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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Hepatitis C virus (HCV) infection induces the endogenous interferon (IFN) system in the liver in some but not all patients with chronic hepatitis C (CHC). Patients with a pre-activated IFN system are less likely to respond to the current standard therapy with pegylated IFN-α. Mitochondrial antiviral signaling protein (MAVS) is an important adaptor molecule in a signal transduction pathway that senses viral infections and transcriptionally activates IFN-β. The HCV NS3-4A protease can cleave and thereby inactivate MAVS in vitro, and, therefore, might be crucial in determining the activation status of the IFN system in the liver of infected patients. We analyzed liver biopsies from 129 patients with CHC to investigate whether MAVS is cleaved in vivo and whether cleavage prevents the induction of the endogenous IFN system. Cleavage of MAVS was detected in 62 of the 129 samples (48%) and was more extensive in patients with a high HCV viral load. MAVS was cleaved by all HCV genotypes (GTs), but more efficiently by GTs 2 and 3 than by GTs 1 and 4. The IFN-induced Janus kinase (Jak)-signal transducer and activator of transcription protein (STAT) pathway was less frequently activated in patients with cleaved MAVS, and there was a significant inverse correlation between cleavage of MAVS and the expression level of the IFN-stimulated genes IFI44L, Viperin, IFI27, USP18, and STAT1. We conclude that the pre-activation status of the endogenous IFN system in the liver of patients with CHC is in part regulated by cleavage of MAVS. (HEPATOLOGY 2010.)

Infection with the hepatitis C virus (HCV) leads to chronic hepatitis C (CHC) in 50% to 80% of individuals. The recognition of HCV by the host triggers pathways that lead to type I interferon (IFN) (IFN-α and IFN-β) production and to the induction of an antiviral state.1, 2 To establish persistent infection, HCV has evolved numerous strategies to evade and counteract the immune response of the host.3–6 Recent studies have identified the HCV NS3-4A serine protease as a key viral protein blocking innate immune pathways. NS3-4A cleaves and thereby inactivates the caspase recruitment domain–containing essential adaptor protein mitochondrial antiviral signaling protein (MAVS)7 (also known as caspase recruitment domain adaptor inducing IFN-β,8 interferon-β promoter stimulator protein 1,9 and virus-induced signaling adaptor10) in the retinoic acid-inducible gene-I (RIG-I) viral RNA-sensing pathway.8 MAVS is located at the outer mitochondrial membrane and associates with RIG-I through its caspase recruitment domain. This interaction, on activation of a kinase complex comprising TRAF family member-associated NF-κB activator (TANK)-binding kinase 1 (TBK1) and inhibitor of nuclear factor kappaB ε, results in the phosphorylation of the cytoplasmic IFN regulatory factor 3 and IFN regulatory factor 7, which dimerize and translocate to the nucleus, where they induce the transcription of IFN-α and IFN-β.11–15 Cleavage of MAVS occurs at cysteine 508 within an almost canonical NS3-4A cleavage site and results in dislocation of the protein from the outer mitochondrial membrane.8, 16 HCV NS3-4A also targets TIR-domain-containing adapter-inducing interferon-β (TRIF), a key adaptor molecule in the Toll-like receptor 3 (TLR3) double-strand RNA-sensing pathway.16 Hence, HCV may establish persistent infection by cleaving and inactivating cellular proteins essential for the induction of the first-line immune defense.

Despite its ability to inactivate key components of the viral sensory pathways, HCV triggers an ongoing IFN response during chronic infections in chimpanzees1 and humans.2, 17, 18 Importantly, there is a large variation in the level of IFN-stimulated gene (ISG) expression among patients with CHC. Moreover, activation of the endogenous IFN system is linked to the response to the current standard treatment with pegylated IFN-α and ribavirin. Patients with high expression of ISGs in liver biopsy specimens taken before therapy are poor responders to treatment, whereas a lack of ISG pre-activation correlates with a favorable response to therapy.2, 17, 18

