Impaired antiviral activity of interferon alpha against hepatitis C virus 2a in Huh-7 cells with a defective Jak-Stat pathway

Huh-7.5 cells, obtained from the laboratory of Dr. Charles M. Rice (Center for the Study of Hepatitis C, The Rockefeller University, New York), were cultured at 37°C in Dulbecco's modified Eagle's medium supplemented with 2 mM l-glutamine, nonessential amino acids, 100 U/ml of penicillin, 100 μg/ml of streptomycin and 10% fetal bovine serum, under 5% CO₂ conditions. Interferon resistant (R-24/1) replicon cells were generated in our laboratory by prolonged treatment of low inducer replicon cell lines (Con-15, Con-17, and Con-24) with IFN-α as described previously [21, 22]. A cured Huh-7 cell line with defective Jak-Stat pathway (R-Huh-7) was prepared from IFN-α resistant replicon cell line (R-24/1) after repeated treatment with cyclosporine-A (1 μg/ml) as described previously [22]. Interferon sensitive cured Huh-7 cells (S-Huh-7) were derived from the 5-15 replicon cell line after treatment with IFN-α. Interferon sensitive and interferon resistant phenotypes in the cured S-Huh-7 and R-Huh7 cells were examined by measuring their ability to activate the ISRE-luciferase promoter in the presence of exogenous IFN-α. The expression of functional Jak-Stat signaling proteins in these cells after IFN-α treatment was examined by western blot analysis of phosphorylated Stat1 and Stat2. All the resistant cell lines have defects in the phosphorylation of Stat1 and Stat2 protein, whereas the S-Huh-7 clone showed a high level of phosphorylation of Stat1 and Stat2 proteins within 30 minutes of IFN-α treatment [22]. All Huh-7 cell lines were maintained in Dulbecco's modified Eagle's medium supplemented with 2 mM l-glutamine, nonessential amino acids, 100 U/ml of penicillin, 100 μg/ml of streptomycin and 5% fetal bovine serum.

The JFH1 full-length cDNA clone of HCV 2a strain which was isolated from a chronically infected Japanese fulminant hepatitis patient was obtained from Wakita and his coworkers [14]. Chimeric clones between JFH1 and enhanced green fluorescent protein (EGFP) were constructed in our laboratory by the standard overlapping PCR amplification and cloning methods. The coding sequence of GFP was amplified from pEGFP-N1 plasmid and inserted in-frame of the NS5A coding sequence of the JFH1 cDNA clone between 2394 and 2395 amino acids position (between 417 and 418 amino acids of the NS5A protein). The PCR amplification of recombinant DNA and cloning was performed in four different steps. In the first step, the 228 bp (F1) recombinant DNA fragment containing 70 amino acids of NS5A (nts.7339-7546) fusion with the first 6 amino acids of EGFP-N1 was amplified using a sense primer (S/7336-7360/HCV-5'-CCTCCCCCAAGGAGACGCCGGACA-3') and anti-sense primer (AS/7529/HCV- 5'CTCGCCCTTGCTCACCATG GGGGGCATAGAGGAGGC-3'). In the second step, the 719 bp (F2) recombinant DNA fragment containing the total EGFP-N1 open reading frame (ORF) fused with the N- and C-termini of HCV NS5A was amplified using sense and anti-sense overlapping primers (S/7529/GFP- 5'-GCCTCCTCTATGCCCCCCATGGTGAGC AAGGGCGAG-3' and (AS/7547-7564/GFP 5'-TCCAGGCTCCCCCTCGAGCTTGTACA GCTCGTCCAT-3'). In the third step, the recombinant 531 bp DNA fragment (F3) containing last 6 amino acids of EGFP-N1 and 177 amino acids of NS5A (nt. 7547-8077) was amplified by using sense primers (S/7547/HCV- 5'-ATGGACGAGCTGTACAAG CTCGAGGGGGAGCCTGGA-3') and anti-sense primer (AS/8059-8077/HCV-5'-GTCTTCCAGGAGGTCCTTCCACAC-3'). In fourth step, the F1, F2 and F3 PCR fragments were assembled into the 1478 bp DNA fragment through overlapping PCR. In the final step, the recombinant DNA was digested with restriction enzyme RsrII and HpaI, gel purified and then ligated with pJFH1 clone using the unique RsrII and HpaI restriction sites present in the NS5A gene. The resulting plasmid was named pJFH1-GFP. The recombinant plasmid was amplified and the construction was confirmed by sequence analysis. A full-length pJFH1-GFP plasmid clone was prepared with a GDD to GND mutation in the NS5B gene to use as a control (pJFH1-GND-GFP) in the replication assay. A full-length pJFH1-GFP plasmid was also prepared with a deletion in the E1-E2 gene (pJFH1-ΔE1E2-GFP) to use as a control in the infectivity assay. To generate a sub genomic GFP replicon clone of HCV 2a, the recombinant plasmid containing the NS5A and EGFP-fusion was excised from full-length pJFH1-GFP plasmid using the NsiI and HpaI enzyme and re-cloned into the pSGR replicon [23]. This chimeric clone was named pSGR-GFP. As a control, we created a mutant construct pSGR-GND-GFP clone with a point mutation that changes a GDD motif to GND, abolishing the RNA polymerase activity of the NS5B protein. All the recombinant plasmids constructs were confirmed by DNA sequence analysis.

