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Abstract

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

Interferon (IFN) α inhibits hepatitis C virus (HCV) replication both clinically and in vitro; however, the complete spectrum of interferon-stimulated genes (ISGs) expressed in the HCV-infected liver or the genes responsible for control of HCV replication have not been defined. To better define ISG expression in the chronically infected HCV liver, DNA microarray analysis was performed on 9 individuals with chronic hepatitis C (CHC). A total of 232 messenger RNAs were differentially regulated in CHC compared with nondiseased liver controls. A significant proportion of these were potential ISGs that were transcriptionally elevated, suggesting an ongoing response to endogenous IFN and/or double-stranded RNA. One ISG significantly elevated in all patients was viperin, an evolutionary conserved ISG that has antiviral activity against human cytomegalovirus. Stimulation of Huh-7 and HepG2 cells with IFN-α or -γ revealed viperin is predominantly a type I ISG. Furthermore, viperin expression could also be induced following transfection of Huh-7 cells with either poly(I:C) or HCV RNA. Transient expression of viperin in cells harboring the HCV genomic replicon resulted in a significant decrease in HCV replication, suggesting that viperin has anti-HCV activity. In conclusion, even in the face of a persistent HCV infection, there is an active ISG antiviral cellular response, highlighting the complexity of the host viral relationship. Furthermore, ISG viperin has anti-HCV activity in vitro; we postulate that viperin, along with other ISGs, acts to limit HCV replication. (HEPATOLOGY 2005.)

Hepatitis C virus (HCV) is the leading cause of chronic hepatitis and liver disease–related morbidity worldwide. A significant proportion of infected individuals escape the host antiviral response and develop a chronic infection, which can result in progressive liver disease such as cirrhosis and hepatocellular carcinoma.1 The current and only therapy for chronic hepatitis C (CHC) infection is interferon (IFN) α/ribavirin combination therapy that results in response rates to sustained viral clearance in at best 50% of patients. Clearly a significant number of individuals cannot be cured of HCV infection, many of whom will progress to serious liver disease.

Viral infection activates the host innate antiviral response, which results in the induction of cellular protective genes, including type I IFNs (IFN-α and IFN-β), proinflammatory cytokines, and a subset of interferon-stimulated genes (ISGs) that directly limit viral replication.2, 3 Microarray studies in vitro have revealed that hundreds if not thousands of ISGs may be expressed in response to stimulation with IFN, some of which are known to have direct antiviral and immunomodulatory action.4 Of the best-characterized antiviral ISGs, MxA, double-stranded RNA (dsRNA)–activated protein kinase R, and 2′-5′-oligoadenylate synthesase (OAS) are most renowned; however, the complete spectrum of ISGs has not been identified, thus ISGs with potential antiviral action remain unknown. The ISGs that limit HCV replication at the molecular level are ill-defined, although recent work has shown that ISG6-16 can enhance the anti-HCV activity of IFN-α,5 and ISG-56 has direct anti-HCV activity through its ability to suppress HCV IRES translation.6 Interestingly, both ISG6-16 and ISG-56 are significantly expressed in the livers from acutely and chronically experimentally HCV-infected chimpanzees, suggesting that these along with other ISGs may play a role in limiting virus replication or spread.7–9

In this study, we used microarray analysis of percutaneous liver biopsies to better define the spectrum of ISG expression in the chronically infected HCV liver and to identify potential anti-HCV effector molecules. Similar to experimentally infected chimpanzees, we demonstrate significant upregulation of ISGs, suggesting an ongoing host cellular response to viral infection. Furthermore, analysis of this data set identified the ISG viperin as significantly expressed in all cases of CHC. Viperin is an evolutionary conserved type I ISG, and recent work suggests it has antivial activity against human cytomegalovirus (HCMV) in vitro.10 However, its expression in the liver has not been characterized, nor has its anti-HCV activity been investigated. We show that viperin is expressed in the human hepatoma cell line Huh-7 in response to IFN-α stimulation and, furthermore, that it can limit the replication of a HCV replicon in vitro. We postulate that viperin in combination with other antiviral ISGs plays an important role in the limiting of HCV replication.

Materials and Methods

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

Cell Lines.

The human hepatoma cell line Huh-7 and Huh-7 cells harboring the HCV genomic replicon, NNeo/C-5B(RG),11 were kindly supplied by Stanley M. Lemon and were maintained as previously described.12 HepG2 cells were maintained in minimal essential media (Invitrogen, Carlsbad, CA) under the same conditions as for Huh-7 cells.

Tissue and RNA Isolation.

Liver biopsy samples were collected from patients with CHC and from nonviral hepatitis patients attending the Royal Adelaide Hospital liver clinic (collection of samples was approved by the hospital's ethics committee) or the outpatient clinic at the University of Texas Medical Branch (informed consent was obtained and the study was approved by the university's institutional review board; see Table 1 for clinical features of each patient). A portion of each biopsy was collected into RNAlater (Ambion, Austin, TX) for RNA extraction, and the remainder was formalin-fixed for histological examination. All patients were negative for hepatitis B surface antigen, and other causes of chronic liver disease were excluded. All patients were IFN treatment–naïve. Nondiseased liver (n = 5) was obtained from macroscopically and histologically normal areas of liver during hepatic metastasis resection.

