Hepatitis C virus NS5A-regulated gene expression and signaling revealed via microarray and comparative promoter analyses

Authors


Abstract

Most individuals exposed to hepatitis C virus (HCV) become chronically infected and are predisposed to liver disease. The mechanisms underlying viral persistence and disease progression are unknown. A role for the HCV NS5A protein in viral replication and interferon resistance has been demonstrated. To identify mechanisms affected by NS5A, we analyzed the gene expression of Huh7 cells expressing NS5A and control cells using oligonucleotide microarrays. A set of 103 genes (43 up-regulated, 60 down-regulated) whose expression was modified by at least twofold was selected. These included genes involved in cell adhesion and motility, calcium homeostasis, lipid transport and metabolism, and genes regulating immune responses. The finding of modulated expression of genes related to the TGF-β superfamily and liver fibrosis was observed. Interestingly, both the tumor necrosis factor and lymphotoxin beta receptors were down-regulated by NS5A. Similar data were obtained following expression of four NS5A mutants obtained from patients who were not responsive or were sensitive to interferon therapy. Through computational analysis, we determined that 39 of the 43 genes up-regulated by NS5A contained one or more nuclear factor κB (NF-κB) binding sites within their promoter region. Using the Gibbs sampling method, we also detected enrichment of NF-κB consensus binding sites in the upstream regions of the 43 coexpressed genes. Activation of NF-κB by NS5A was subsequently demonstrated in luciferase reporter assays. Adenovirus-mediated expression of IκBα reverted NS5A mediated up-regulation of gene expression. In conclusion, this study suggests a role of NS5A and NF-κB in HCV pathogenesis and related liver disease. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html). (HEPATOLOGY 2004;40:708–718.)

Hepatitis C virus (HCV) is a highly prevalent pathogen associated with a high subsequent risk of progression to liver cirrhosis and hepatocellular carcinoma (HCC).1 The HCV genome is a positive-stranded RNA of 9.6 kb that encodes for a polyprotein precursor, which is co- and posttranslationally processed to yield 10 structural and nonstructural proteins (C, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B).2

Several studies have suggested a role of NS5A in resistance to interferon (IFN)-α treatment.3–7 We and others reported an altered cellular response to IFN-α and a concomitant up-regulation of interleukin (IL)-8 by NS5A.8, 9 It has also been reported that NS5A participates in HCV viral replication10–14 and is a potent transcriptional activator.15 Therefore, identification of cellular genes modified by NS5A in hepatocytic cells may provide insight into the role of NS5A in HCV replication and pathogenesis. We used microarrays to compare the gene expression profile between control Huh7 cells and Huh7 cells expressing a NS5A mutant isolated from a patient infected with HCV-1b and resistant to IFN-α therapy. We subsequently compared the effects of four NS5A mutants isolated from patients who were responsive or nonresponsive to IFN-α therapy upon transient expression in Huh7 cells.

The computational identification of transcription factor binding sites through the analysis of DNA sequence data has recently emerged as a major technology for the elucidation of transcription regulatory networks. We used computer-based approaches such as the Gibbs sampling method to determine common transcription factor motifs in the promoters of the genes up-regulated by NS5A.

Abbreviations

HCV, hepatitis C virus; NF-κB, nuclear factor κB; HCC, hepatocellular carcinoma; IFN, interferon; IL, interleukin; GFP, green fluorescence protein; SDS-PAGE, SDS–polyacrylamide gel electrophoresis; LTBR, lymphotoxin beta receptor; IκBα, inhibitor κBα; cDNA, complementary DNA; RT-PCR, reverse-transcriptase polymerase chain reaction; PCR, polymerase chain reaction; mRNA, messenger RNA; LTBP1, latent TGF-β binding protein 1; IL-1RA, interleukin 1 receptor antagonist; TNFR, tumor necrosis factor receptor; BMP2, bone morphogenic protein 2; PLTP, phospholipid transfer protein.

Patients and Methods

Cell Culture and Transient NS5A Expression.

NS5A sequences were isolated from 4 patients infected with HCV 1b, treated with standard IFN-α therapy (3 to 6 MU, 3 times per week for 6 months). Three NS5A sequences from 1 responder (R1) and 2 nonresponder (NR1 and NR2) patients to IFN-α treatment were previously described.7 The fourth NS5A sequence, isolated from a responder (R2) patient, was amplified, sequenced, and cloned in the pCDNA3.1 vector (Invitrogen, Carlsbad, CA) using the same methodology.7 We established Huh7 clones expressing the R1, NR1, and NR2 NS5A mutants.7 Herein, we used the control clones T1 and T2 and two clones, F1 and F11, expressing the NS5A mutant NR1.7 For transient NS5A expression, Huh7 cells were transfected via addition of 30 υL of lipofectamine (GIBCO BRL, Gaithersburg, MD) and 15 υg of the plasmid containing NR1, NR2, R1, and R2 NS5A sequences or the empty vector in OPTIMEM I medium (GIBCO BRL). In additional experiments, Huh7 cells were cotransfected with 4 υg of the pEGFP vector encoding the green fluorescence protein (GFP) (Clontech, La Pont de Claix, France) as a monitor for transfection efficiency. The efficiency of transfection was approximately 50%–60%.

SDS–Polyacrylamide Gel Electrophoresis and Western Blot Analysis.

