined by an in vitro phosphatase assay (Figure 1C). Since PP2Ac expression and activity are tightly regulated² , the observed differences between HCV transgenic and control mice are likely to be biologically significant. An influence of the protein phosphatase PP2A on serine or tyrosine phosphorylation of STATs would be a possible mechanism of how PP2A might interfere with Jalc-STAT signaling. It was reported that pharmacological inhibition of PP2A induced serine/threonine phosphorylation of STAT3 in human T cells and T lymphoma cell lines and resulted in a functional inhibition of STAT3²³. Conversely, in 32Dcl3 myeloid progenitor cells, inhibition of PP2A increased tyrosine phosphorylation and nuclear translocation of STAT5 and resulted in a increased STAT5 signaling²⁴, hi HCV transgenic mice, the overexpression of PP2Ac in liver cells has no effect on STATl phosphorylation. Both the tyrosine and serine phosphorylation status of STATl from liver extracts of HCV transgenic mice was the same as in control mice (Figure ID). Finally, PIASl is an obvious candidate protein for interference with STATl DNA binding²⁵. We therefore analyzed the expression level of PIASl in liver extracts of transgenic B6HCV mice and control mice, but found no difference (data not shown).

Example 2: Expression of a constitutively active PP2Ac in human hepatoma cells inhibits Jak-STAT signaling To further investigate the significance of PP2Ac overexpression in STATl signaling we stably transfected human hepatoma derived Huh7 cells with a constitutive active form of PP2Ac, HA-PP2Ac. HA-PP2Ac has been generated by the fusion of an influenza hemagglutinin derived nine-amino-acid epitope tag to the amino terminus of the full length PP2Ac¹⁵. Transfection of Huh7 cells with HA-PP2Ac resulted in stable expression of the fusion protein without changing the expression level of the endogenous PP2Ac. Although the amount of HA-PP2Ac was less than 10% of the endogenous PP2Ac (Figure 2A), the phosphatase activity was increased more than twofold (Figure 2B). IFNα induced DNA-binding of STATl was inhibited in these cells (Figure 2C). Interestingly, the inhibition could be partially reversed by addition of the PP2Ac inhibitor okadaic acid (Figure 2C). Of note, okadaic acid had no effect on STATl signaling in untransfected cells (Figure 2C). Again, we found no difference in the tyrosine or in the serine phosphorylation status of STATl after IFNα treatment in HA-PP2Ac transfected versus untransfected Huh7 cells (Figure 2D). These findings suggested an active role of PP2A in STAT dephosphorylation in the nucleus. Indeed, it has been reported that the addition of purified PP2Ac to nuclear extracts from IFNγ treated U937 cells inhibits DNA binding of the STATl dimer to the GAS oligonucleotide²⁶. We therefore tested if the addition of purified, active PP2Ac to nuclear extracts from IFNα treated Huh7 cells could inhibit STATl DNA binding in a cell-free system, but found no direct effect of PP2Ac on activated STATl in an EMSA with the m67 oligonucleotide (Figure 2E). Therefore, we conclude that dephosphorylation of STATl by PP2A is not the mechanism responsible for inhibition of Jak-STAT signaling.

Example 3: Expression of PP2Ac results in arginine hypomethylation of STATl and increased association of STATl with PIASl The association of STATl with the inhibitor PIASl is regulated by arginine methylation of STATl on arginine 31¹¹. We therefore determined the arginine methylation status of STATl in HA-PP2Ac transfected Huh7 cells by the method described by Mo wen et al. (immunoprecipitation of arginine methylated proteins followed by a Western blot with STATl antibodies). Indeed, expression of HA-PP2Ac resulted in a decrease of STATl methylation (Figure 3A). Importantly, STATl was also hypomethylated in liver extracts of HCV transgenic mice when compared to control mice (Figure 3B and 3C). Methylation of arginine was shown to inhibit the association of STATl with PIASl¹¹. Our observation of a decreased level of STATl methylation suggests that an enhanced association of PIASl with STATl might be responsible for the inhibition IFNα induced Jak-STAT signaling. Indeed, the PIASl -STATl association was increased in HA-PP2Ac transfected Huh7 cells (Figure 3D). The effect was almost as pronounced as when PRMTl was pharmacologically inhibited by 5'-methyl-thioadenosine (MTA) (Figure 3D). We used here MTA treatment as a positive control, since it has been shown that pretreatment with MTA significantly reduced the amount of STATl that is immunoreactive with DMA antibodies¹¹. Finally, enhanced binding of PIASl to STATl was also found in liver cell extracts from HCV transgenic mice when compared to controls (Figure 3E). These experiments demonstrate that PP2A is a negative regulator of arginine methylation of STATl. As a consequence, hypomethylated STATl associates with PIASl, a negative regulator of STATl induced gene transcription. This negative regulation is partially dependent on the phosphatase activity of PP2Ac, since inhibition of the catalytic activity of PP2Ac with okadaic acid increased STATl methylation (Figure 3F).