Interference of HCV with the innate immune response, by cleaving MAVS or TRIF, could explain the variability of ISG pre-activation in CHC patients. There is evidence from biochemical analyses and from cell culture experiments that HCV triggers IFN-β expression through the RIG-I pathway,19 and, as outlined previously, there is strong in vitro evidence that HCV interferes with the RIG-I pathway by NS3-4A–mediated cleavage of MAVS.8, 16 Cleavage of MAVS has been reported in liver biopsy specimens from four patients with CHC.20 In the current study, we (1) validated and extended the observations on MAVS cleavage in a large panel of well-characterized liver biopsy specimens from patients infected with different HCV genotypes (GTs), (2) determined whether the extent of MAVS cleavage correlates with activation of the endogenous IFN system in vivo, and (3) correlated differences in cleavage and inactivation of this crucial adaptor molecule with treatment response, HCV viremia, and GT as well as histological grading and staging. Our results support a role of MAVS cleavage in the HCV-mediated control of antiviral responses in vivo in the liver of patients with CHC.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Antibodies.

An anti-MAVS rabbit polyclonal antiserum and mouse monoclonal antibodies (mAbs) were raised against an Escherichia coli–expressed recombinant protein representing amino acids 160 through 450 of MAVS. The immunoglobulin G2b mAb designated as IID12 was selected for this study. This mAb and the polyclonal antiserum are now available from ELS AG (Lausen, Switzerland) under the designation Adri-1 and AT107, respectively. MAb AC-15 against beta-actin was from Sigma (St. Louis, MO).

Cell Lines.

Huh-7.5 cells21 and a subgenomic HCV replicon that served as controls for detection of MAVS in its full-length (FL) and cleaved forms were provided by Charles M. Rice (The Rockefeller University, New York, NY).

Liver Biopsies and Patient Data.

Liver biopsy specimens from patients with CHC (n = 150) and controls (n = 46) were obtained in the context of routine diagnostic workup. Grading and staging of CHC was performed according to the Metavir classification. A specimen was frozen for research purposes only if sufficient material was obtained for histopathological examination and the patient gave his/her written informed consent in accordance with local ethical committees. Serum HCV RNA was quantified using the COBAS AmpliPrep/COBAS Taqman HCV-Test and the Cobas Amplicor Monitor from Roche Molecular Systems. Patient characteristics are shown in Table 1.

Table 1. Patient Characteristics
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Immunoblot Analyses.

Proteins were extracted by homogenization of biopsy samples in a lysis buffer containing 50 mM Tris. Cl pH 8.0, 150 mM NaCl, 1% NP40, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate, 1 mM sodium orthovanadate, 10 mM NaF, and a cocktail of protease inhibitors (Complete Protease Inhibitor, Roche Diagnostics, Mannheim, Germany). Ten micrograms protein was loaded onto each lane and separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, followed by immunoblot as previously described,22 using horseradish peroxidase–coupled secondary antibodies and the ECL Advanced Western Blotting Detection Kit (Amersham, Dübendorf, Switzerland). Densitometric scanning was performed with an ImageScanner (Amersham), and the bands corresponding to FL MAVS (aa 1-540) and cleaved MAVS (aa 1-508) as well as beta-actin were quantified with the ImageQuant TL software (Amersham).

Immunohistological Staining.

Standard indirect immunoperoxidase procedures were used for immunohistochemistry (ABC-Elite, Vectra Laboratories, Burlingame, CA). Four-mm-thick sections were cut from paraffin blocks, rehydrated, pretreated for 20 minutes in ER2 solution, incubated with a rabbit mAb against phosphorylated STAT1 (p-STAT1) (Cell Signaling, Bioconcept, Allschwil, Switzerland), and counterstained with hematoxylin. The entire procedure was performed with an automated stainer from Vision BioSystems (Newcastle upon Tyne, UK). For quantification of the p-STAT1 staining, samples were divided into four categories according to the proportion of positive hepatocyte nuclei (−: <5%; +: 5%-33%; ++: 34%-66%; +++: >66% of hepatocytes with positive nuclear staining).

Measurement of ISG Messenger RNA Levels.