Total proteins (50 μg) were diluted in Laemmli sample buffer, resolved via 10% SDS–polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto nitrocellulose membranes (Schleicher and Schuell, Dasel, Germany). The membranes were incubated with monoclonal antibodies against NS5A (Biogenesis Ltd., Poole, United Kingdom) (1:1,000 dilution) and actin (ICN, Costa Mesa, CA) (1:20,000 dilution) or with polyclonal antibodies against lymphotoxin beta receptor (LTBR) (R&D systems, Minneapolis, MN) (1:5,000 dilution) and IκBα (Santa Cruz Biotechnology, Santa Cruz, CA) (1:1,000 dilution). Immunodetection was realized using enhanced chemiluminescence reagents (Amersham, Buckinghamshire, United Kingdom).

Adenovirus Infection.

F1 and F11 cells were plated at a density of 4 × 105 per well in 6-well tissue culture plates. Twenty-four hours later, the cells were infected with the adenoviruses AdIκBα and AdGFP, with a cytomegalovirus (CMV) promoter driving expression of full-length IκBα complementary DNA (cDNA) and GFP, respectively. Fluorescence microscopy confirmed that adenovirus-transferred genes were expressed in nearly 100% of cells 18 hours after infection.

Luciferase Reporter Assays.

Luciferase assays were performed using an E-selectin luciferase plasmid and reagents from Promega (Madison, WI) in the single and dual format by monitoring the transfection efficiency with pRL-TK.

Preparation of Complementary RNA and Gene Chip Hybridization.

Total RNA was isolated and used to generate complementary RNA probes. Preparation of complementary RNA, hybridization, and scanning of the HuGeneFL Arrays (Affymetrix, Inc., Santa Clara, CA) were performed as described previously.8 Data analysis was performed using GeneChip 4.0 software (Affymetrix, Inc.). Genes differently modified by NS5A were compared in F1 and F11 clones with each control cell line (T1 and T2) and then selected (fold change ≥2, P < .05). For each gene, the fold change between NR1 and control clones was calculated for 2 experiments using Affymetrix software (Affymetrix, Inc.).

Reverse Transcription Followed by Polymerase Chain Reaction.

Single-stranded cDNA was synthesized from total RNA using Superscript II reverse transcriptase (GIBCO-BRL) and random hexamers (GIBCO-BRL) as primers. For regular reverse-transcriptase polymerase chain reaction (RT-PCR), the reaction mixture was composed of 1 μL of cDNA template obtained from 1 μg extracted RNA; 25 pmol primers; 25 nmol each deoxyribonucleoside triphosphate; 2.5 U Taq DNA polymerase (GIBCO-BRL); and 5 μL 10× polymerase chain reaction (PCR) buffer in a final volume of 50 μL. The nucleotide sequences of primers and corresponding annealing temperatures used are as indicated (Table 1). Primers for real-time PCR were designed using the PRIMER3 program (http://www.genome.wi.mit.edu) (see Table 1). As an internal reference, actin was amplified in parallel with the genes of interest. All reactions were carried in 20 μL volumes containing 10 μL of iQ SYBR Green Supermix (Bio-Rad, Hercules, CA), 1 μL (25 pmol) of each primer and 5 μL of cDNA product. Reactions were performed in the Bio-Rad iCycler iQ Optical detection system. Meltcurves were also performed to determine unique amplification products.

Table 1. Primer Sequences
GeneSequencesProduct Size (bp)Annealing Temperature (°C)
Actin5′-ATCATGTTTGAGACCTTCAA-3′64460
 5′-TTGCGCTCAGGAGGAGCAAT-3′  
Actin5′-TGGACTTCGAGCAAGAGATGG-3′13860
 5′-GGAAGGAAGGCTGGAAGAGTG-3′  
Neurotensin5′-GCATGCTACTCCTGGCTTTC-3′13160
 5′-CCAGAGGGACATGTGCTT-3′  
Osteopontin5′-CCAAGTAAGTCCAACGAAAGCC-3′27358
 5′-GTACTGGATGTCAGGTCTGCG-3′  
Aldore reductase5′-AGCAGCCAGGATATGACCAC-3′8960
 5′-GGGTAATCCTTGTGGGAGGT-3′  
LTBP15′-CTTCCGCTGCCTCTGTTATC-3′12560
 5′-ACCCTTCCACGTTTTCACAG-3′  
GRO-α5′-GAAAGCTTGCCTCAATCCTG-3′10760
 5′-CSCCSGTGSGCTTCCTCCTC-3′  
IL-1RA5′-GCAGTCACCTAATCACTCTCC-3′22860
 5′-CCAAGAACAGAGCATGAGGC-3′  
LTBR5′-CCATATGCGTCGGAGAACCAG-3′34959
 5′-CAGGCGGGCAGTCAGAAAGTAG-3′  
TNFR1A5′-GACCAGGCCGTGATCTCTATGC-3′56160
 5′-ATGCCGGTACTGGTTCTTCCTG-3′  
Carbonic anhydrase IV5′-AGTCCCCCATCAACATCGTCAC-3′34560
 5′-GCACCGCAATTTCGTCTTCAG-3′  
Trefoil factor 15′-GGTCGCCTTTGGAGCAGAGA-3′20559
 5′-CGTCGAAACAGCAGCCCTTATT-3′  
CRBP15′-TGTCCCGGCAGTCTCCAG-3′38059
 5′-CCACGCCCCTCCTTCTCA-3′  
PLTP5′-GGCACCACCATCTCTGTCACTG-3′34459
 5′-AATCCCGCATGGTTCGTCAC-3′  
TGF-β15′-GCGCCCATCTAGGTTATTTC-3′28459
 5′-CCAGGCGTCAGCACCAGTAG-3′  
Collagen IV-α55′-TCCAGCTCTGGAAGGACCTA-3′8960
 5′-CCAGGATTTCCCTTCTCTCC-3′  
PLAB5′-GACCTATGATGACTTGTTAGCC-3′14360
 5′-CAGGAATCGGGTGTCTCAGG-3′  
BMP25′-AACACTGTGCGCAGCTTCC-3′10160
 5′-CTCCGGGTTGTTTTCCCAC-3′  
Decorin5′-TCAATGGACTGAACCAGATGA-3′14560
 5′-CCTTGAGGAATGCTGGTGAT-3′  

Quantitative Enzyme-Linked Immunosorbent Assay for TGF-β1.