Example 4: IFNα induced signaling through STATl is inhibited in liver biopsies of patients with chronic hepatitis C To test if our results obtained in cell lines and transgenic mice are relevant to human disease, we developed a semiquantitative method to measure IFNα induced Jak- STAT signaling in liver biopsies of patients with chronic hepatitis C. Since patients could not be treated with IFNα before liver biopsies were performed, we incubated biopsy samples ex vivo with IFNα. We first tested if IFNα was able to readily diffuse in such tissue cylinders and if diffusion was dependent on the degree of liver tissue fibrosis. 5-10 mm long tissue cylinders obtained by percutaneous liver biopsy with a 1.4mm biopsy needle were incubated immediately after retrieval in phosphate buffered saline (PBS) alone or in PBS with lOOOU/ml human IFNα. Nuclear translocation of phosphorylated STATl was detected throughout the entire biopsy after 10 min, reached a maximum after 20 min, and disappeared after one hour (Figures 4A and 4B). Diffusion of IFNα into the biopsy cylinder was independent of the fibrosis stage of the liver tissue, since the same pattern of uniform STATl nuclear translocation was observed in biopsies of patients with liver tissue fibrosis ranging from minimal fibrosis to complete cirrhosis. The next 44 consecutive biopsies of patients with chronic hepatitis C were used for semiquantitative assessment of IFNα induced STATl activation using gel shift assays. Again, biopsy cylinders were incubated in vitro with or without IFNα in PBS for 20 min. Cytoplasmic and nuclear extracts were prepared as detailed under experimental procedures. The samples of 19 of the 44 patients could not be used, because the biopsies were to small and the protein concentration in the nuclear extracts was less than 0.5 mg/ml. From the remaining 25 patients, untreated and IFNα treated samples were analyzed with EMSA (Figure 4C). Signal intensity of the STATl gel shift was quantified for each sample, and divided by the protein concentration of the nuclear extracts (normalized gel shift intensity). We then calculated the fold induction of STATl gel shifts for each patient by dividing the normalized gel shift intensity values of the IFNα treated biopsy by the normalized value of the untreated biopsy (Figure 4C). Control samples were obtained from 12 consecutive patients who underwent ultrasound-guided biopsy of focal lesions in otherwise healthy livers (mainly liver metastasis of carcinomas). From the 12 samples, two could not be used because of an insufficient protein concentration in the nuclear extract. Some samples showed activated STATl already before stimulation with IFNα (for example patient number 111 in Figure 4C). It is possible that in these patients the Jalc-STAT pathway was already activated by endogenous JEN. Alternatively, other STATl activating cytokines might have been present in the liver at the time of the biopsy. Some patients with chronic hepatitis C showed no increase in STATl DNA binding after treatment (for example patient number 115 in Figure 4C), whereas others had a good response (patient number 73 in Figure 4C). The results are summarized in table 1. Despite this heterogeneity of our findings, as a group, patients with chronic hepatitis C had a poorer response to IFNα treatment than the controls (Figure 4D). Statistical analysis with the Mann- Whitney U test showed a significant difference between controls and HCV samples at p = 0.03. We conclude that HCV infection inhibits IFNα induced Jalc-STAT signaling in liver cells.