Table 1. Clinical Features of Patients With CHC
Liver BiopsyAgeSexRaceHCV GenotypeFibrosis ScoreInflammation Score
  1. Abbreviations: F, female; AA, African American; M, male; C, Caucasian.

145FAA1A01
254MAA1B12
351MC1A22
446FC3A11
561MAA2B10
645FC1B10
746FC1A43
836MC1A32
948FC1B11

RNA Extraction and Microarray Analysis.

Total cellular RNA was isolated from liver biopsy samples and nondiseased liver using the RNAqueous RNA extraction kit (Ambion). Biotinylated, single-stranded, antisense RNA probes were prepared from 8 to 15 μg of total RNA using Affymetrix protocols (Affymetrix, Santa Clara, CA) and were hybridized to a HG-U95A Affymetrix Human GeneChip, each of which contains 12,625 probe sets. The DNA arrays were scanned using an Affymetrix confocal scanner, and the data were analyzed with the GeneChip Analysis Suite 3.3 software package using Affymetrix statistical algorithms. All microarray assays were performed at the University of Texas Medical Branch Molecular Genomics Core Facility. Gene expression profiles of HCV-infected liver biopsies were compared with the pooled nondiseased livers as baseline; those genes that showed a more than 2.5-fold increase in 5/9 patients were considered significant and were selected for further analysis. Assigning ISG status to differentially expressed genes was determined from the published literature4, 13 and Affymetrix NETAFFX Analysis Centre (http://www.affymetrix.com), GeneCards (http://bioinfo.weizmann. ac.il/cards/) and OMIM (http://www.ncbi.nlm.nih.gov).

Complementary DNA Synthesis and Polymerase Chain Reaction.

First-strand complementary DNA (cDNA) was synthesized from total RNA, and standard polymerase chain reaction (PCR) was performed using previously published protocols and reaction conditions.12 The primers used are described in Table 2. Real-time PCR analysis was utilized to quantitate the relative levels of HCV RNA in the NNeo/C-5B(RG) cells transiently transfected with pRC-CMV-vip5. Each reaction was performed in duplicate, and all samples were standardized using the control ribosomal gene RPLPO.12 Reactions were set up using 2× Sybr green master mix (Applied Biosystems, Foster City, CA), cDNA template, and 20 pmoles of each primer in a final volume of 20 μL. Reactions were performed on an ABI 7000 prism (Applied Biosystems) with the following reaction conditions: 95°C for 10 minutes, followed by 30 cycles of 95°C for 15 seconds, 60°C for 1 minute, and a disassociation protocol to obtain a melt curve for all samples running from 60°C to 90°C.

Table 2. Primers
Primer NameNucleotide Sequence
Viperin 
 Sense5′-CGTGGAAGAGGACATGACGGAAC
 Antisense5′-CCGCTCTACCAATCCAGCTTC
IFN-α 
 Sense5′-CCTCCTGGTGCTCAGCTGCAAG
 Antisense5′-GGTCTCATCCCAAGCAGCAGATG
IFN-β 
 Sense5′-CTATTGTTGAGAACCTCCTGGCTA
 Antisense5′-CCACTCTGACTATGGTCCAGGCAC
ITAC 
 Sense5′-GCTATAGCCTTGGCTGTGATAT
 Antisense5′-GCCTTGCTTGCTTCGATTTGGG
IP-10 
 Sense5′-AGTGGCATTCAAGGAGTACCT
 Antisense5′-ATCCTTGGAAGCACTGCATCG
Mig 
 Sense5′-TTCAGCAGATGTGAAGGAACTG
 Antisense5′-AGCATGATGAAATTCAACTGGT
2-5a 
 Sense5′-CCTCTCCCTGCTTCAAGGATG
 Antisense5′-CGAGTCTTTAAAAGCGATTGCCAGAT
HCV 
 Sense5′-TCTTCACGCAGAAAGCGTCTAG
 Antisense5′-GGTTCCGCAGACCACTATGG
RPLPO 
 Sense5′-AGATGCAGCAGATCCGCAT
 Antisense5′-GGATGGCCTTGCGCA

Treatment of Huh-7 Cells With IFN and dsRNA.

Huh-7 cells were treated with various concentrations of either IFN-α2b (Intron A: Schering Plough, Kenilworth, NJ) or IFN-γ (Sigma, St. Louis, MO). Time course experiments were all performed using 500 U of either IFN-α2b or IFN-γ. Following treatment, total RNA was isolated using Trizol reagent (Invitrogen). The ability of dsRNA to induce viperin messenger RNA (mRNA) expression was assessed by transfection of Huh-7 cells with either 2 μg of T7 RNA polymerase-derived HCV subgenomic replicon RNA11 or poly(I)-poly(C) RNA (Sigma) for 8, 24, and 48 hours using Transmessenger transfection reagent (Qiagen, Valencia, CA). Total RNA was isolated as described above.

Immunoblot and Immunofluorescent Detection of Viperin.

Huh-7 cells stimulated with IFN-α were lysed and protein-separated via SDS-PAGE and transferred to nitrocellulose as previously described.14 Membranes were blocked with 5% skim milk and incubated for 1 hour at room temperature with a 1/4,000 dilution of anti-rabbit viperin antibody (a gift from Dr. Peter Cresswell, Yale University, New Haven, CT), followed by a 1/5,000 dilution of an anti-rabbit-HRP antibody (Rockland, Gilbertsville, PA). Protein bound to antibody was visualized via chemiluminescence (ECL; Amersham Biosciences, Piscataway, NJ).