The amount of TGF-β1 present in the culture media of the T1, T2, F1, and F11 clones were measured with Quantikine TGF-β1 enzyme-linked immunosorbent assay (R&D Systems). The supernatants were activated with 1 N HCl for 10 minutes and neutralized with 1.2 N NaOH and 0.5 M hydroxyethylpiperazine-N-2 ethanesulfonic acid. The captured TGF-β1 proteins were quantitated at 450 nm in a spectrophotometer. Two independent experiments as well as two independent measures were performed for each sample.

Computational Promoter Analysis.

Human genomic sequences were obtained from http://genome.ucsc.edu. Corresponding human messenger RNA (mRNA) sequences encoding for the N terminus of each protein were obtained from the National Center for Biotechnology Information GenBank RefSeq database (available at http://www.ncbi.nlm.nih.gov/Genbank/index.html) and subsequently aligned with the genomic sequence to identify the transcription start site. Promoter regions were then defined from this transcription start site—1 kbp upstream and 1 kbp downstream—and analyzed using MATCH, a weight matrix–based tool for searching putative transcription factor biding sites in DNA sequences.16, 17 MATCH assigns matrix and core similarity values for each transcription factor based on the position weight matrices. The cutoff values for core and matrix similarities used were 0.85. Promoter region comparison was performed by entering the defined promoter sequences into the Gibbs Motif Sampler (http://bayesweb.wadsworth.org/gibbs/gibbs.html). The Gibbs Motif Sampler is a software for locating multiple transcription factor binding sites for multiple transcription factors simultaneously in unaligned DNA sequences.18 Motif searches were specified between 14–25 bp in length. Sequences with score values of 1.000 for core and 0.500 for matrix were selected. The internal Wilcoxon signed rank test was also used in the assessment of statistical significance of our data sets. Pictograms of the Gibbs output were generated using software available at http://genes.mit.edu/pictogram.html to show the relative frequency of each base within the returned motifs.

Results and Discussion

Gene Expression Profile of Huh7 Cells Expressing NS5A.

Hepatocytic Huh7 cells expressing NS5A mutants isolated from patients infected with HCV-1b were generated as described previously.7 We wished to compare the gene expression profile of clones F1 and F11 expressing the NS5A NR1 mutant isolated from a nonresponder to IFN-α therapy with control clones T1 and T2 expressing the empty vector using oligonucleotide arrays complementary to 6,800 genes. Expression levels of NS5A in these cells are shown in Fig. 1. Two independent experiments were performed. RNA transcript levels from both NS5A expressing cell lines F1 and F11 were compared with control cell lines T1 and T2. We selected genes that differed in their expression levels by twofold or greater in each comparison pair and in both experiments, with a P value of less than .05. The 103 genes that were identified are presented in Tables 2 and 3 for up-regulated and down-regulated genes, respectively. Changes included differential expression of genes related to cell adhesion and motility. Expression of trefoil factors 1 and 2, galectin 2, LI-cadherin, and hepatocyte growth factor–like protein transcripts were decreased with a concomitant up-regulation of genes of the extracellular matrix such as matrilin 3, collagen type IV, Cyr61, fibrillin-like protein, and latent TGF-β binding protein 1 (LTBP1). Several up-regulated genes were related to immune functions and inflammation, such as osteopontin, interleukin 1 receptor antagonist (IL-1RA), and the chemokines MIP-3α/CCL20, IL-8, and GRO-α. In contrast, we observed a down-regulation by NS5A of STAT6 and two members of the tumor necrosis factor receptor (TNFR) family: TNFR1A and LTBR. Finally, the regulation of a large group of genes encoding for lipid-binding proteins or enzymes involved in lipid homeostasis was observed.

Figure 1.

NS5A expression in Huh7 cell clones. Total proteins from Huh7 clones stably expressing NS5A (F1 and F11) and from control clones (T1 and T2), were separated via SDS-PAGE, transferred to nitrocellulose membranes, and probed with anti-NS5A and anti–β-actin antibodies.

Table 2. Genes Up-Regulated in NS5A-Expressing Huh7 Clones F1 and F11
DescriptionGene Bank Accession No.Fold Change
  1. NOTE. mRNA from control clones T1 and T2 and from NS5A-expressing clones F1 and F11 were hybridized onto Affymetrix oligonucleotide arrays as indicated in Patients and Methods. For each gene, the fold change was calculated with Affymetrix software, and up-regulated genes were selected. The ∼ indicates a fold change calculation for which the smaller value is replaced by an estimate of the minimum value for detectable transcripts. Genes up-regulated following transient expression of NSSA are shown in bold.