Example 5: Overexpression of PP2A and hypomethylation of STATl in chronic hepatitis C Finally, we extended the analysis of signaling through the Jak-STAT in liver biopsy samples. As in our cell line and transgenic mouse models, we found no inhibition of STATl phosphorylation in HCV infected liver cells (Figure 5A). Interestingly, the STATl expression level was higher in samples from HCV patients as compared to controls (Figure 5B and 5C). Analyzed with the Mann- Whitney U test, the difference was significant at p < 0.0001. PP2Ac was also found to be induced in liver cells of HCV patients (Figure 5D and 5E). The difference was significant at p = 0.043 (Mann- Whitney U test). We then measured the PP2A phosphatase activity and found a strong correlation with PP2Ac protein expression levels (data not shown). We conclude that HCV infection induces an upregulation of the catalytically active PP2Ac. Next, the methylation of STATl was tested in large biopsy specimens obtained from two HCV infected patients and two controls. As mentioned, STATl expression levels are increased in HCV samples. Nevertheless, less arginine methylated STATl could be immunoprecipitated from HCV samples (Figure 5F and 5G). These large amounts of hypomethylated STATl avidly bind to PIASl, as shown in a coimmunoprecipitation of the two proteins (Figure 5H). Thus, despite normal activation of STATl by the Jak kinases at the IFN receptors, IFNα induced signaling is severely impaired in HCV infected liver cells, because PIASl prevents STATl from binding to the promoters of IFN target genes. To complete our analysis, the expression of the STATl phosphatase TC-PTP was determined. No difference was detected between extracts from biopsies of HCV infected patients and controls (Figure 51).

Example 6: Treatment with a methyl group donor restores STATl methylation and IFN signaling. Huh7 cells and Huh7 cells stably transfected with HA-PP2Ac were treated with various combinations of hlFNa and AdoMet (SAMe). In the HA-PP2Ac cells IFN signaling and STATl methylation were impaired in comparison with the untransfected cells. Moreover, the HA-PP2Ac cells showed increased association between STATl and Piasl, indicating inhibition of STATl -mediated signaling. Treatment with the methyl group donor SAMe (or AdoMet) at concentrations as low as 1.7 nM essentially eliminated the effect of HA-PP2A transfection with regard to STATl methylation, STATl association with Piasl, and signaling via hlFNa. Thus, SAMe, by restoring STATl methylation, restored responsiveness to IFN in this model of hepatitis C infection. While the present invention has been described with reference to certain preferred embodiments, one of ordinary skill in the art will recognize that additions, deletions, substitutions, modifications and improvements can be made while remaining within the spirit and scope of the present invention as defined by the appended claims.

References:

1. Biron CA, Sen GC. Interferons and Other Cytokines. In: Knipe DM, Howley PM, eds. Fundamental Virology. 4th ed. Philadelphia: Lippincott Williams & Wilkins, 2001:321- 351.

2. Sen GC. Viruses and interferons. Annu Rev Microbiol 2001;55:255-81.

3. Darnell JE, Jr, Kerr J_M, Stark GR. Jalc-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science 1994;264:1415-21.

4. Shuai K, Stark GR, Kerr DVI, Darnell JE, Jr. A single phosphotyrosine residue of Stat91 required for gene activation by interferon-gamma. Science 1993;261:1744-6.

5. Uile JN. Stats- Signal Transducers and Activators of Transcription. Cell 1996;84:331-334.

6. Horvath CM. STAT proteins and transcriptional responses to extracellular signals. Trends Biochem Sci 2000;25:496-502.

7. Darnell JE, Jr. STATs and Gene Regulation. Science 1997;277: 1630-5.

8. O'Shea JJ, Gadina M, Schreiber RD. Cytokine signaling in 2002: new surprises in the Jak/Stat pathway. Cell 2002;109 Suppl:S121-31.

9. Krebs DL, Hilton DJ. SOCS proteins: negative regulators of cytokine signaling. Stem Cells 2001;19:378-87.

10. Shuai K. Modulation of STAT signaling by STAT-interacting proteins. Oncogene 2000;19:2638-44.

11. Mowen KA, Tang J, Zhu W, Schurter BT, Shuai K, Herschman HR, David M. Arginine methylation of statl modulates ifhalpha/beta-induced transcription. Cell 2001;104:731-41.

12. ten Hoeve J, de Jesus Ibarra-Sanchez M, Fu Y, Zhu W, Tremblay M, David M, Shuai K. Identification of a Nuclear Statl Protein Tyrosine Phosphatase. Mol Cell Biol 2002;22:5662-5668.