Cellular localization of viperin was performed via indirect immunoflourescence microscopy essentially as described previously14 with the exception that cells were incubated in a 1/250 dilution of the anti-viperin antibody and a goat anti-rabbit immunoglobulin G Alexa 488-conjugated secondary antibody (Molecular Probes, Eugene, OR). Cells were visualized using an Olympus Provis AX70 microscope.

Assessment of Viperin Antiviral Function.

Full length viperin cDNA was amplified from a human liver library (Clontech, San Jose, CA) using the primers 5′-ATATAAGCTTATCTGCCACAATGTGGGTGCTTAC-3′ and 5′-CAATTCTAGACCGCTCTACCAATCCAGCTTC -3′ and cloned into the mammalian expression vector pRC-CMV (Invitrogen) to generate pRC-CMV-Vip5b. Huh-7 cells transfected with pRC-CMV-Vip5b were assessed for viability at 24, 48, and 72 hours utilizing a trypan blue exclusion assay. NNeo/C-5B(RG) cells were seeded at 9 × 105 for 24-hour experiments, or 6 × 105 for 48- and 72-hour experiments in 10-cm2 dishes and transfected with either a plasmid expressing enhanced green fluorescent protein (pE-GFP-N1:, BD Biosciences-Clontech, Palo Alto, CA) alone or in combination with pRC-CMV-Vip5b. Cells were harvested following transfection at 24, 48, and 72 hours and resuspended in Dulbecco's modified Eagle medium supplemented with 3% fetal calf serum. Cells were sorted on a Facstar plus (Becton Dickinson, San Jose, CA) with CELLQuest 3.3 for green fluorescent protein (GFP) expression. Total RNA was extracted from the sorted cells as described above, and real-time PCR was performed to assess HCV replication levels as mentioned previously. All experiments were performed at least in triplicate.

Results

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

ISGs are Upregulated in CHC.

To gain a better understanding of the host response to HCV infection, we performed Affymetrix GeneChip analysis on liver biopsy samples from 9 patients with CHC. According to the criteria outlined in Materials and Methods, 232 mRNA transcripts were differentially regulated in CHC compared with nondiseased liver. Although many different functional groups of genes were identified (results not shown), a significant number (39) were known ISGs (Table 3), indicating an ongoing response to endogenous IFN or the dsRNA response to viral infection. This gene set is remarkably similar to that identified in the HCV experimentally infected chimpanzee7–9; however, it represents the most comprehensive list of potential ISGs differentially regulated in the human HCV-infected liver to date. The majority of ISGs are regulated by either type 1 IFN or by type 1 and type 2 IFN and include some of the classical ISGs such as OAS, MxA, STAT-1, p56, ISG-54, and ISG-15, all of which are increased in expression in chronically infected chimpanzees.8, 9 Only six genes were IFN-γ (type 2) only response genes, of which the CXCR3 chemokine family was the most prominent. The CXCR3 chemokines IP-10, Mig, and I-TAC are predominantly expressed in response to IFN-γ12, 15, 16 and are important in the chemoattraction of memory and activated T cells to the HCV-infected liver.15, 16 Consistent with the immunomodulatory function of IFN and previous studies,7, 9 members of the major histocompatibility complex family and proteosome- and antigen-processing pathways were also upregulated (Table 3). Apart from our previous studies investigating I-TAC,12 we noted no significant association of gene expression with a particular patient group of histological grade of liver disease. The similarity of gene expression seen in previous studies and in our results suggests that similar host IFN responses occur in chronically HCV-infected chimpanzees and in humans and thus validates our data set. Nevertheless, standard reverse-transcriptase PCR (RT-PCR) was used to confirm microarray data for the ISGs I-TAC, IP-10, Mig, 2′-5′-OAS (71-kd species), and viperin. In all cases, RT-PCR revealed very little expression of the genes in nondiseased liver, apart from IP-10, while there were significant increases in mRNA expression seen in the HCV-infected liver (Fig. 1A).

Table 3. IFN-Stimulated Gene Expression in CHC
Gene NameFold ChangeIFN Type I/IIFunction
  • Abbreviation: MHC, major histocompatibility complex.

  • *

    Genes shown to be previously upregulated in microarray analysis performed on liver taken from HCV-infected chimpanzees.7–9