Hepatocarcinoma-associated  
 NeurotensinU9161812
 Epidermal growth factor receptorX005882.5
 Aldose reductaseX154142.3
Signal transduction/cell proliferation  
 Adrenal-specific protein pG2X17544∼8
 GravinU81607∼4.5
 CDK inhibitor p57/Kip2U22398∼3.5
 SH3-domain binding protein 2AB000462∼3
 Bone morphogenetic protein 2M224893
 Pim-2 oncogeneU77735∼2.5
 Phosphodiesterase 3A, cGMP-inhibitedU36798∼2.5
 Ephrin receptor EphA5L36644∼2.5
 Thromboxane A2 receptorD38081∼2.3
 Chimerin 1X514082.3
 Mitogen-activated protein kinase 1/extracellular signal–regulated kinase 2Z116952.2
 Homeobox protein Cdx1U51095∼2.2
 Guanine nucleotide binding proteinJ03260∼2.2
 Activin A receptor, type IIM93415∼2.2
 Dual-specificity protein kinase 4Y09305∼2.2
 RhoBM121742.1
 TGF-Beta superfamily protein PLAB/GDF-15/MIC-1AB0005842
 Dual-specificity phosphatase 1X682772
Immune response  
 Interleukin 8M281305
 OsteopontinU20758∼5
 GRO-αX544892.2
 Interleukin 1 receptor antagonistX53296∼2.2
 MIP-3α/CCL20U641972
 ISG20U889642
 TNF alpha-induced protein 2M923572
 Major histocompatibility complex class II (HLA-DQA1)M34996∼2
Extracellular matrix  
 Matrilin 3AJ0010473.5
 Cysteine-rich, angiogenic inducer 61U620153
 Fibrillin-like proteinU03877∼3
 Latent TGF-β binding protein 1M34057∼2.5
 Type IV collagen α5 chainU04520∼2.3
Cell adhesion and motility  
 α-TubulinX017034.5
 Integrin-associated protein/CD47Z255212.5
 VimentinZ195542.2
Miscellaneous  
 Reticulon 1L10333∼3
 Mg81L08240∼3
 Chloride channel 5X81836∼2.5
 Cytochrome P450, subfamily IX026122.3
 Glycogen phosphorylase BU470252.1
 Nova1U048402
Table 3. Genes Down-Regulated in NS5A-Expressing Huh7 Clones F1 and F11
DescriptionGene Bank Accession No.Fold Change
  1. NOTE. mRNA from control clones T1 and T2 and from NS5A expressing clones F1 and F11 were hybridized onto Affymetrix oligonucleotide arrays as indicated in Patients and Methods. For each gene, the fold change was calculated with Affymetrix software, and down-regulated genes were selected. The ∼ indicates fold change calculation for which the smaller value is replaced by an estimate of the minimum value for detectable transcripts. Genes down-regulated following transient expression of NSSA are shown in bold.

Cell adhesion and motility  
 Trefoil factor 2 (SP)X51698 −8.6
 Galectin 2M87860∼ −5.7
 Hepatocyte growth factor-like protein/MSPU37055∼ −3.3
 Trefoil factor 1 (pS2)X52003−2.9
 Urokinase-type plasminogen activator receptorU09937∼ −2.6
 LI-cadherinX83228−2.5
 Carcinoma-associated antigen GA733-2M93036−2.3
 CD97U76764 −2.3
 Lymphocyte antigen 6 complex, locus D (E48)X82693 −2.2
Signal transduction/cell cycle  
 Transcription elongation factor S-IID50495∼ −3.9
 Frizzled-5U43318−3
 Growth factor receptor-bound protein 14L76687−2.8
 ELK3/net transcription factorZ36715 −2.4
 Phosphoinositide-3-kinaseY10055∼ −2.4
 Cyclin FZ36714∼ −2.3
 Mitogen-activated protein kinase kinase 3D87116−2.2
 Calpain 1X04366−2.2
 DecorinM14219−2
Immune response  
 Lymphotoxin β receptorL04270 −7.6
 Tumor necrosis factor receptor 1AM58286−7.3
 Complement component 4-binding protein, βL11244−2.6
 Complement component 4-binding protein, αM62486−2.6
 Omega Light Chain, prot 14.1M34515∼ −2.4
 STAT6U16031∼ −2
Calcium-binding proteins  
 Inositol 1,4,5-triphosphate receptor, type 3U01062 −2.8
 Reticulocalbin 1D42073−2.4
 GrancalcinM81637−2.3
 SERCA1U96781∼ −2.2
 S100 calcium binding protein PX65614−2
Metabolism  
 Cellular retinol binding protein 2U13831−9
 Carbonic anhydrase IVL10955 −6
 Glutathione S-transferase 2X65727−4.3
 Nuclear receptor SHPL76571−3.1
 Phospholipid transfer proteinL26232 −2.9
 Hepatocyte nuclear factor 1β (TCF2)X58840 −2.5
 Fatty acid binding protein 1M10050−2.5
 Carboxylesterase 1L07765−2.5
 Lipoprotein-associated phospholipase A2U24577−2.4
 HistidaseD16626∼ −2.4
 Nuclear orphan liver X receptor αU22662∼ −2.3
 Glutamine transaminase KX82224∼ −2.3
 Solute carrier family 7, member 6D87432−2.3
 Galactokinase 1L76927 −2.2
 Arylacetamide deacetylaseL32179−2.2
 Hydroxyacyl-CoA dehydrogenase, type II (ERAB)U73514−2
 Apolipoprotein E receptor 2Z75190−2
 Cellular retinol binding protein 1M11433−2
Miscellaneous  
 Neuronal pentraxin IIU29195−4.3
 Giant larvae (Drosophila) homologX87342 −4
 GTP-binding protein hsr1X66436∼ −3.4
 Predicted osteoblast protein (GS3786)D87120−2.9
 H2A histone family, member AM60752−2.4
 Carboxypeptidase N2J05158∼ −2.3
 Guanylate cyclase 2CM73489−2.3
 Cold shock domain protein AM24069−2.2
 Fibrinogen-like protein 1D14446−2.2
 Hypertension-associated SAD16350∼ −2.1
 KininogenM11437−2.1
 LIM homeobox protein 1U14755∼ −2
 KIAA0140D50930−2