13. Heim MH, Moradpour D, Blum HE. Expression of hepatitis C virus proteins inhibits signal transduction through the Jak-STAT pathway. J Virol 1999;73:8469-75.

14. Blindenbacher A, Duong FH, Hunziker L, Stutvoet ST, Wang X, Terracciano L, Moradpour D, Blum HE, Alonzi T, Tripodi M, La Monica N, Heim MH. Expression of hepatitis c virus proteins inhibits interferon alpha signaling in the liver of transgenic mice. Gastroenterology 2003;124:1465-75.

15. Wadzinski BE, Eisfelder BJ, Peruski LF, Jr., Mumby MC, Johnson GL. NH2-terminal modification of the phosphatase 2 A catalytic subunit allows functional expression in mammalian cells. J Biol Chem 1992;267:16883-8.

16. Hytiroglou P, Thung SN, Gerber MA. Histo logical classification and quantitation of the severity of chronic hepatitis: keep it simple! Semin Liver Dis 1995;15:414-21.

17. Cayla X, Van Hoof C, Bosch M, Waelkens E, Vandekerckhove J, Peeters B, Merlevede W, Goris J. Molecular cloning, expression, and characterization of PTPA, a protein that activates the tyrosyl phosphatase activity of protein phosphatase 2 A. J Biol Chem 1994;269:15668-75.

18. Sontag E. Protein phosphatase 2A: the Trojan Horse of cellular signaling. Cell Signal 2001;13:7-16.

19. McCright B, Rivers AM, Audlin S, Virshup DM. The B56 family of protein phosphatase 2A (PP2A) regulatory subunits encodes differentiation-induced phosphoproteins that target PP2A to both nucleus and cytoplasm. J Biol Chem 1996;271:22081-9.

20. Millward TA, Zolnierowicz S, Hemmings BA. Regulation of protein kinase cascades by protein phosphatase 2A. Trends Biochem Sci 1999;24:186-91.

21. Janssens V, Goris J. Protein phosphatase 2 A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling. Biochem J 2001;353:417-39.

22. Baharians Z, Schonthal AH. Autoregulation of protein phosphatase type 2A expression. J Biol Chem 1998;273:19019-24. 23. Woetmann A, Nielsen M, Christensen ST, Brockdorff J, Kaltoft K, Engel AM, Skov S, Brender C, Geisler C, Svejgaard A, Rygaard J, Leiclc V, Odum N. Inhibition of protein phosphatase 2A induces serine/threonine phosphorylation, subcellular redistribution, and functional inhibition of STAT3. Proc Natl Acad Sci USA 1999;96:10620-5.

24. Yokoyama N, Reich NC, Miller WT. Involvement of protein phosphatase 2A in the interleulcin-3-stimulated Jak2-Stat5 signaling pathway. J Interf Cytolc Res 2001;21:369-78.

25. Liu B, Liao J, Rao X, Kushner SA, Chung CD, Chang DD, Shuai K. Inhibition of Statl - mediated gene activation by PIASl. Proc Natl Acad Sci USA 1998;95:10626-31.

26. Eilers A, GeorgeUis D, Klose B, Schindler C, Ziemiecki A, Harpur AG, Wilks AF, Decker T. Differentiation-regulated serine phosphorylation of STATl promotes GAF activation in macrophages. Mol Cell Biol 1995;15:3579-86.

27. Altschuler L, Woolc JO, Gurari D, Chebath J, Revel M. Involvement of receptor-bound protein methyltransferase PRMTl in antiviral and antiproliferative effects of type I interferons. J Interf Cytolc Res 1999;19:189-95.

28. Lee J, Stock J. Protein phosphatase 2A catalytic subunit is methyl-esterified at its carboxyl terminus by a novel methyltransferase. J Biol Chem 1993;268:19192-5.

29. De Baere I, Derua R, Janssens V, Van Hoof C, Waelkens E, Merlevede W, Goris J. Purification of porcine^" brain protein phosphatase 2A leucine carboxyl methyltransferase and cloning of the human homologue. Biochemistry 1999;38:16539-47.

30. Nishikawa M, Omay SB, Toyoda H, Tawara I, Shima H, Nagao M, Hemmings BA, Mumby MC, Deguchi K. Expression of the catalytic and regulatory subunits of protein phosphatase type 2A may be differentially modulated during retinoic acid-induced granulocytic differentiation of HL-60 cells. Cancer Res 1994;54:4879-84.