IP-10*21.1-512I/IIChemokine
I-TAC*27.9-147I/IIChemokine
Mig*12.1-39.3IIChemokine
71 kd 2-5a oligoadenylate synthetase*4.0-13.9INucleic acid metabolism/immune response
59 kd 2-5a oligoadenylate synthetase*4.9-19.7INucleic acid metabolism/immune response
2-5 oligo A synthetase E gene*3.5-6.5INucleic acid metabolism/immune response
2-5A synthetase (1.6 kb mRNA)*5.7-36.8INucleic acid metabolism/immune response
PSMB9 proteosome beta subunit (LMP2)*2.8-9.8IIProteolysis and peptidolysis/immune response
Diubiquitin*3.2-26.0IIProtein modification/proteolysis and peptidolysis
IFN-inducible 56-kd protein*3.2-13.9IImmune response
ISG-54K*3.7-8.0I/IIImmune response
IFIT4*2.6-26.0IImmune response
IFN-inducible peptide 6-16*3.7-13.0IImmune response
Thyroid receptor interactor (TRIP14)*3.5-13.0IImmune response
GBP-2*2.6-6.5I/IIImmune response
MHC complex class 1, G (HLA6)*2.6-4.3I/IIImmune response
MHC complex class 1, F (CDA12)*2.8-6.1I/IIImmune response
MHC class 1 fragment gi5617258.6-42.2I/IIImmune response
Monocyte protein 513.9-52.0IIImmune response/response to virus
Histone H1(0)5.7-24.3I/IINucleosome assembly/chromosome organization and biogenesis
Histone 2A–like protein (gi2062703)*2.6-4.6INucleosome assembly/chromosome organization and biogenesis
Eif-2 gamma subunit13.0-48.5IProtein biosynthesis
Ubiquitin-conjugating enzyme*15.0-32.0IProtein modification
Desmosome-associated protein pinin2.8-6.1IPhosphoprotein
Glutamate dehydrogenase*3.7-8.6IMetabolism
P5317.1-39.4IIInhibitor of growth/invasion
XAF1*6.5-27.9I/IIInhibitor of apoptosis
MxA*3.2-18.3IInduction of apoptosis/immune response/signal transduction/pathogenesis
Stat 1 (90/84 kDa)*3.0-512I/IIImmune transcription factor
Stat23.0-8.6IIImmune transcription factor
IRF-24.6-18.4I/IIImmune transcription factor
Cyclin G1*5.3-12.1ICell cycle
Ear-1r26.0-59.7IThyroid/steroid hormone receptor superfamily
O-linked GlcNAc transferase12.1-22.6IProtein glycosylation
T-cluster binding protein3.5-6.1IRNA-binding protein
Hepatitis C-associated protein p44*4.9-14.9IUnknown
IFN-induced 17 kd/15 kd protein*4.9-22.6I/IIUnknown
Staf50*5.7-14.9IUnknown
Viperin*18.4-128I/IIUnknown (potential antiviral)
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Figure 1. RT-PCR reveals ISG mRNA is increased in CHC. (A) ISG mRNA for viperin, I-TAC, Mig, IP-10, and OAS is substantially upregulated in HCV liver biopsies when compared with pooled nondiseased liver. Lane 1, pooled nondiseased liver; lanes 2-8, HCV core liver biopsies. (B) Viperin mRNA expression is not significantly increased in core liver biopsies taken from nonviral hepatitis patients. Lane 1, pooled nondiseased liver; lanes 2-5, nonviral hepatitis core liver biopsies.

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Viperin mRNA Is Upregulated in the HCV-Infected Liver.

Viperin is a recently recognized type I ISG,4, 10 and its mRNA was significantly expressed in all HCV-infected livers examined via microarray analysis (Table 3) and RT-PCR analysis (Fig. 1A). In contrast, we noted no increase in viperin mRNA levels in four liver biopsy samples representing nonviral hepatitis (nonalcoholic steatohepatitis and autoimmune hepatitis) (Fig. 1B), suggesting that its expression is specific for the virus-infected liver. Viperin has antiviral activity against HCMV when constitutively expressed in human fibroblasts10; however, its expression and the stimuli responsible in liver-derived cells has not been investigated. We therefore sought to determine the kinetics of viperin expression in Huh-7 cells and to investigate its anti-HCV activity.

Viperin Expression Is Induced in Huh-7 Cells by Stimulation With IFN-α/γ.

Previous reports have shown viperin expression is stimulated preferentially by type I IFNs in a variety of cell types, including primary macrophages, monocytes, and primary foreskin fibroblasts, but not in HeLa cells.10 To determine if hepatocyte-derived cell lines can express viperin mRNA, and to gain further insight into molecular mechanisms regulating its expression, Huh-7 and HepG2 cells were stimulated with varying concentrations of IFN-α or IFN-γ, total RNA extracted and cell lysates were harvested for examination of viperin mRNA and protein, respectively. No expression of viperin was detectable at the mRNA or protein level in unstimulated Huh-7 cells (Fig. 2A-C) or HepG2 cells (results not shown). However, stimulation with either IFN-α or IFN-γ resulted in an increase in viperin mRNA expression, with expression greatest for IFN-α stimulation, consistent with reports in other cell lines.10 Time course experiments for both IFN-α and IFN-γ revealed viperin mRNA expression as early as 4 hours after stimulation (Fig. 2B) that persisted to 72 hours. However, protein production was transient, with viperin first detected at 8 hours after IFN-α stimulation (Fig. 2C) with maximal expression at 24 hours. We also investigated the cellular distribution of viperin in Huh-7 cells stimulated with IFN-α using a rabbit polyclonal antibody directed against viperin.10 Consistent with previous reports in human fibroblasts, viperin was localized in a diffuse cytoplasmic pattern consistent with endoplasmic reticulum distribution (Fig. 2D). These results suggest that hepatocytes, at least in culture, have the ability to express viperin in response to stimulation with IFN-α.