Some genes reported to be differentially expressed in HCV-infected liver tissue changed their expression accordingly in our analysis. This group included the TGF-β superfamily member PLAB, HLA-DQA1, and Rho-B, which were up-regulated during acute HCV infection in chimpanzees,19 and the CDK inhibitor Kip2, IL-8, and epidermal growth factor receptor, which were up-regulated in HCV cirrhotic liver tissue.20

To validate the microarray data, real-time RT-PCR analysis was undertaken. Transcript levels were determined for neurotensin, LTBP1, bone morphogenic protein 2 (BMP2), collagen IV-α5, IL-1RA, carbonic anhydrase IV, and decorin in clones T1, T2, F1, and F11 (Fig. 2). Real-time RT-PCR and microarray data were highly concordant for all of these randomly selected genes (see Fig. 2). Transcript levels were also determined using semiquantitative RT-PCR for osteopontin, aldose reductase, GRO-α, trefoil factor 1, cellular retinol binding protein 1, and phospholipid transfer protein (PLTP) in clones T1, T2, F1, and F11. In agreement with the microarray data, transcripts for osteopontin, aldose reductase, and GRO-α were increased in F1 and F11 compared with control clones, while transcripts for trefoil factor 1, cellular retinol binding protein 1, and PLTP were decreased in NS5A-expressing clones (data not shown). Modification by NS5A of the mRNA levels for MIP-3α/CCL20, IL-8, and STAT6 was previously confirmed via PCR.8

Figure 2.

Effects of NS5A on Huh7 gene expression. Total RNA from T1, T2, F1, and F11 Huh7 clones was reverse-transcribed, and real-time PCR amplification was performed for neurotensin, LTBP1, BMP2, collagen IV-α5, IL-1RA, carbonic anhydrase IV, and decorin as described in Patients and Methods. Transcript levels in F1 and F11 were normalized using actin mRNA levels and compared with T1 and T2 controls. Data are shown as mean ± SEM of the independent microarray experiments. Abbreviations: LTBP1, latent TGF-β binding protein 1; BMP2, bone morphogenic protein 2; IL-1RA, interleukin 1 receptor antagonist; PCR, polymerase chain reaction.

Genes Dysregulated in HCC.

The strongest effect of NS5A observed was a 12-fold up-regulation of neurotensin transcript. Neurotensin is transiently expressed in the fetal liver, suppressed in the adult liver, and re-expressed in certain liver cancers.21 We identified additional genes up-regulated by NS5A that were previously reported to be overexpressed in HCC, such as epidermal growth factor receptor, aldose reductase, and osteopontin.22–24 Decorin expression suppressed in HCC25 was down-regulated by NS5A. Modulation of neurotensin, aldose reductase, osteopontin, and decorin by NS5A was validated via RT-PCR analysis (see Fig. 2 and data not shown).

Intrahepatic metastasis of HCC is observed frequently. Loss of cell–cell contact caused by increased cell motility is a critical step in cancer metastasis. Many genes involved in cell adhesion, motility, and cytoskeleton remodeling were regulated by NS5A. This group included vimentin, α-tubulin, scaffold protein gravin, LI-cadherin, trefoil factors 1 and 2, and RhoB. It has been reported that RhoB is involved in intrahepatic metastasis of human HCC.26

Finally, we observed a 2.2-fold increase of mitogen-activated protein kinase 1/extracellular signal–regulated kinase 2 mRNA expression. Mitogen-activated protein kinase/extracellular signal–regulated kinase activation may play an important role in the progression of HCC.27

TGF-β Superfamily Members and Modulators of Fibrosis.

TGF-β is a potent modulator of liver fibrogenesis. In our microarray analysis, TGF-β1 mRNA levels remained unchanged between control and NS5A-expressing cells. Similar results were obtained via RT-PCR assay (Fig. 3A).

Figure 3.

Effect of NS5A on the TGF-β1 gene expression and protein secretion in Huh7 cells. (A) Total RNA from T1, T2, F1, and F11 Huh7 clones was reverse-transcribed and PCR amplification was performed for TGF-β1. (B) Secretion of TGF-β1 protein in NS5A-expressing cells as determined with enzyme-linked immunosorbent assay. T1, T2, F1, and F11 Huh7 clones were cultured for 2 days in OPTIMEM I medium (GIBCO BRL), and TGF-β1–secreted protein was measured in the supernatants every 24 hours.

Remarkably, expression of LTBP1, a component of the latent TGF-β1 complex, was increased by 2.5-fold. LTBP1 is involved in the assembly, secretion, and targeting of TGF-β1 to sites at which it is stored and/or activated.28 Positive staining for TGF-β and LTBP was observed in liver biopsies from HCV patients in areas with inflammation and fibrosis.29 To determine if higher levels of LTBP1 mRNA in NS5A-expressing cells correlated with a modification of TGF-β1 protein secretion, TGF-β1 expression was determined with enzyme-linked immunosorbent assay in controls, T1, T2, and NS5A-expressing clones F1 and F11. The pattern of TGF-β1 protein expression was not significantly different in the culture supernatants between T1, T2, F1, and F11 clones, reaching 323 ± 39 (mean ± SEM) pg/mL, 327 ± 20 (mean ± SEM) pg/mL, 249 ± 28 (mean ± SEM) pg/mL, and 392 ± 121 (mean ± SEM) pg/mL in T1, T2, F1, and F11 respectively, at 48 hours (Fig. 3B).