RNA extracted from human liver tissue was used for quantification of STAT1, IP10, USP18, IFI27, Viperin and IFI44L messenger RNAs (mRNAs). Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Basel, Switzerland) according to manufacturer's instructions. RNA was aliquoted and stored at −75°C. RNA was reverse transcribed by Moloney murine leukemia virus reverse transcriptase (Promega Biosciences, Wallisellen, Switzerland) in the presence of random hexamers (Promega) and deoxynucleoside triphosphates. The SYBR-PCR reactions were performed using the SYBR green PCR master mix (Applied Biosystems) and primers spanning the exon-intron junctions to avoid amplification of genomic DNA. The following primers were used: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5′-GCTCCTCCTGTTCGACAGTCA-3′ and 5′-ACCTTCCCCATGGTGT- CTGA-3′; STAT1, 5′-TCCCCAGGCCCTTGTTG-3′ and 5′-CAAGCTGCTGAAGTTGGTACCA-3′; IP10, 5′-CGATTCTGATTTGCTGCCTTAT-3′ and 5′-GCAGGTACAGCGTACGGTTCT-3′; USP18, 5′-CTCAGT- CCCGACGTGGAACT-3′ and 5′-ATCTCTCAAGCGC- CATGCA-3′; IFI27, 5′-CCTCGGGCAGCCTTGTG-3′ and 5′-AATCCGGAGAGTCCAGTTGCT-3′; Viperin, 5′-CTTTGTGCTGCCCCTTGAG-3′ and 5′-TCCATACCAGCTTCCTTAAGCAA-3′; IFI44L, 5′-GCTGCGGGCTGCAGAT-3′ and 5′-CTCTCTCAATTGCACC- AGTTTCC-3′. The difference in the cycle threshold (ΔCt) value was derived by subtracting the Ct value for GAPDH, which served as internal control, from the Ct value for transcripts of interest. All reactions were run in duplicate, using an Applied Biosystems Prism 7000 Sequence Detection System. Messenger RNA expression levels were calculated relative to GAPDH from the ΔCt values, using the formula 2−ΔCt.

Quantification of Intrahepatic HCV RNA.

One microgram total RNA isolated from biopsy specimens of 44 CHC patients was reverse transcribed according to the manufacturer's instructions using a pathogen-specific RT primer mix (PrimerDesign, Southampton, UK) designed for the in vitro quantification of all HCV genotypes. HCV RNA was quantified using a pathogen-specific primer/probe mix (PrimerDesign) and the Taqman Universal PCR Master Mix (Applied Biosystems, manufactured by Roche, NJ). Fluorescence was detected through the FAM channel of the Applied Biosystems 7000 Sequence Detection System, and copy number of HCV RNA per microgram total RNA was calculated according to the standard curve obtained using control template (PrimerDesign).

Statistical Methods.

Correlations were assessed using the Spearman coefficient. Comparisons between two groups or between multiple groups were performed with the Mann-Whitney test and one-way analysis of variance, respectively, using GraphPad Prism version 4.00 for Macintosh (GraphPad Software, San Diego, CA). A P ≤ 0.05 was considered as statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

MAVS Is Cleaved in Liver Tissue from Patients with Chronic Hepatitis C.

The clinical characteristics of all CHC patients and controls who were part of this study are summarized in Table 1. Liver biopsy samples from 129 patients with CHC and 39 controls were analyzed by western blot using mAb IID12 (Adri-1) specific for MAVS. Both forms of MAVS, FL and cleaved, were detected in liver biopsy specimens from patients with CHC, whereas only FL MAVS was detected in controls (Fig. 1A, B). For all analyses, signal intensities of FL and cleaved MAVS were normalized to β-actin. The percentage of MAVS cleavage (cleaved/total MAVS × 100) varied widely among the 129 CHC patients, ranging from 0 to 76% (mean, 18%; standard deviation 21%) (Fig. 1B). Cleavage of MAVS was detected in 62 of the 129 patients (48%), and the percentage of cleavage in these 62 patients showed a normal distribution ranging from 7% to 76% (mean, 37%; standard deviation 16%). Only FL MAVS was detected in the remaining 67 patients.

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Figure 1. MAVS is cleaved specifically in patients with chronic hepatitis C. (A) Lysates from liver biopsies of patients with CHC (lanes 4-7) as well as a control (lane 3) were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and analyzed by immunoblot using mAb IID12 (Adri 1) against MAVS or mAb AC-15 against beta-actin. Arrows indicate full-length (FL) and cleaved MAVS (CL). Naïve Huh-7.5 cells (lanes 1 and 8) and Huh-7.5 cells harboring a subgenomic HCV replicon (lanes 2 and 9) served as controls for FL and cleaved MAVS. (B) MAVS cleavage in patients with CHC and in controls. Western blot signal for FL and cleaved MAVS were quantified and normalized to beta-actin. Cleaved MAVS was calculated as percentage of total MAVS. (C) FL MAVS normalized to beta-actin in patients with CHC and in controls. The difference between the two patients groups is statistically significant (P = 0.0007, Mann-Whitney test). (D) Genotype (GT) 2 and 3 patients have significantly fewer FL MAVS compared with GT 1 and 4 patients (P = 0.009, Mann-Whitney test). N = number of patients in each group.