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Figure 2. IFN induction of viperin in Huh-7 cells. (A) RT-PCR analysis of viperin mRNA induction with increasing concentrations of IFN-α and IFN-γ. (B) RT-PCR time course analysis of viperin induction following stimulation with either 500 U/mL of IFN-α or IFN-γ. (C) Western blot time course of viperin expression in Huh-7 cells stimulated with 500 U/mL of IFN-α. (D) Viperin expression was visualized in Huh-7 cells via immunofluorescence microscopy without IFN-α treatment (A) or with 500 U/mL IFN-α treatment for 24 hours (B). IFN, interferon.

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Viperin Expression in Response to HCV and dsRNA.

To test the possibility that HCV RNA and/or protein expression may stimulate viperin expression, we compared viperin mRNA levels in Huh-7 cells harboring either the subgenomic or genomic HCV replicons11 with parent Huh-7 cells. No stimulation of viperin mRNA was noted with either cell line (results not shown). This is not surprising considering these highly selected Huh-7 cells are impaired in their innate response to viral infection.17 In contrast, transfection of parent Huh-7 cells with either HCV RNA (subgenomic replicon) or the dsRNA analogue poly(I:C) to mimic intracellular dsRNA generated during viral replication resulted in transient induction of viperin mRNA (Fig. 3). Viperin mRNA expression was significantly induced as early as 24 hours after transfection in the case of HCV RNA and poly(I:C) and was sustained for 48 hours for poly(I:C) (Fig. 3). IFN-α mRNA was only weakly induced with poly(I:C) stimulation and not with HCV RNA, whereas IFN-β mRNA was significantly upregulated at all time points for poly(I:C) stimulation and only at 8 hours following HCV RNA stimulation. The fact that viperin mRNA was strongly induced following transfection with HCV RNA (24 h) in the absence of detectable IFN-α or IFN-β production suggests as previously described that viperin can be directly induced through dsRNA activation of IRF-3, independent of IFN-α/β.18 However, it is also possible that transient expression of IFN-β at 8 hours is sufficient to induce viperin mRNA expression at 24 hours.

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Figure 3. Viperin mRNA expression is upregulated in response to dsRNA. Huh-7 cells were transfected with 2 μg of either RNA derived from the HCV subgenomic replicon or the dsRNA analogue, poly(I:C) for 8, 24, and 48 hours. RT-PCR analysis of both viperin and IFN-α and β mRNA demonstrates viperin induction by HCV RNA and poly(I:C). HCV, hepatitis C virus; IFN, interferon.

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Viperin Is Able to Limit HCV Replication in a Replicon Model.

The strong induction of viperin expression by IFN-α and its documented antiviral activity against HCMV10 prompted us to examine its anti-HCV activity. cDNA encoding the entire open reading frame of viperin was cloned from a human cDNA liver library, and its expression was confirmed following transient expression in Huh-7 cells. A similar pattern of staining to that of IFN-α stimulated Huh-7 cells (Fig. 4A) was noted. Attempts to produce a Huh-7 cell line constitutively expressing viperin were unsuccessful, suggesting that long-term overexpression of viperin was toxic. In contrast, short-term expression of viperin resulted in no adverse effects on Huh-7 cells (Fig. 4B). We therefore employed a transient expression approach; however, the low transfection efficiency (<10%) of the HCV replicon cell line NNeoC-5B(RG) precluded a direct analysis of the effects of viperin on HCV replication. This was overcome by cotransfection experiments in which NNeoC-5B(RG) cells were transfected with pRC-CMV-Vip5b and a plasmid encoding for E-GFP. Twenty-four, 48, and 72 hours after transfection, cells were FACS sorted on the basis of GFP production (Fig. 5B, fraction M2). Viperin mRNA and protein expression were confirmed in the sorted fraction via RT-PCR and immunoblot analysis (Fig. 5C-D) with no expression noted in unsorted cells. Viperin expression was barely detectable at 24 hours but peaked at 48 and 72 hours after trasfection (Fig. 5D). Total RNA was extracted from the GFP-sorted fraction, and HCV RNA levels were analyzed using real-time PCR and compared with cells transfected with E-GFP alone (to control for the effect of exogenous protein expression on HCV replication) at similar time points. Cells expressing viperin were able to significantly reduce HCV RNA levels by approximately 50% at the 48- and 72-hour time points (P = .007 and P < .001, respectively), with no effect observed for the 24-hour time point (Fig. 5E). These data suggest viperin expression can in part directly suppress HCV replication.

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Figure 4. Transient viperin expression is not toxic to Huh-7 cells. (A) Immunofluorescence analysis of viperin protein expression in Huh-7 cells (a) and Huh-7 cells 24 hours after transfection with pRC-CMV-vip5b (b). (B) Transient transfection of the viperin-encoding plasmid did not affect cellular viability at 24, 48, and 72 hours after transfection.