Full-length (pJFH1-GFP) and sub-genomic replicon (pSGR-GFP) plasmids were linearized with XbaI restriction enzyme and purified by phenol chloroform extraction and precipitated by ethanol. The HCV full length and sub-genomic RNAs were transcribed from XbaI linearized plasmid DNA templates using the MEGA-script T7 kit (Ambion, Austin, TX, USA). In vitro transcribed RNA was treated with DNase I to eliminate any residual plasmid DNA, extracted with phenol and chloroform, and then precipitated with absolute ethanol. The RNA pellet was re-suspended in nuclease free water and 10 μg aliquots of this RNA were stored at -80°C. The integrity of in vitro transcribed RNA was verified by agarose gel electrophoresis.

Huh-7.5, S-Huh-7 and R-Huh-7 cells were electroporated with in vitro transcribed HCV RNA using a standard protocol described previously [17]. Briefly, cells were harvested using trypsin-EDTA, pelleted by centrifugation and washed in 10 ml of phosphate buffered saline (PBS). The cell pellet was suspended in PBS (10⁷ cells per ml). Ten micrograms of in vitro transcribed RNA was mixed with 400 μl of Huh-7 cell suspension in a cuvette (0.4 cm, Bio-Rad) and subjected to an electric pulse at 960 μF and 270 volts using a gene pulser apparatus (Bio-Rad). Following electroporation, cells were diluted in 10 ml of complete medium and plated in a 100-mm diameter cell culture dish.

To study replication of full-length HCV-GFP chimeric RNA, the electroporated Huh-7 cells were cultured in a 100-mm plate with regular growth medium. The expression of GFP in the transfected Huh-7 cells was recorded at 0, 24, 48, 72 and 96 hours post-transfection. To study the replication of HCV sub-genomic RNA, stable Huh-7 cells replicating sub-genomic RNA were prepared. Cured Huh-7 cells derived from interferon sensitive (S-Huh-7) and resistant replicon cell lines (R-Huh-7) in our laboratory were used. Huh-7 cells electroporated with sub-genomic RNA were cultured in a growth medium supplemented with 500 μg/ml G-418 drug. These cells were maintained with a regular medium change at every three days for 3-4 weeks until distinct G-418 resistant cell colonies were developed. To make a stable cell line replicating HCV 2a sub-genomic RNA, multiple G-418 resistant cell clones were isolated and cultured in medium supplemented with 1 mg/ml G-418. In these stable cell lines absence of HCV plasmid DNA integration was confirmed by direct PCR followed by Southern blot analysis for the neo gene (sense 5'-ATCGAATTCATCGTGGCTGGCCA-3'; anti-sense 5'-CTAGAATTCGGCGCGAGCCCCTG-3'; probe 5'-GCTTGGTGGTCGAATGGGCAG GTAGCCGGA-3'.