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Because only the FL form of MAVS is functional, and because it is unknown whether and how fast the cleaved MAVS product is degraded in cells, we also performed all our analyses using the FL MAVS/beta-actin signal intensity ratio. The amount of FL MAVS showed strong negative correlation with the percentage of cleaved MAVS (Spearman r =-0.57, P < 0.0001; data not shown). There was a wide variation in the amount of functional FL MAVS within both the CHC and control patient groups (Fig. 1C), with the median value being significantly lower in CHC compared with control samples (0.48 versus 0.77; P = 0.0007). Immunoblotting with the polyclonal MAVS antiserum AT107 yielded results similar to those obtained with mAb IID12 (Adri 1) (data not shown). We conclude that HCV-induced cleavage of MAVS reduces the amount of the functional FL MAVS in a considerable proportion of patients with CHC.

Cleavage of MAVS was independent from the Metavir activity grade or fibrosis score (data not shown). All investigated HCV GTs were able to cleave MAVS. Samples from patients infected with the easier-to-treat GTs 2 or 3 had significantly lower amounts of FL MAVS compared with the difficult-to-treat GTs 1 and 4 (P = 0.009; Fig. 1D). There was no significant difference between GT 2/3 and GT 1/4 patients with respect to viral load (VL), Metavir activity grade, or fibrosis score. Therefore, the difference most likely reflects different intrinsic properties of HCV GTs with respect to MAVS cleavage.

Correlation of Viral Load and Cleavage of MAVS.

Because in vitro studies demonstrated a very efficient cleavage of MAVS by the HCV NS3-4A protease,8, 16, 20 we assessed whether patients with a high VL show more MAVS cleavage in the liver. Indeed, there was a weak but statistically significant inverse correlation between the amount of FL MAVS and serum VL (Spearman r =-0.22, P = 0.011; Fig. 2A) and a strong positive correlation between the percentage of cleaved MAVS and serum VL (Spearman r = 0.53, P < 0.0001; Fig. 2B). The correlation remained significant when only the 62 samples with some detectable degree of MAVS cleavage were included in the analysis (Fig. 2C). The mean VL was significantly higher in the group of 62 patients with detectable MAVS cleavage than in 67 patients without any detectable cleavage product in the liver (Fig. 2D). We also measured intrahepatic HCV VL in a subset of 46 patients, and, in agreement with published data,23, 24 there was a very strong correlation between intrahepatic and serum VL (Fig. 2E). As a confirmation of the results obtained with serum VL, the intrahepatic VL correlated negatively with the amount of functional FL MAVS and positively with the percentage of cleaved MAVS (Fig. 2F). Taken together, we provide strong evidence that high viral replication is correlated with increased cleavage of MAVS and reduced amounts of the functional FL form of MAVS.

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Figure 2. Correlation of HCV viral load (VL) with cleavage of MAVS. (A) Western blot signals for FL MAVS were normalized to the beta-actin bands and then correlated with the serum HCV VL of 129 patients with CHC. There was a weak significant negative correlation (r = −0.22, P = 0.012). (B) The percentage of cleaved MAVS is positively correlated with serum VL in 129 patients with CHC (r = 0.53, P < 0.0001). (C) Correlation of serum VL with the percentage of cleaved MAVS in the 62 of the 129 patients with MAVS cleavage. The correlation remains statistically significant after exclusion of the 67 patients without any cleavage of MAVS (r = 0.35, P = 0.005). (D) Serum VL is significantly higher in the group of patients with cleaved MAVS compared with patients with only FL MAVS in the liver biopsy samples (P < 0.0001, t test). (E) Intrahepatic HCV RNA (measured in a subgroup of 46 patients) strongly correlates with serum VL (r = 0.71, P < 0.0001). (F) Intrahepatic VL positively correlates with the percentage of cleaved MAVS (r = 0.58, P < 0.0001).