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Figure 5. Transient expression of viperin can limit HCV replication. HCV replicon cells were either (A) untransfected or (B) transfected with the plasmids pE-GFP and pRC-CMV-vip5b and FACs sorted to isolate GFP-transfected cells. Cells in the M2 fraction were harvested and used to assess HCV RNA replication. (C,D) Viperin mRNA expression was confirmed in the M2 fraction via (C) RT-PCR and (D) immunoblot analysis. Note that there is no viperin mRNA expression in the unsorted fraction (GFP−). Viperin expression was barely detectable via immunoblotting at 24 hours and maximal at 48 and 72 hours after trasfection. (E) The effect of viperin expression on HCV RNA replication was determined via real-time PCR and was compared with replicon cells containing GFP alone to account for exogenous protein expression on HCV replication. Note the decrease in HCV RNA levels 48 and 72 hours after transfection with viperin. GFP, green fluorescent protein; HCV, hepatitis C virus.

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Discussion

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

The only treatment for CHC is IFN-α2b/ribavirin combination therapy; however, not all individuals respond, highlighting the complexity of host–viral interactions. However, upward of 50% do respond, suggesting that in part IFN can induce an antiviral state. This is further validated in vitro, in which treatment of cells harboring HCV replication with IFN-α results in a dose-dependent decrease in HCV RNA.19, 20 Furthermore, long-term treatment of HCV replicon cells with IFN-α can “cure” these cells completely of HCV replication.21 However, despite this, the complete spectrum of ISGs in the HCV-infected liver has not been identified, nor have the ISGs responsible for the control of HCV replication been defined. We therefore sought to identify and characterize ISGs expressed in liver biopsy samples obtained from patients with CHC using oligonucleotide microarray analysis.

The majority of ISGs identified in our study have also been identified in the experimental chimpanzee model of acute and chronic HCV infection7–9 and in several small studies on human CHC.22–27 Taken together, these studies suggest that the HCV chronically infected liver is capable of mounting an antiviral response. It is therefore intriguing to postulate why HCV is not cleared from these individuals given a number of classic antiviral genes such as OAS and MxA and genes with anti-HCV activity such as ISG6-165 and ISG-566 are increased in expression. It is possible that in those individuals with CHC, the cellular response to IFN is inefficient compared with those individuals that clear the virus. Although this is difficult to test in humans, the ISG response in experimentally infected chimpanzees is more robust in acute compared with chronic infection.7, 9 Weak induction of the ISG antiviral state coupled with an ineffective cellular immune response could therefore promote chronicity. An alternative scenario is the possibility that HCV can block the host antiviral response to IFN. Several HCV-encoded proteins (E2, NS3, and NS5A) have been shown to block ISG function in vitro. E2 and NS5A bind and inhibit protein kinase R function in vitro,24, 28, 29 while the NS3/4A protein complex has recently been shown to block phosphorylation of IRF-3,30 a key player in the induction of IFN-β and IRF-3 target genes. However, it is most likely that all these processes work simultaneously to promote chronicity. As proposed by Bigger and colleagues,9 it is possible to envisage that newly infected hepatocytes, through viral activation of IRF-3, results in IRF-3–dependent gene expression and the production of IFN-α and IFN-β, before the expression of viral proteins that inhibit ISG induction and/or function. Neighboring hepatocytes may then become resistant to infection as a result of the paracrine action of IFN-induced ISG expression. However, the ineffective T cell response results in emergence of escape mutants and a dynamic equilibrium is reached between newly infected hepatocytes, IFN secretion, modulation of ISG expression, and function by HCV proteins and regenerating hepatocytes that are susceptible to infection culminating in chronic infection. In contrast, the ISG response in acute infection limits HCV replication to a level that allows resolution of infection by a robust T cell response.

Chemokines play a pivotal role in the recruitment of activated/memory T cells to the HCV-infected liver; however, although they are important for viral clearance, they are also associated with both lobular and portal inflammation and disease progression.12, 31 Although several chemokines have been implicated in CHC, the results of this study suggest that the CXCR3 chemokine family members IP-10, I-TAC, and Mig play a dominant role in recruitment of T cells to the HCV-infected liver. IP-10, I-TAC, and Mig were among the most significantly upregulated (12- to 512-fold) in all of the liver biopsies examined, which together with previous studies12, 31 confirms their importance in CHC. Furthermore, they are predominantly stimulated by IFN-γ, indicating that in addition to a type I IFN response, there is also an active type II IFN response in the chronic HCV-infected liver.

One of the earliest responses of cells to viral infection is activation of IRF-3, which cooperates with other transcriptional activators to induce production of IFN-α and IFN-β and downstream ISG expression in an effort to limit viral replication and spread.32 However, work by Grandvaux et al.18 has recently demonstrated that activated IRF-3 is able to directly upregulate a small subset of ISGs before the induction of IFN. Among these are many known antiviral proteins, including ISG15, ISG56, ISG54, and OAS, all of which are increased in expression in our microarray analysis of CHC, suggesting that in addition to an active type I and II IFN response, there is also an active response to dsRNA. Our studies identified viperin, also an IRF-3 responsive gene,18 as significantly increased (18.4- to 128-fold) in expression in CHC. Considering viperin has antiviral activity against HCMV,10 this prompted us to examine if the same was true for HCV. Viperin is a relatively newly described ISG and is highly conserved among different animal species, indicating the potential importance of this protein.10, 33–35 Enforced transient expression of viperin in Huh-7 cells harboring the complete HCV genome resulted in an approximate 50% reduction in HCV RNA levels as early as 48 hours after transfection, suggesting that as with HCMV, viperin has anti-HCV activity. Furthermore, we demonstrated that viperin mRNA and protein expression can be induced in Huh-7 cells predominantly by stimulation with IFN-α as well as in response to dsRNA. The exact nature of how viperin exerts its anti-HCV effect is unknown, and further studies are required to determine the molecular mechanisms associated with its antiviral effect on HCV. However, the presence of a leucine zipper domain commonly involved in protein–protein interactions, and its ER localization,10 suggests that it may interact with HCV proteins, potentially disrupting polyprotein cleavage/maturation and replication complex formation.