An infectivity assay for HCV was performed using a published protocol [15]. Huh-7.5 cells were transfected with 20 μg of in vitro transcribed full-length JFH1-GFP RNA by electroporation method. After 72 h, cells were collected by scraping and then lysed by four rounds of freeze-thaw cycles. The cell lysates were clarified by centrifugation at 3400 rpm for five minutes. The clear supernatant was collected and the titer of HCV in the supernatant was determined by real-time RT-PCR using a primer set targeted to the 5'UTR. A tissue culture infective dose (TCID50) was determined using 10-fold serial dilution of the virus containing supernatant on 2-well Lab-Tek chamber slides (Nalge Nunc International, Rochester, New York). Briefly, Huh-7.5, S-Huh-7 and R-Huh-7 cells were seeded on a 2-well glass chamber slide at a density of 1 × 10⁴ cells per well. The next day, the culture medium was removed and 1-ml of serial dilutions of culture supernatant containing infectious virus was added to the wells. The cells were incubated overnight at 37°C. On the following day the culture medium was removed, and the cells were washed once using PBS and then incubated in fresh complete medium. After 96 hours post-transfection, the cells were removed, washed in PBS, fixed in 4%-parformaldehyde for 30 minutes and then counter stained with Hoechst dye (H33342, Calbiochem, Darmstadt, Germany) for 15 minutes at 37°C. Cells were examined at 484 nm using a fluorescence microscope (Olympus) for expression of green fluorescence. Cells were then examined at 340 nm for blue nuclear staining. For each area, two sets of pictures were taken. The final image was generated by superimposing blue nuclear fluorescence of Hoechst dye with green fluorescence of GFP using Abode Photoshop software (V 7.0). The numbers of green positive cells in ten different fields were counted and the percentage of green fluorescence positive cells in the culture was determined. The dilution of virus-containing supernatant that showed 50% GFP positive cells 96 hours after infection in the culture (called the TCID50) was determined. The MOI of the infectious culture supernatants was determined by dividing the TCID50 with the cell number used in the infectivity assay.

To study the effect of interferon on the full-length HCV 2a clone, transfected or infected Huh-7 cells were treated with increasing concentrations of IFN-α(Intron A, Schering-Plough, NJ, USA). The antiviral effect of IFN-α against HCV using different Huh-7 clones was confirmed by observing GFP expression under a fluorescence microscope or by flow cytometric analysis, and HCV RNA levels was measured by RPA.