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Induction of the Endogenous IFN System in Hepatocytes of a Subset of Patients with CHC.

CHC patients with a nonresponse (NR) to therapy with pegylated interferon alpha and ribavirin show an up-regulated IFN system in the liver before treatment initiation when compared with patients with a complete early virological response (cEVR).2, 17 This activation of the endogenous IFN system is specific to CHC and is not found in patients or in chimpanzees with chronic hepatitis B (Fig. 3A and Wieland and Chisari25). Expression levels of four selected ISG mRNAs (STAT1, IP10, USP18, IFI27) were high in pretreatment liver biopsy specimens of CHC patients with a primary nonresponse (PNR; less than 2 log10 drop of VL at week 12 of treatment), as compared with patients with a cEVR, or with patients with chronic hepatitis B and controls (Fig. 3A; Kruskal-Wallis test, P < 0.0001). The mechanisms responsible for a preactivated IFN system in the liver seen in a subgroup of HCV patients remain unknown, and it is not known in which cells the ISG up-regulation occurs. To elucidate whether the increase in ISG transcripts, measured using RNA extracted from a heterogeneous liver biopsy, results from an activated type I IFN-induced Jak-STAT signaling pathway specifically in hepatocytes, we assessed levels of p-STAT1 by immunohistochemistry in 80 CHC patients and eight controls (histologically confirmed healthy liver tissue). Nuclear p-STAT1 signal in hepatocytes was quantified, and each biopsy sample was assigned to one of four categories according to the number of stained nuclei (<5%, 5%-33%, 34%-66%, >66%). There was a significant correlation of nuclear p-STAT1 staining in hepatocytes and the mRNA expression of selected ISGs (Fig. 3B). We therefore propose that the elevated ISG levels observed in livers of patients with CHC originate from hepatocytes with an IFN-α/IFN-β-induced activation of the Jak-STAT signal transduction pathway.

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Figure 3. Analysis of selected ISGs in liver biopsy specimens from patients with chronic hepatitis C or chronic hepatitis B and from subjects without chronic liver disease. (A) The mean expression levels of selected ISG mRNAs (STAT1, IP10, USP18, IFI27) in pretreatment liver biopsy specimens of patients are significantly different in the four study groups: patients with CHC and complete early virological response (cEVR), patients with CHC and primary nonresponse (PNR; less than 2 log10 drop of VL at week 12), patients with chronic hepatitis B (CHB) and controls (Kruskal-Wallis test, P < 0.0001). The mean expression of all four ISGs was significantly different between cEVR and PNR patients (Dunn's Multiple Comparison test, P < 0.01) and between PNR patients and controls (Dunn's Multiple Comparison test, P < 0.001). There was no significant difference between CHB patients and controls. N = number of patients in each group. (B) Quantification of p-STAT1 nuclear staining in hepatocytes by immunohistochemical analysis of liver biopsy specimens of CHC patients (−: 5% of hepatocytes; +: 5%-33%; ++: 34%-66%; +++ = >66%) and subjects without liver disease (controls = C) and correlation to mRNA levels of four selected ISGs (STAT1, IP-10, USP18, IFI27; expression values relative to GAPDH). There were significant differences of the mean ISG expression between the five groups (shown are the P-values obtained with the Kruskal-Wallis test). In Dunn's Multiple Comparison test, significant (P < 0.01) differences were found between the +++ group (>66% nuclear p-STAT1 staining) and the control group and between the +++ and the −group (<5% nuclear p-STAT1 staining) for all four ISGs, and between the +++ group and the + group (5%-33% nuclear p-STAT1 staining) for STAT1, IP10, and USP18. N = number of patients in each group.

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MAVS Cleavage Negatively Correlates with the Activation Status of the Endogenous IFN System.