Concomitant to the increase of LTBP1, we observed a twofold decrease of the gene coding for decorin. Decorin neutralizes TGF-β bioactivity and reduces fibrosis. In addition, transcripts for 2 members of the TGF-β superfamily, BMP2 and PLAB, were up-regulated by three- and twofold, respectively, in the NS5A-expressing clones compared with controls. PLAB is up-regulated during acute liver injury and liver regeneration.30 The HBV pX protein enhances TGF-β, BMP2, and activin transcriptional activity, contributing to HBV-associated liver fibrosis.31 It must be noted that the receptor of activin A, another member of the TGF-β superfamily increased in fibrotic liver, was also up-regulated by 2.2-fold in NS5A-expressing cells.

We also observed an increase in type IV collagen. High type IV collagen expression was reported in large fibrotic areas and serum type IV collagen levels correlated with fibrotic grades of HCV liver patients.32, 33 Finally, we and others reported previously that NS5A induces IL-8 expression.8, 9 Elevated IL-8 expression correlated with liver fibrosis in HCV patients.34

Regulation by NS5A of LTBP1, decorin, PLAB, BMP2, collagen IV-α5, and IL-8 was confirmed via RT-PCR analysis (see Fig. 2 and data not shown).9

Thus, the increased levels of LTBP1, PLAB, BMP2, activin A receptor, type IV collagen, and IL-8 on the one hand and the decrease of decorin on the other may contribute to hepatic fibrosis associated with HCV. In addition, NS5A may play critical roles in controlling TGF-β activity, by modulating LTBP1 and decorin gene expression.

Inflammation and Immune Response.

Expression of genes implicated in inflammatory process and immune response was modulated by NS5A. TNF-related cytokines are recognized as crucial effectors of the innate and adaptive immune defenses, and targeting members of the TNF/lymphotoxin superfamily and their receptors is a strategy used by many viruses. Of great interest, therefore, was the observation that expression of two members of the TNF receptor family, TNFR1A and LTBR, were strongly down-regulated (7.6- and 7.3-fold, respectively) in the NS5A-expressing clones compared with controls. This effect was subsequently confirmed via PCR analysis (Fig. 4A). We wished to determine the protein expression levels for both genes. Although the sensitivity of Western blotting was not sufficient to detect the TNFR1A protein in Huh7 cells, the analysis of LTBR protein expression indicated that LTBR protein levels were strongly reduced in both NS5A-expressing clones F1 and F11 compared with control cells T1 and T2 (Fig. 4B).

Figure 4.

Effect of NS5A on gene and protein expression of the TNFR family members LTBR and TNFR1A. (A) Total RNA from T1, T2, F1, and F11 Huh7 clones was reverse-transcribed and PCR amplification was performed for LTBR and TNFR1A. Lane 0 represents the negative control, which was the product of RT-PCR where RNA was omitted. (B) Total proteins extracted from T1, T2, F1, and F11 Huh7 clones were separated via SDS-PAGE, transferred to nitrocellulose membranes, and probed with anti-LTBR and anti–β-actin antibodies. Abbreviations: LTBR, lymphotoxin beta receptor; TNFR1A, tumor necrosis factor receptor 1A.

Our data, which are in agreement with those of others,35, 36 support a role for NS5A as a negative regulator in TNF-α signaling cascade. Interestingly, an up-regulation of TNFR1A was observed in chimpanzee liver during acute HCV infection, correlating with viral clearance.19 Therefore, we could speculate that the down-regulation of TNFR1A and LTBR by NS5A may confer to HCV the ability to escape immune suppression leading to persistent infection.

IL-1β is one of the most important cytokines with respect to the liver. IL-1RA counterbalances the biological activity of IL-1β by a competitive binding to IL-1 receptors. Increased levels of IL-1β and IL-1RA have been found in the sera of patients with chronic liver diseases. We observed a 2.2-fold increase in IL-1RA mRNA levels, which was confirmed via real-time RT-PCR (see Fig. 2). Other genes that are involved in inflammation and are modulated by NS5A included osteopontin, hepatocyte growth factor–like protein (which was implicated in hepatic responses to inflammatory stimuli), calpain 1, and lipoprotein-associated phospholipase A2. We also observed a down-regulation of STAT6 by NS5A. STAT6 is a mediator of IL-12 and IL-4 functions, and activation of STAT6 may regulate liver inflammation injury.37 The NS5A–up-regulated genes also included the proinflammatory chemokines IL-8 and GRO-α.

Appropriate induction of a T helper 1 response is required for effective eradication of intracellular pathogens. IL-12, which was first described for its ability to stimulate IFN-γ production and enhance CD8 cytotoxicity, is a dominant factor in T helper 1 phenotype development. We observed an up-regulation of CD47 and a down-regulation of mitogen-activated protein kinase kinase 3, 2 regulators of IL-12 production. CD47 ligation down-regulates IL-12 production,38 and mitogen-activated protein kinase activating kinase 3 deficiency results in a severe reduction of IL-12 production.39 In addition, T cells from mice deficient in mitogen-activated protein kinase activating kinase 3 have a defect in IFN-γ production. It is worth noting that the potential of dendritic cells generated from HCV-infected patients to stimulate allogeneic CD4 T cells has been reported to be lower than that of dendritic cells from healthy donors, potentially because of low IL-12 expression.40

Complement also plays a protective role in the acute host response against several viruses. The host uses a family of proteins called regulators of complement activation to prevent damage to host cells by activated complement; these include the complement component 4–binding protein, an important regulator of the classical pathway of the complement system. Component 4–binding protein acts as a cofactor in degradation of complement protein C4b. Component 4–binding protein is mainly expressed in the liver and is composed of the alpha and beta subunits. Its expression increases during inflammation, infection, or tissue damage. We observed a 2.6-fold decrease of component 4–binding protein α and β in NS5A-expressing cells compared with controls.