Total RNA was prepared from the cell pellet by the GITC method and subjected to RPA for the detection of genomic positive-strand HCV RNA. For RPA experiments, 25 μg of the total RNA was mixed with a negative-strand RNA probe targeted to the 5'UTR of HCV (1 × 10⁶ cpm) in a 10 μl hybridization solution, denatured for 3 minutes at 95°C and then hybridized overnight at 50°C. RNase digestion was performed in 200 μl of RNase digestion buffer (10 mM Tris, pH 7.5, 5 mM EDTA and 0.3 M NaCl) containing RNaseA/T1 cocktail at 1:100 dilutions (Ambion Inc., Austin, TX) for an hour at 37°C. Then the sample was treated with 2.5 μl of 25% SDS and 10 μl of proteinase K (20 mg/ml) for 15 minutes. Samples were extracted with phenol and chloroform and then precipitated after addition of 2.5 volumes of absolute ethanol. The pellet was obtained by centrifugation for 30 minutes at 12,000 rpm. The RNA pellet was washed with 70% ethanol, suspended in 8 μl of gel loading buffer, boiled for one minute and separated on a 6% polyacrylamide TBE-Urea gel (Invitrogen, Carlsbad, CA). The gel was dried and exposed to X-ray film (Kodak Biomax-XAR, Rochester, NY). We prepared a plasmid construct called pCR-II (2a), which contained the 79-297 nucleotides of the 5'UTR sequence of the JFH1 clone (pCR-II NT-218) (Invitrogen). This plasmid was linearized with HindIII restriction enzyme and a positive strand RNA probe was prepared using T7 RNA polymerase in the presence of 32p labeled CTP. Likewise, this plasmid was linearized with XbaI restriction enzyme and Sp6 RNA polymerase was used to prepare a negative strand RNA probe for the detection of a positive strand HCV RNA. The same amounts of the RNA extracts were subjected to RPA for GAPDH mRNA. We used a linearized pTRI-GAPDH-human anti-sense control template to prepare a probe to detect GAPDH mRNA using Sp6 RNA polymerase (Ambion Inc., Austin, TX). The appearance of 218 (HCV 2a) and 317 nts protected fragments indicated the presence of the HCV positive-strand and the GAPDH mRNA, respectively.

Real time RT-PCR was performed to quantify HCV RNA levels in the infected cell culture using a published protocol [24]. The 243 bp HCV DNA was amplified from the RNA extract by reverse transcription polymerase chain reaction using the outer sense (OS) primer 5'-GCAGAAAGCGCCTAGCCATGGCGT-3' (67-90) and outer anti-sense (OAS) primer 5'-CTCGCAAGCGCCCTATCAGGCAGT-3' (287-310). First the complementary DNA synthesis was performed from positive strand HCV-RNA using an outer anti-sense primer (OAS) targeted to the highly conserved 5'UTR region of HCV in 20 μl volume. Briefly, 2 μgm of total cellular RNA were mixed with 1 μl OAS primer (200 ng/μl), denaturized at 65°C for 10 minutes and annealed at room temperature. Avian myeloblastosis virus (AMV) reverse transcriptase (10 U) (Promega, Madison, WI) was added and incubated at 42°C for 60 minutes in the presence of 50 mmol/L Tris, pH 8.3, 50 mmol/L ethylenediaminetetraacetic acid (EDTA), 500 nmol/L dNTP, 250 nmol/L spermidine, and 40 U RNasin (Promega). The cDNA was stored at -20°C until use. SYBR Green real time PCR amplification was performed in 20 μl of volume containing 10 μl of SYBR Green ER qPCR SuperMix, 1 μl (250 ng/ul) of sense and antisense primer with 4 μl of cDNA and 4 μl of distilled water. All samples were run in triplicate. The amplification was carried out using the standard program recommended by Bio-Rad Laboratory that includes: 50°C for 2 minutes, 95°C for 8 minutes, then additional 50 cycles wherein each cycle consists of a denaturation step at 95°C for 10 seconds, and annealing and extension step at 60°C for 30 seconds. At the end of the amplification cycles, melting temperature analysis was performed by a slow increase in temperature (0.1°C/s) up to 95°C. Amplification, data acquisition, and analysis were performed on CFX96 Real Time instrument (Bio Rad) using CFX manager software (Bio Rad).