The observed interindividual differences in MAVS cleavage in patients with CHC (Fig. 1A, B) provide an attractive hypothesis to explain the differences in preactivation of the endogenous IFN system in the liver of these patients. Extensive cleavage of MAVS in some patients could prevent the transcriptional induction of IFN-β in HCV-infected hepatocytes, thereby preventing the autocrine and paracrine activation of the Jak-STAT pathway and the up-regulation of ISGs. These patients would not have preactivation, whereas patients with little MAVS cleavage would have a strong induction of the endogenous IFN system. To test this hypothesis, we performed correlation analyses of MAVS cleavage with nuclear p-STAT1 staining in hepatocytes or with induction of ISGs in the liver (Fig. 4). The extent of MAVS cleavage differed significantly between the four categories of nuclear p-STAT1 staining (one-way analysis of variance, P = 0.023, R2 = 0.153), with a significant linear trend between the groups (slope = −4.58, R2 = 0.149, P = 0.0025; Fig. 4A). The percentage of MAVS cleavage also correlated with the induction of the ISGs IFI44L, Viperin, IFI27, USP18, and STAT1 (Fig. 4B, C, D, E, and F), but not IP10 (Fig. 4G). FL MAVS showed significant correlation only with IFI27 mRNA, but not with the other five investigated ISGs (data not shown). Interestingly, we found more cleavage of MAVS in the livers of patients that later had an early virological response (EVR) to pegylated IFN-α/ribavirin treatment compared with PNR patients (Fig. 4H). This difference was independent of GT, because it persisted even after stratification of the data according to GT (Supporting Fig. 1). As outlined, pre-activation of the endogenous IFN system is a strong predictor of NR to therapy. The significant difference in the extent of MAVS cleavage between EVR and PNR patients, together with the correlation of MAVS cleavage with pre-activation, therefore supports an important role of MAVS-dependent signaling for the induction of the endogenous IFN system in patients with CHC.

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Figure 4. Activation of the endogenous IFN system in the liver and cleavage of MAVS. (A) Quantification of p-STAT1 nuclear staining in hepatocytes of CHC patients (−: <5%, +: 5%-33%, ++: 34%-66%, +++: >66%) and correlation to the percentage of cleaved MAVS. Shown are the mean values with standard error of the mean. One-way analysis of variance analysis showed a significant difference between the four categories of p-STAT1 staining (P = 0.023, R2 = 0.153) and a significant linear trend (slope = −4.58, R2 = 0.149, P = 0.0025). N = number of patients in each group. (B-G) Correlation of percentage of cleaved MAVS with the mRNA expression of six ISGs. There was a significant negative correlation with IFI44L, Viperin, IFI27, USP18, and STAT1. Sixty-two samples from patients with CHC were analyzed. (H) CHC patients with an EVR have significantly more cleavage of MAVS compared with patients with a PNR. Shown are the mean values with standard error of the mean. The P-values were obtained with the t test. N = number of patients in each group.

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Activation of the Endogenous IFN System and HCV VL.

We next analyzed whether the HCV VL correlates with the activation status of the endogenous IFN system. Because high VL positively correlates with MAVS cleavage (Fig. 2) and MAVS cleavage negatively correlates with pre-activation (Fig. 4), one could predict that patients with high VL have less pre-activation of the IFN system. However, knowing that high VL is associated with poor response to therapy,26 and poor response correlates with pre-activation,2, 17 patients with high VL should more often have a pre-activated endogenous IFN system.

Consistent with these conflicting observations, we found no significant differences in serum or intrahepatic VL and the amount of nuclear p-STAT1 staining (Fig. 5) and no correlation of VL with ISG expression (data not shown). Our data, therefore, support neither a negative nor a positive correlation of VL with pre-activation of the endogenous IFN system. Most likely, this is reflecting the fact that multiple factors affect the balance between HCV and the viral sensory pathways of the host.

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Figure 5. Preactivation of the endogenous IFN system and HCV viral load. Serum (A) or intrahepatic (B) HCV VLs did not significantly differ between patients showing <5% (−), 5%-33% (+), 34%-66% (++), or >66% nuclear (+++) p-STAT1 staining in hepatocytes (Kruskal-Wallis test, P = 0.26 for serum VL and P = 0.81 for intrahepatic VL).

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

The cleavage and inactivation of MAVS by the HCV NS3-4A protease provides a conceptual framework that could explain why many CHC patients do not activate their endogenous IFN system in the liver. Such a mechanism would predict that HCV infections with a very efficient cleavage of MAVS do not (or only weakly) induce the transcriptional activation of the IFN-β gene, thereby preventing the autocrine and paracrine positive feedback loop through the Jak-STAT pathway that typically limits the replication and spread of many viruses. The work presented in this paper supports a role of MAVS cleavage in the HCV-mediated control of antiviral responses in vivo. However, we also provide evidence that MAVS cleavage cannot be the only factor affecting the activation status of the endogenous IFN system in the liver of patients with CHC.