31. Altiolc S, Xu M, Spiegelman BM. PPARgamma induces cell cycle withdrawal: inhibition of E2F/DP DNA- binding activity via down-regulation of PP2A. Genes Dev 1997;11:1987-98.

32. Wilson NJ, Moss ST, Csar XF, Ward AC, Hamilton JA. Protein phosphatase 2A is expressed in response to colony-stimulating factor 1 in macrophages and is required for cell cycle progression independently of extracellular signal-regulated protein kinase activity. Biochem J 1999;339:517-24.

Table 1

Liver biopsies from patients with chronic hepatitis C and from controls

Claims

What is claimed is: 1. A compound increasing methylation of a STATl in vivo.

2. A compound according to claim 1, wherein said compound acts by increasing or decreasing an activity of a protein selected from the group consisting of: PP2A2, PRMT1, and PIASl.

3. A therapeutic composition comprising said compound of claim 2 and a pharmacologically-acceptable carrier.

4. A compound decreasing activity of a PP2A.

5. A therapeutic composition comprising said compound of claim 4 and a pharmacologically-acceptable carrier.

6. A method of selecting a drug to treat hepatitis C, comprising the steps of: a. providing a plurality of drug candidates, b. assaying the candidates to ascertain a change in a level of STATl methylation upon application of the candidates to a model system, c. selecting candidates that increase methylation of STATl , and d. selecting a candidate from among those selected in step c that is effective in treating hepatitis C.

7. The method of claim 6, wherein said model system is selected from the group consisting of a protein binding assay, a protein activity assay, a cell culture model, and an animal model.

8. The method of claim 6, wherein step d comprises testing said drug candidate in an animal model of hepatitis C infection.

9. A drug developed by the method of claim 8.

10. A method of selecting a drug to treat hepatitis C, comprising the steps of: a. providing a plurality of drug candidates, b. assaying the candidates to ascertain a change in a level of PP2A activity upon application of the candidates to a model system, c. selecting candidates that change the level of PP2A activity, and d. selecting a candidate from among those selected in step c that are effective in treating hepatitis C.

11. The method of claim 10, wherein said model system is selected from the group consisting of a protein binding assay, a protein activity assay, a cell culture model, and an animal model.

12. The method of claim 10, wherein step d comprises testing said drug candidate in an animal model of hepatitis C infection.

13. A drug selected by the method of claim 12.

14. A method of treating hepatitis C infection, comprising enhancing a level of methylation of STATl .

15. The method of claim 14, further comprising increasing or decreasing an activity of a protein selected from the group consisting of: PPA2, PRMTl, and PIASl.

16. The method of claim 15, wherein said increasing or decreasing step is carried out by administering a therapeutic composition.

17. A method of treating hepatitis C infection, comprising reducing an activity ofPP2A.

18. The method of claim 17, wherein said reducing is carried out by administering a therapeutic composition.

19. A method according to claim 14, further comprising the step of administering a drug selected by the method of claim 6.

20. A method according to claim 14, further comprising the step of administering a drug selected by the method of claim 10.

21. The use of methyl group donor for the manufacture of a pharmaceutical composition, wherein the pharmaceutical composition comprises a methyl-group donor in an amount effective to increase a level of STATl methylation in a patient.

22. The use of a methyl group donor to increase a level of STATl methylation in a patient, comprising (1) selecting a patient, and (2) treating the patient with a methyl group donor in an amount effective to increase the level of STATl methylation in the patient.

23. The method of claim 22, further comprising the step of monitoring said level of STATl methylation in said patient.

24. A pharmaceutical composition consisting essentially of interferon, ribavarin, a methyl group donor, and a pharmacologically-acceptable carrier.

25. Any of claims 21-24 wherein said methyl group donor is SAMe.

26. Any of claims 21-25 wherein betaine is used in conjunction with said methyl group donor.

27. A method of monitoring a patient, comprising the steps of (1) selecting a patient undergoing treatment for viral infection, and (2) testing one or more samples from the patient to ascertain a level of STATl methylation.

28. The method of claim 27, wherein said viral infection is a hepatitis C infection.

29. A pharmaceutical composition comprising pegylated interferon, ribavarin, SAMe, betaine, and a pharmacologically-acceptable carrier.