In conclusion, our understanding of the dynamics between the host response to HCV infection at the level of ISG expression and the virus itself is not well understood. These results add to our knowledge of the response of the liver to infection with HCV and demonstrate that a type I/II IFN and dsRNA response is active in the chronically infected HCV liver. Furthermore, we have identified the ISG viperin as significantly increased in expression in CHC and that it has anti-HCV activity. It is likely that a cascade of ISGs act synergistically to reduce HCV replication, and we propose that viperin is one of these. The challenge will now be to identify other ISGs induced during HCV replication and to determine which ones play a role in limiting HCV replication. Furthermore, it will also be important to determine the spectrum and magnitude of ISG expression in individuals that spontaneously clear infection and those that respond to therapy compared with those that do not. This will hopefully lead to a better understanding of the function of IFN and ISG action that will provide insight into mechanisms of HCV persistence and hopefully identify novel therapeutic approaches for CHC.

Acknowledgements

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

The authors thank Stanley M. Lemon for the HCV replicon cell lines, Peter Cresswell for the viperin antibody, and Ming Qiao for critical reading of the manuscript.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  • 1
    Theodore D, Fried MW. Natural history and disease manifestations of hepatitis C infection. Curr Top Microbiol Immunol 2000; 242: 43-54.
  • 2
    Katze MG, He Y, Gale M Jr. Viruses and interferon: a fight for supremacy. Nat Rev Immunol 2002; 2: 675-687.
  • 3
    Sen GC. Viruses and interferons. Annu Rev Microbiol 2001; 55: 255-281.
  • 4
    de Veer MJ, Holko M, Frevel M, Walker E, Der S, Paranjape JM, et al. Functional classification of interferon-stimulated genes identified using microarrays. J Leukoc Biol 2001; 69: 912-920.
  • 5
    Zhu H, Zhao H, Collins CD, Eckenrode SE, Run Q, McIndoe RA, et al. Gene expression associated with interferon alfa antiviral activity in an HCV replicon cell line. HEPATOLOGY 2003; 37: 1180-1188.
  • 6
    Wang C, Pflugheber J, Sumpter R Jr, Sodora DL, Hui D, Sen GC, et al. Alpha interferon induces distinct translational control programs to suppress hepatitis C virus RNA replication. J Virol 2003; 77: 3898-3912.
  • 7
    Bigger CB, Brasky KM, Lanford RE. DNA microarray analysis of chimpanzee liver during acute resolving hepatitis C virus infection. J Virol 2001; 75: 7059-7066.
  • 8
    Su AI, Pezacki JP, Wodicka L, Brideau AD, Supekova L, Thimme R, et al. Genomic analysis of the host response to hepatitis C virus infection. Proc Natl Acad Sci U S A 2002; 99: 15669-15674.
  • 9
    Bigger CB, Guerra B, Brasky KM, Hubbard G, Beard MR, Luxon BA, et al. Intrahepatic gene expression during chronic hepatitis C virus infection in chimpanzees. J Virol 2004; 78: 13779-13792.
  • 10
    Chin KC, Cresswell P. Viperin (cig5), an IFN-inducible antiviral protein directly induced by human cytomegalovirus. Proc Natl Acad Sci U S A 2001; 98: 15125-15130.
  • 11
    Ikeda M, Yi M, Li K, Lemon SM. Selectable subgenomic and genome-length dicistronic RNAs derived from an infectious molecular clone of the HCV-N strain of hepatitis C virus replicate efficiently in cultured Huh7 cells. J Virol 2002; 76: 2997-3006.
  • 12
    Helbig KJ, Ruszkiewicz A, Semendric L, Harley HA, McColl SR, Beard MR. Expression of the CXCR3 ligand I-TAC by hepatocytes in chronic hepatitis C and its correlation with hepatic inflammation. HEPATOLOGY 2004; 39: 1220-1229.
  • 13
    Der SD, Zhou A, Williams BR, Silverman RH. Identification of genes differentially regulated by interferon alpha, beta, or gamma using oligonucleotide arrays. Proc Natl Acad Sci U S A 1998; 95: 15623-15628.
  • 14
    Li K, Prow T, Lemon SM, Beard MR. Cellular response to conditional expression of hepatitis C virus core protein in Huh7 cultured human hepatoma cells. HEPATOLOGY 2002; 35: 1237-1246.
  • 15
    Bonecchi R, Bianchi G, Bordignon PP, D'Ambrosio D, Lang R, Borsatti A, et al. Differential expression of chemokine receptors and chemotactic responsiveness of type 1 T helper cells (Th1s) and Th2s. J Exp Med 1998; 187: 129-134.
  • 16