MAVS cleavage can be detected in almost half of the patients with CHC and is found in infections with all HCV GTs tested (Fig. 1A). Cleavage of MAVS is specific for hepatitis C, because it was never detected in patients with other chronic liver diseases, including chronic hepatitis B (Table 1, and Fig. 1A, B). Easier-to-treat GTs 2 and 3 cleave MAVS more extensively than the difficult-to-treat GTs 1 and 4 (Fig. 1D). Accordingly, MAVS cleavage was detected in a larger proportion of patients infected with GTs 2 and 3 than with GTs 1 and 4 (56.6% versus 42.6%, data not shown). Given the role of MAVS in IFN-β induction, one would predict that GT 2 and 3 infections would less often induce activation of the endogenous IFN system. Indeed, we recently reported a lower rate of ISG induction in pretreatment biopsy specimens of patients infected with GTs 2 and 3 when compared with GTs 1 and 4.2 In agreement with this, p-STAT1 nuclear staining was less extensive in GT 2 and 3 patients than in GT 1 and 4 patients (Supporting Fig. 2).

HCV GTs 2 and 3 may also be more successful in establishing a persistent infection, because they more efficiently cleave MAVS and thereby hamper innate immune responses. However, the limited data that are available are controversial. GT 3 infections are more often spontaneously cleared during the acute phase than infections with GT 1,27 but another study reported higher spontaneous resolution in genotype 1–infected patients.28 The limitations of the type I IFN induced innate immune response in clearing HCV infections are also reflected by the fact that many chronically infected patients have a strong up-regulation of hundreds of ISGs in the liver.2 There is apparently no simple correlation between the degree of ISG up-regulation and viral elimination in hepatitis C.

Our model predicts an inverse correlation between MAVS cleavage and the activation of the endogenous IFN system. Indeed, we found that the mean percentage of MAVS cleavage was significantly lower in patients showing a strong activation of the Jak-STAT pathway, as assessed by nuclear p-STAT1 staining in hepatocytes (Fig. 4A). Also, the individual expression levels of five classical ISGs showed inverse correlations with MAVS cleavage (Fig. 4B-F). IFI44L, Viperin, IFI27, and USP18 were chosen for the analysis because high expression of these genes in liver biopsy specimens of CHC patients is predictive of NR to pegylated IFN-α/ribavirin treatment2, 17, 18 and reflects an up-regulation of the endogenous IFN system. We also analyzed STAT1, because it is the central signal transducer of type I IFN signaling, and IP-10 because it has been used as a serum marker for response to pegylated IFN-α/ribavirin,29 although both genes are not among the best predictors. The absence of significant correlation of IP10 with MAVS cleavage (Fig. 4G) may be attributable to the generally weak induction of IP10, only 2.6-fold, in the human liver in response to pegylated IFN.2 The correlations with the other five ISGs were significant, albeit weak with small correlation coefficients. This could be explained by the fact that cleavage of MAVS occurs only in hepatocytes infected with HCV, whereas activation of ISGs involves all liver cells because of the paracrine effects of secreted IFNs. Clearly, analyses of MAVS cleavage and ISG induction at the single cell will be required to address this issue; however, this is still a technical challenge. Alternatively, the weak correlation between cleavage of MAVS and ISG induction could be explained by MAVS cleavage being only one of several factors that determine the activation status of the endogenous IFN system. Other factors with a possible impact on pre-activation include NS3-4A–mediated cleavage of TRIF,16 inhibition of IFN regulatory factor-3,30, 31 and cleavage of T-cell protein tyrosine phosphatase, a recently identified cellular substrate of the NS3-4A protease.32

Cleavage of MAVS was more extensive in patients who subsequently showed EVR to therapy with pegylated IFN-α and ribavirin (Fig. 4H). Given the known correlation between treatment NR and pre-activation of the endogenous IFN system,2, 17, 18 this finding supports a role of MAVS cleavage in regulating the activation status of the endogenous IFN system. However, many patients with cleaved MAVS do not respond to therapy (and vice versa), and quantification of MAVS cleavage in pretreatment biopsy specimens therefore cannot accurately predict response to treatment. Furthermore, we did not find a significant correlation of MAVS cleavage with final treatment outcomes (data not shown).