Calcium Homeostasis and Cellular Signaling.

It was reported recently that NS5A alters calcium homeostasis.41 Expression of several members of the family of proteins known as EF-hand calcium-binding proteins such as calpain 1, grancalcin, S100P, and the endoplasmic resident protein reticulocalbin 1 were reduced in NS5A-expressing cells. Other down-regulated genes included inositol 1,4,5-triphosphate receptor type 3—an intracellular channel that mediates the release of calcium from intracellular stores—and sarco/endoplasmic reticulum calcium adenosine triphosphatase 1, the latter of which pumps calcium from the cytosol to the endoplasmic reticulum and therefore plays a major role in the control of calcium signaling.

Lipid Metabolism.

Remarkably, microarray analysis revealed a dysregulation upon NS5A expression of numerous genes involved in lipid transport and metabolism. Several lines of evidence suggest that the infection cycle of HCV might involve lipoproteins and apolipoproteins. HCV associates with low-density lipoprotein, including those containing apolipoproteins E and B. Recent studies have indicated that lipoprotein-associated HCV particles may infect cells via the low-density lipoprotein receptor.42 Furthermore, binding of lipo-viro particles to the cell relies on apolipoproteins and their receptors, and this binding is efficiently inhibited by normal low-density lipoprotein or very low-density lipoprotein or antibodies to apolipoprotein B and apolipoprotein E.43 Remarkably, carriage of an apolipoprotein E-ϵ4 allele may be protective against liver damage caused by HCV.44 HCV infection is also often associated with the occurrence of liver steatosis. Interestingly, NS5A colocalizes with the HCV core protein on lipid droplets and interacts with apolipoprotein A1.45

Genes down-regulated by NS5A included PLTP (which mediates phospholipid transfer, high-density lipoprotein conversion, and reverse cholesterol transport), fatty acid binding protein 1, lipoprotein-associated phospholipase A2, the nuclear liver X receptor α, arylacetamide deacetylase, and apolipoprotein E receptor 2. Lipoprotein-associated phospholipase A2 is secreted by liver cells and is associated with low- and high-density lipoprotein. Liver X receptor α, PLTP, and arylacetamide deacetylase have all been associated with the production and assembly of very low-density lipoprotein. ApoE receptor 2 belongs to the low-density lipoprotein receptor family and functions in the uptake of apolipoprotein E–containing lipoproteins.

These findings support an important role for NS5A in modulating lipid homeostasis in hepatic cells that may affect the viral life cycle and/or contribute to the development of liver steatosis.

Comparison of 4 NS5A Sequences Isolated From Responders and Nonresponders to IFN-α Therapy.

To discriminate between early and late events or potentially clonal effects, transient transfection assays were performed in Huh7 cells with the plasmid-expressing NR1 NS5A mutant. NS5A protein expression was determined 48 hours posttransfection via Western blot analysis (Fig. 5B, lane 2). Microarray analysis was performed for two independent experiments. Out of the 103 genes described above, 34 genes were also identified as up- or down-regulated following transient expression of NS5A (P < .05) as shown in bold in Tables 2 and 3. These genes included IL-8, IL-1RA, fibrillin-like protein, and vimentin, which were up-regulated, and galectin 2, trefoil factor 2, hepatocyte growth factor–like protein, inositol 1,4,5-triphosphate receptor, type 3, calpain 1, and PLTP, which were down-regulated upon NS5A expression. It is interesting to note that both TNFR1A and LTBR were also down-regulated following transient expression of NS5A. The genes identified in the clones but not verified with the transient expression system may be modulated by NS5A by less than fourfold at 48 hours and therefore are not selected because of the transfection efficiency of 50%–60%, or they may be targets whose expression is modulated by NS5A at a later time. A clonal effect resulting in the selection of these genes cannot be excluded.

Figure 5.

NS5A expression in Huh7 cells following transient transfections. (A) Amino acid sequence alignments of NS5A sequences. Full-length NS5A sequences were cloned from 2 responders (R1 and R2) and 2 nonresponders (NR1 and NR2) to IFN-α treatment. Amino acid residues are indicated by the standard single-letter codes; dashes indicate the identical amino acid residues, with the consensus 1b sequence shown at the top. The position of the interferon-sensitivity determining region (ISDR) is depicted in black. (B) Huh7 cells were transfected with NR1, NR2, R1, and R2 NS5A-expressing plasmids or with the empty vector. After 48 hours, total proteins were separated via SDS-PAGE, transferred to nitrocellulose membranes, and probed with anti-NS5A and anti–β-actin antibodies.