Replication of the full-length HCV 2a genome is possible due to the availability of the JFH1 cDNA clone. However, the highly sensitive RT-PCR and immunodetection methods are most often needed to detect replication of HCV in the transfected cells. To overcome the technical difficulties associated with the detection of the full-length viral RNA replication, we constructed chimeric clones of the JFH1 clone and GFP so that replication of whole viral genome in the transfected cells could be examined by fluorescence microscopy. Previous reports suggest that heterologous sequences can be inserted into the HCV genome without altering its ability to replicate [25‐28]. The coding sequence of GFP was inserted into C-terminus of the NS5A protein of HCV at the 2394 amino acid position. Chimeric clones of GFP and full-length, and a sub-genomic replicon of HCV 2a were prepared (Fig. 1). The N-terminal and C-terminal fusion of HCV NS5A with EGFP protein was confirmed by sequence analysis. To study the replication of full-length virus, in vitro transcribed RNA derived from wild type and GND-mutant clone were electroporated into Huh-7.5 cells. The expression of GFP was recorded in a kinetic study. The replication of full-length JFH1-GFP chimera in the transfected Huh-7.5 cells was seen as early as 24 hour post-transfection and the number of GFP positive cells in the culture increased gradually at 48, 72 and 96 hours (Fig 2A). In contrast, replication of the JFH1-GND-GFP mutant RNA in Huh-7.5 cells was not observed at 48, 72 or 96 hours post-transfection, while only a very faint GFP signal was seen in Huh-7.5 cells at 24 hours post-transfection (Fig. 2B). The efficiency of replication of chimeric clones in Huh-7.5 cells after RNA transfection was observed in approximately 8% of cells as examined by flow cytometry (Fig. 2C and 2D). Replication of full-length JFH1-GFP chimera clone was confirmed by examining HCV positive and negative strand RNA levels by RPA assay. The levels of HCV RNA in the full-length transfected cells and GND mutant transfected cells were clearly different (Fig 3A). As expected, the levels of mutant RNA dropped below the input level and remained undetected at 48, 72 and 96 hours post-transfection. The level of HCV positive strand RNA seen in the RPA assay appeared to be higher at an earlier time point in the full-length transfected cells at a later time point. This may be due to an input RNA carryover during the transfection step. There was a good correlation between the amount of HCV RNA and expression of GFP at later time points.

HCV is a positive strand RNA virus that replicates via the synthesis of negative strand RNA. To demonstrate that the replication of transfected RNA resulted in the production of negative strand RNA in the transfected cells, RPA for negative strand HCV RNA was performed in the transfected cells at 0, 24, 48, 72 and 96 hours post-transfection. Negative strand HCV RNA was not detectable at the zero-time point but appeared at 24 hour post-transfection (Fig. 3B). Negative strand RNA was undetectable in Huh-7.5 cells transfected with GND mutant RNA. The presence of negative strand HCV RNA in the full-length transfected cells confirmed active replication of virus in the culture. Based on the results of these experiments we conclude that the chimeric JFH1-GFP clone is replication competent.

To examine infectious virus particle production from cells transfected with JFH1-GFP chimera RNA, an infectivity assay was performed. Culture supernatants were collected from transfected cells, clarified by centrifugation and inoculated to Huh-7.5 cells. The infectivity of HCV was confirmed by direct examination of infected cells under a fluorescence microscope and HCV RNA levels were measured by real-time RT-PCR assay. Infectivity of culture supernatants from cells transfected with full-length and E1-E2 deleted mutant clone was determined by measuring intracellular GFP expression. There was an increase in the number of GFP positive cells after 24, 48 and 72 hours suggesting the replication of HCV RNA after natural infection (Fig. 4A). No GFP expression was observed in Huh-7.5 cells infected with supernatants derived from cells transfected with E1-E2 deleted mutant HCV RNA (Fig. 4B). To confirm that the expression of HCV in the infected cells is associated with the increase in viral RNA, the titer of HCV positive strand RNA was measured using a real-time RT-PCR. The level of HCV RNA in the infected cell cultures was increased from 24 to 72 hours suggesting the replication of HCV-RNA genome in the infected culture (Fig. 4C). Thus, JFH1-GFP-tagged HCV RNA genome is able to replicate in Huh-7.5 cells after transfection and generates an infectious virus.