Not only HCV GT but also serum and intrahepatic VL significantly correlated with cleavage of MAVS. Patients with high VL showed more cleavage of MAVS in the liver (Fig. 2) and might be expected to have a weaker activation of the endogenous IFN system. Such a correlation would be conceptually very attractive, because a weak activation of the endogenous IFN system could allow an increased viral replication, resulting in a high VL. However, we could not confirm this notion, neither by measuring the activation of the Jak-STAT pathway by quantification of nuclear p-STAT1 staining (Fig. 5), nor by correlation analysis between VL and ISG expression levels (data not shown). There are several explanations for the lack of inverse correlation between baseline VL and pre-activation. First of all, our analysis with 129 patients might be underpowered to show a significant correlation between baseline VL and pre-activation. In our model, these two parameters are indirectly linked through MAVS cleavage, and the correlations between VL and MAVS cleavage as well as between MAVS cleavage and ISG induction are weak. Second, high VL increases the risk of NR to treatment with pegylated IFN-α and ribavirin by yet unknown mechanisms.26 Because a pre-activation of the endogenous IFN system also strongly correlates with NR,2, 17, 18 VL should positively correlate with the activation status of the endogenous IFN system. However, as shown in Fig. 5, there is neither a positive nor a negative correlation between VL and activation of the IFN system in the liver. Apparently, the effect of high VL on cleavage of MAVS is abrogated by yet unknown effects of VL on the induction of the endogenous IFN system, resulting in the lack of correlation between VL and pre-activation of the IFN system shown in Figure 5.

The number of infected hepatocytes in CHC has not been determined unequivocally, because the spread of HCV infection in the liver is difficult to assess because of inherent technical difficulties.33 Whereas some reports argue that a limited proportion of hepatocytes harbor replicating HCV,33–35 others suggest a more widespread infection at least in some patients.36–38 Strikingly, we observed up to 76% MAVS cleavage (Fig. 1B), suggesting that in some patients HCV infection is widespread, because cleavage of MAVS is expected to occur only in hepatocytes harboring HCV. This notion is supported by experiments in which Huh-7 cells harboring HCV replicons were co-cultured with Huh-7 cells expressing the green fluorescent protein; cleavage of MAVS was detected only in replicon-harboring cells (P.B. and D.M., unpublished data).

In conclusion, our data demonstrate an important role of HCV-induced cleavage of MAVS in the interaction between virus and host. MAVS cleavage can be detected in approximately half of patients with CHC and results in a reduced activation of the endogenous IFN system in the liver. Patients with high VL and GT 2 and 3 infections have MAVS cleaved more often and more extensively. However, the correlation of MAVS cleavage with pre-activation of the endogenous IFN system and with response to treatment with pegylated IFN-α and ribavirin is not strong enough to use this parameter for patient management. Our results indicate that MAVS is just one of probably many factors that control virus–host interactions in CHC. Although this is currently debated,39 there might be important effects of therapies with NS3-4A protease inhibitors on the innate immune system in the liver, and they should be studied in the future by analyzing MAVS cleavage, IFN signaling, and ISG induction in liver biopsy specimens of patients undergoing such novel treatments.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
HEP_23426_sm_SupFig1.tif126KSupporting Fig. 1. MAVS cleavage is more extensive in patients with an EVR independent of GT.Activation of the endogenous IFN system in the liver and cleavage of MAVS. CHC patients with an EVR have significantly more cleavage of MAVS compared to patients with a PNR. Shown are the mean values with SEM separately for GT 1 or 4 and for GT 2 or 3 infected patients as indicated. The p-values were obtained with the Mann Withney test. N = number of patients in each group.
HEP_23426_sm_SupFig2.tif100KSupporting Fig. 2. Nuclear pSTAT1 staining according to HCV genotype (GT). 45 liver biopsies from patients with GT 1 or 4 and 35 patients with GT 2 or 3 were stained with antibodies to phosphorylated STAT1. Biopsies were classified in one of four groups according to the number of nuclear p-STAT1 signals in nuclei of hepatocytes: < 5% (−), 5–33% (+), 34–66% (++) or > 66% nuclear (+++). The median was ++ and + for the 45 GT1/4 and the 35 GT2/3 samples, but the difference was not statistically significant using the non-parametric Mann Withney test (p = 0.18).

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