    Sallusto F, Lenig D, Mackay CR, Lanzavecchia A. Flexible programs of chemokine receptor expression on human polarized T helper 1 and 2 lymphocytes. J Exp Med 1998; 187: 875-883.
  • 17
    Li K, Chen Z, Kato N, Gale M Jr, Lemon SM. Distinct poly(I-C) and virus-activated signaling pathways leading to interferon-beta production in hepatocytes. J Biol Chem 2005; 280: 16739-16747.
  • 18
    Grandvaux N, Servant MJ, tenOever B, Sen GC, Balachandran S, Barber GN, et al. Transcriptional profiling of interferon regulatory factor 3 target genes: direct involvement in the regulation of interferon-stimulated genes. J Virol 2002; 76: 5532-5539.
  • 19
    Lanford RE, Guerra B, Lee H, Averett DR, Pfeiffer B, Chavez D, et al. Antiviral effect and virus-host interactions in response to alpha interferon, gamma interferon, poly(i)-poly(c), tumor necrosis factor alpha, and ribavirin in hepatitis C virus subgenomic replicons. J Virol 2003; 77: 1092-1104.
  • 20
    Guo JT, Bichko VV, Seeger C. Effect of alpha interferon on the hepatitis C virus replicon. J Virol 2001; 75: 8516-8523.
  • 21
    Frese M, Schwarzle V, Barth K, Krieger N, Lohmann V, Mihm S, et al. Interferon-gamma inhibits replication of subgenomic and genomic hepatitis C virus RNAs. HEPATOLOGY 2002; 35: 694-703.
  • 22
    MacQuillan GC, Mamotte C, Reed WD, Jeffrey GP, Allan JE. Upregulation of endogenous intrahepatic interferon stimulated genes during chronic hepatitis C virus infection. J Med Virol 2003; 70: 219-227.
  • 23
    Patzwahl R, Meier V, Ramadori G, Mihm S. Enhanced expression of interferon-regulated genes in the liver of patients with chronic hepatitis C virus infection: detection by suppression-subtractive hybridization. J Virol 2001; 75: 1332-1338.
  • 24
    Smith MW, Yue ZN, Korth MJ, Do HA, Boix L, Fausto N, et al. Hepatitis C virus and liver disease: global transcriptional profiling and identification of potential markers. HEPATOLOGY 2003; 38: 1458-1467.
  • 25
    Ji X, Cheung R, Cooper S, Li Q, Greenberg HB, He XS. Interferon alfa regulated gene expression in patients initiating interferon treatment for chronic hepatitis C. HEPATOLOGY 2003; 37: 610-621.
  • 26
    Fernandez M, Quiroga JA, Martin J, Herrero M, Pardo M, Horisberger MA, et al. In vivo and in vitro induction of MxA protein in peripheral blood mononuclear cells from patients chronically infected with hepatitis C virus. J Infect Dis 1999; 180: 262-267.
  • 27
    Yu SH, Nagayama K, Enomoto N, Izumi N, Marumo F, Sato C. Intrahepatic mRNA expression of interferon-inducible antiviral genes in liver diseases: dsRNA-dependent protein kinase overexpression and RNase L inhibitor suppression in chronic hepatitis C. HEPATOLOGY 2000; 32: 1089-1095.
  • 28
    Gale MJ Jr, Korth MJ, Tang NM, Tan SL, Hopkins DA, Dever TE, et al. Evidence that hepatitis C virus resistance to interferon is mediated through repression of the PKR protein kinase by the nonstructural 5A protein. Virology 1997; 230: 217-227.
  • 29
    Taylor DR, Shi ST, Romano PR, Barber GN, Lai MM. Inhibition of the interferon-inducible protein kinase PKR by HCV E2 protein. Science 1999; 285: 107-110.
  • 30
    Foy E, Li K, Wang C, Sumpter R Jr, Ikeda M, Lemon SM, et al. Regulation of interferon regulatory factor-3 by the hepatitis C virus serine protease. Science 2003; 300: 1145-1148.
  • 31
    Harvey CE, Post JJ, Palladinetti P, Freeman AJ, Ffrench RA, Kumar RK, et al. Expression of the chemokine IP-10 (CXCL10) by hepatocytes in chronic hepatitis C virus infection correlates with histological severity and lobular inflammation. J Leukoc Biol 2003; 74: 360-369.
  • 32
    Fredericksen BL, Smith M, Katze MG, Shi PY, Gale M Jr. The host response to West Nile Virus infection limits viral spread through the activation of the interferon regulatory factor 3 pathway. J Virol 2004; 78: 7737-7747.
  • 33
    Boudinot P, Massin P, Blanco M, Riffault S, Benmansour A. vig-1, a new fish gene induced by the rhabdovirus glycoprotein, has a virus-induced homologue in humans and shares conserved motifs with the MoaA family. J Virol 1999; 73: 1846-1852.
  • 34
    Boudinot P, Riffault S, Salhi S, Carrat C, Sedlik C, Mahmoudi N, et al. Vesicular stomatitis virus and pseudorabies virus induce a vig1/cig5 homologue in mouse dendritic cells via different pathways. J Gen Virol 2000; 81: 2675-2682.
  • 35
    Sun BJ, Nie P. Molecular cloning of the viperin gene and its promoter region from the mandarin fish Siniperca chuatsi. Vet Immunol Immunopathol 2004; 101: 161-170.