Since the JFH1 2a clone replicates to a high level in a cell culture without adaptive mutations, we attempted to develop stable replication competent Huh-7 cells containing GFP labeled sub-genomic HCV RNA. The availability of these cell lines allowed us to reliably quantify the antiviral effect of IFN-α. A chimeric clone combining GFP and sub-genomic clone was prepared. As a control, GND mutant (pSGR-GND-GFP) for the replicon clone was also prepared. The full-cycle replication of pSGR-GFP RNA and unmodified pSGR-RNA in Huh-7 cells were compared for their ability to form cell colonies when cultured in the presence of a medium containing G-418 (500 μg/ml). In this assay, the cells supporting HCV RNA replication survived G-418 drug selection and formed cell colonies. No noticeable differences in the efficiency of replication of the sub-genomic RNA with or without GFP insertion in the NS5A region were observed based on the number of G-418 resistant cell colonies that appeared on the plate (Fig. 5). No colonies developed in the culture transfected with the GND mutant sub-genomic HCV RNA. Individual cell colonies were picked and stable Huh-7 cell lines supporting replication of HCV-GFP sub-genomic RNA were developed. The absence of stable DNA integration in these cell lines was confirmed by direct PCR analysis for neo gene followed by southern blot analysis. High levels of GFP expression due to replication of sub-genomic HCV 2a clone was seen in sensitive and resistant Huh-7 clones (Fig. 6A). The expression of HCV-GFP chimera protein was seen exclusively in the cytoplasm in the majority of Huh-7 cells in the culture. These cell lines have maintained stable GFP expression over more than one year when cultured in a growth medium supplemented with G-418 (500 μg/ml). Two types of stable replicon cell lines were prepared using Huh-7 cells with or without functional Jak-Stat pathway. Stable HCV-GFP replicon cell lines prepared using IFN sensitive (S-Huh-7) cells were named as S3-GFP and S10-GFP replicons. Replicon cell lines, also prepared using IFN resistant Huh-7 cell lines (R-Huh-7), were named as R4-GFP and R8-GFP replicons. The level of GFP expression in the IFN sensitive and resistant replicon Huh-7 cell lines was quantitatively determined by flow analysis. The results of these experiments suggest that more than 80% of replicon cells express GFP (Fig. 6B).

The development of JFH1-GFP chimera using the HCV 2a clone allowed us to quantify the antiviral properties of IFN-α in Huh-7 cells. One important predictive factor associated with IFN response is the viral genotype. It has been reported by a number of investigators that the sustained virological response in patients infected with HCV genotype 2 is much higher than in patients infected with genotype 1 virus. We conducted experiments to determine whether the replication of an HCV 2a strain could be inhibited in liver cells (R-Huh-7) having a defective Jak-Stat pathway. Both S-Huh-7 and R-Huh-7 cells were transfected individually with full-length JFH1-GFP RNA and then treated with an increasing concentration of IFN-α. We first determined that both S-Huh-7 and R-Huh-7 cells developed in our laboratory supported HCV 2a replication and infection. The ability of IFN-α to inhibit full-length HCV 2a replication in these two different Huh-7 clones was examined in a kinetic study at 24 to 96 hours. Results shown in the upper panel of Fig. 7A suggest that GFP expression can be efficiently inhibited in S-Huh-7 cell clones. There was no reduction in GFP expression in the R-Huh-7 cell clones with a defective Jak-Stat pathway at all time points (lower panel of Fig. 7A). The antiviral effect of IFN-α against HCV 2a in these two cell clones (S-Huh-7 and R-Huh-7) was also quantified by flow cytometric analysis. We found a time dependent effect of IFN-α on HCV 2a replication in S-Huh-7 cells and the number of GFP positive cells was decreased from 4.2% to 0.2% as compared to resistant Huh-7 cell line (Fig. 7B). To verify that the inhibition of GFP is also associated with the reduction of viral RNA in the interferon treated cells, RNA extracts were assayed for HCV RNA by RPA assay using a probe targeted to the 5' UTR region of HCV genome. We found that interferon treatment decreased HCV RNA levels in S-Huh-7 clones and the levels of HCV RNA remained unchanged after interferon treatment in the resistant clone. (Fig. 7C). The ability of IFN-α to stop viral RNA replication in the infected cells was also examined using these two Huh-7 cell clones. IFN-α treatment efficiently inhibited HCV replication as measured by GFP expression in S-Huh-7 cells within 24 hours (Fig 8A). However, antiviral activity of IFN-α against the full-length HCV 2a replication was prevented in R-Huh-7 cells with the defective Jak-Stat pathway (Fig. 8B). The results of these experiments indicate that antiviral activity of IFN-α to inhibit replication of full-length HCV 2a clone was prevented in R-Huh-7 clone with defective Jak-Stat pathway.