We subsequently compared the effects of NS5A mutants isolated from responders and nonresponders to IFN-α therapy. In addition to the NR1 sequence, 3 full-length NS5A sequences were obtained from 1 nonresponder (NR2) and two responders (R1 and R2) (see Fig. 5A). NR1, NR2, R1, and R2 were transiently expressed in Huh7 cells (see Fig. 5B). Total mRNA was prepared from the Huh7 cells expressing transiently each of the 4 NS5A sequences or the empty vector, and transcript levels were determined using microarray analysis. Again, 2 independent experiments were performed. Out of the 34 genes regulated upon both stable and transient expression of NR1 NS5A, 33 were also identified as up- or down-regulated by the 3 other NS5A mutants (NR2, R1, and R2). Lipoprotein-associated phospholipase A2 transcript was found to be down-regulated specifically by NS5A NR1 and NR2 proteins. However, this latter observation was not confirmed in RT-PCR experiments (data not shown). Therefore, no difference was detected among the NS5A sequences derived from responders to IFN-α therapy and the NS5A sequences derived from nonresponders.

Computational Promoter and NF-κB Binding Site Analysis.

We wished to predict putative transcription factors mediating NS5A effects on gene expression. A computational transcript mapping approach was used to locate promoter sequences from the 43 up-regulated genes (see Table 2). The promoter sequences of the 43 up-regulated genes were submitted for analysis to MATCH, a pattern matching program for searching putative transcription factor binding sites in DNA sequences.16, 17 Thirty-nine of the 43 gene promoter regions contained 1 or more binding sites for nuclear factor κB (NF-κB) (Supplementary Table 1), the large majority of them containing multiple NF-κB binding sites. The results included 6 genes known to be target genes of NF-κB: IL-8, GRO-α, vimentin, IL-1RA, MIP-3α/CCl20 and epidermal growth factor receptor (http://people.bu.edu/gilmore/nf-kb/target/).

We subsequently used the Gibbs sampling method18 to derive overrepresented motifs in the upstream regions of these 43 coinduced genes. To determine if this set of genes may be coregulated by NF-κB, motif lengths from 14–25 bp were sampled from the 43 defined promoter regions to search for conserved alignments. Seven returned motifs fit the significant cut-off values (Wilcoxon P < .05), and all 43 genes were returned from the Gibbs sampler with a sequence relating to the specified consensus sequence for all 7 motifs (Supplementary Table 2). Therefore, computational analysis of the 43 coinduced genes predicts that NF-κB mediates NS5A effects on gene expression.

NF-κB Mediates NS5A Effects.

To measure the transcriptional activation of NF-κB in NS5A-expressing cells, Huh-7 cells were transfected with an empty vector or NR1 NS5A expression plasmid, and induction of NF-κB was measured via cotransfection of a luciferase gene driven by the E-selectin promoter. The E-selectin promoter contains three functional NF-κB sites, yields robust induction, and is specific for NF-κB.46 Expression of NS5A leads to activation of NF-κB as measured by the reporter readout (Fig. 6). Significant increase in NF-κB activity was also observed in NS5A-expressing clones F1 and F11 compared with control clones T1 and T2 (P = .012) (data not shown). This is in agreement with a recent report suggesting an activation of NF-κB by NS5A.47 Activation of NF-κB in the liver of patients with chronic hepatitis C was also reported.48 NF-κB is an important mediator of the inflammatory response; however, its role in liver inflammation and fibrosis remains controversial.

Figure 6.

Activation of NF-κB by NS5A. Huh7 cells were cotransfected with NF-κB–controlled luciferase plasmid along with NR1 NS5A expression plasmid or empty vector used as a control. Lysates from transfected cells were prepared for luciferase activity.

To confirm that NF-κB mediates NS5A-dependent gene regulation, we inhibited NF-κB activity by infecting NS5A expressing Huh7 clones F1 and F11, with adenoviruses AdIκBα or AdGFP expressing full-length IκBα cDNA and GFP, respectively. The cells were tested for IκBα expression via Western blotting (Fig. 7A). As shown in Fig. 7B, adenovirus-mediated overexpression of IκBα was effective at inhibiting the up-regulation of genes induced by NS5A for aldose reductase, GRO-α, LTBP1, neurotensin, and BMP2, all randomly selected.

Figure 7.

Adenovirus-mediated IκBα transfer reverts NS5A-dependent gene up-regulation. F1 and F11 Huh7 clones were infected with adenovirus bearing IκBα or GFP, respectively. After 18 hours, total proteins and RNA were extracted. (A) Total proteins were separated via SDS-PAGE, transferred to nitrocellulose membranes, and probed with anti-IκBα and anti-β-actin antibodies. (B) Total RNA was reverse-transcribed and real-time PCR amplification was performed for aldose reductase, GRO-α, LTBP1, neurotensin, BMP2, and actin as described in Patients and Methods. Transcript levels in F1-IκBα and F11-IκBα were normalized using actin mRNA levels and compared with transcript levels in F1-GFP and F11-GFP, respectively. Data are shown as mean ± SEM of at least 3 independent experiments. Abbreviations: IκBα, inhibitor κBα; GFP, green fluorescence protein; LTBP1, latent TGF-β binding protein 1; BMP2, bone morphogenic protein 2.

In conclusion, we identified 103 genes modified by NS5A in the hepatocytic Huh-7 cells, many of which may participate in the pathogenesis of HCV infection. To this regard, the impact of NS5A protein mutations has yet to be elucidated. In addition, activation of NF-κB may play an important role in HCV pathogenesis and development of HCV-related liver diseases.

Acknowledgements

We thank Drs. D. States and R. McEachin (University of Michigan) for productive discussions; C. Logsdon (University of Michigan) for the gift of AdIκBα and AdGFP; and C. Vincenz (University of Michigan) for the gift of E-selectin luciferase. We also thank Alison Elafros, Philip Shalhoub, and Pascal Lescure for their technical support.

Ancillary