The role of the Jak-Stat pathway in the IFN-α response to HCV 2a was also studied using an IFN sensitive (S3-GFP) and IFN resistant (R4-GFP) stable Huh-7 cell line that replicates sub-genomic RNA. Replication of HCV 2a sub-genomic RNA in the S3-GFP after IFN-α treatment was studied by measuring the intracellular GFP expression directly under a fluorescence microscope. It was found that GFP expression in the stable cell line (S3-GFP) diminished over time (Fig 9A). Where as no reduction of the HCV-GFP signal in R4-GFP replicon was observed even when treated with a similar concentration of IFN-α for an extended period. To quantify the IFN antiviral effect intracellular GFP expression was analyzed by flow analysis. The GFP peak disappeared after IFN treatment only in the S3-GFP replicon cell line (53% to 2%). The percentage of GFP positive cells did not decrease (58% to 55%) when similar experiments were carried out using R4-GFP cells (Fig. 9B). To correlate the results of GFP expression, intracellular HCV RNA after IFN-α treatment was also measured by RPA. The results of RPA assays demonstrate that HCV RNA replication is not inhibited by IFN-α treatment in the R4-GFP replicon cell line (Fig. 9C). The level of HCV RNA was also quantified by real-time PCR in these two cell lines after IFN treatment. IFN-α treatment inhibited the HCV RNA level in a dose dependent manner in S3-GFP but the HCV RNA level remained the same in the R4-GFP replicon. There was a significant difference in the level of HCV RNA between the IFN sensitive replicon and resistant replicon after IFN treatment measured by real-time PCR (Fig. 9D). These results suggest that replication of HCV 2a full-length as well as sub-genomic RNA can not be inhibited by IFN-α in R-Huh-7 cells with a defective Jak-Stat pathway.

Experiments were carried out to examine whether infection or replication of HCV in both S-Huh-7 and R-Huh-7 cells could have any impact on the IFN-α induced Jak-Stat signaling. The levels of pStat1 and pStat2 proteins in the lysates of S-Huh-7 and R-Huh-7 cells after 96 hours of HCV infection were examined by western blot analysis. Results shown in Fig. 10A and 10B clearly show that IFN-α treatment induced pStat1 and pStat2 protein in the infected as well uninfected S-Huh-7 only. However, pStat1 or pStat2 protein was not detected in the infected R-Huh-7 cells even after interferon treatment. These results were confirmed by a co-localization of pStat1 protein in the GFP labeled replicon cells after IFN-treatment. We show that pStat1 is induced only in the sensitive replicon (S3-GFP) and localizes to the nucleus. The nuclear translocation of pStat1 is correlated with a decrease in GFP expression after IFN-α at 72 hours in the S-Huh-7 cells only (Fig. 10C). The pStat1 protein was undetectable in R4-GFP cells after IFN-α treatment. To examine if the effect of HCV infection or replication in both S-Huh-7 and R-Huh-7 could alter the overall Jak-Stat signaling, the ISRE-luciferase promoter activity was examined by a transient transfection assay. Interferon induced activity of ISRE-luciferase did not change significantly in R-Huh-7 cells after HCV infection (Fig. 10D).