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    NATAP http://natap.org/ ... Statin Drugs Inhibit HCV in vitro Cholesterol drugs may treat hepatitis C: study Fri Jul 7, 2006 WASHINGTON (Reuters) - Cholesterol
    Message 1 of 1 , Jul 10, 2006
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      Statin Drugs Inhibit HCV in vitro

      Cholesterol drugs may treat hepatitis C: study

      Fri Jul 7, 2006

      WASHINGTON (Reuters) - Cholesterol drugs called statins may help treat hepatitis C infections, Japanese researchers reported on Friday.

      Tests in lab dishes suggest that some statin drugs may help stop the hepatitis C virus from replicating, they wrote in the journal Hepatology, published by the American Association for the Study of Liver Diseases.

      An estimated 170 million people worldwide are infected with the hepatitis C virus. The standard treatment is a combination therapy of interferon and ribavirin but it only helps about 55 percent of patients.

      The rest risk progression to cirrhosis and liver cancer.

      Masanori Ikeda of Okayama University in Japan and colleagues tested several statin drugs against the virus in lab dishes.

      All the drugs except pravastatin interfered with the virus to some degree. Fluvastatin, sold by Novartis under the name Lescol, had the strongest effect, they reported.

      It may be that certain proteins are required for the hepatitis C virus to replicate and that some statins block the action of these proteins, the researchers said.

      They tested the statins along with interferon, and found each worked even better when combined with the second drug.

      "We clearly demonstrated that co-treatment of interferon and fluvastatin was an overwhelmingly effective treatment," the researchers wrote.

      Statins -- which include Pfizer Inc.'s $10 billion-a-year Lipitor, Bristol-Myers Squibb Co.'s Pravachol and Merck and Co. Inc.'s Zocor -- are the world's best-selling drugs, taken by millions to reduce the risk of heart attack.

      But they appear to affect many biological processes. An expert proposed last month that they may affect influenza viruses, including bird flu, and other research has shown they reduce the risk of cataracts.

      Generic statins are available in many countries and have become increasingly inexpensive.

      Different anti-HCV profiles of statins and their potential for combination therapy with interferon

      Hepatology July 2006

      Masanori Ikeda, Ken-ichi Abe, Masashi Yamada, Hiromichi Dansako, Kazuhito Naka, Nobuyuki Kato *
      Department of Molecular Biology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan

      Abstract
      We recently developed a genome-length hepatitis C virus (HCV) RNA replication system (OR6) with luciferase as a reporter. The OR6 assay system has enabled prompt and precise quantification of HCV RNA replication. Pegylated interferon (IFN) and ribavirin combination therapy is the world standard for chronic hepatitis C, but its effectiveness is limited to about 55% of patients. Newer therapeutic approaches are needed. In the present study, we used the OR6 assay system to evaluate the anti-HCV activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, called statins, and their effects in combination with IFN-a. Five types of statins (atorvastatin, fluvastatin, lovastatin, pravastatin, and simvastatin) were examined for their anti-HCV activities. Fluvastatin exhibited the strongest anti-HCV activity (IC50: 0.9 umol/L), whereas atorvastatin and simvastatin showed moderate inhibitory effects. However, lovastatin, reported recently as an inhibitor of HCV replication, was shown to exhibit the weakest anti-HCV activity. The anti-HCV activities of statins were reversed by the addition of mevalonate or geranylgeraniol. Surprisingly, however, pravastatin exhibited no anti-HCV activity, although it worked as an inhibitor for HMG-CoA reductase. The combination of IFN and the statins (except for pravastatin) exhibited strong inhibitory effects on HCV RNA replication. In combination with IFN, fluvastatin also exhibited a synergistic inhibitory effect. In conclusion, statins, especially fluvastatin, could be potentially useful as new anti-HCV reagents in combination with IFN.

      Article Text

      Persistent hepatitis C virus (HCV) infection causes liver fibrosis and hepatocellular carcinoma. Approximately 170 million people worldwide are infected with HCV. The combination of pegylated interferon (IFN) with ribavirin is the current standard therapy for chronic hepatitis C (CHC) and yields a sustained virological response (SVR) rate of about 55%.[1] This means that about 45% of patients with CH C are still threatened by the progression of the disease to cirrhosis and hepatocellular carcinoma. Until 1999, when Lohmann et al. developed the subgenomic replicon of HCV, it was difficult to screen anti-HCV reagents.[2] Many improvements followed that breakthrough, such as a genome-length HCV RNA replication system[3][4] and a subgenomic replicon with a reporter assay system[5]; more recently, Wakita et al. used a genotype 2a strain, JFH1, to produce the infectious virus in cell culture.[6-8]

      Genotype 1 is the major genotype of HCV found in Japan, the United States, and many other countries. Unfortunately, the SVR rate after combination therapy of pegylated IFN with ribavirin is less than 50% for this genotype. To find a more effective therapy especially for CHC patients with genotype 1, we recently developed a genome-length HCV RNA (strain O of genotype 1b) replication reporter system (OR6), which has been an effective screening tool.[9][10]

      Statins, which are 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, are in wide use for the treatment of hypercholesterolemia. Recently, it was reported that lovastatin (LOV) inhibited HCV RNA replication.[11][12] These reports suggested the anti-HCV activity of LOV resulted from inhibition of the geranylgeranylation of cellular proteins rather than the inhibition of cholesterol synthesis.[11][12] More recently, FBL2 has been reported to be a host target protein for geranylgeranylation, which is responsible for HCV replication.[13] Although several types of statins are used clinically to lower cholesterol, thus far only LOV has been tested for anti-HCV activity. In the present study, we used the OR6 assay system to test the anti-HCV activity of five statins: atorvastatin (ATV), fluvastatin (FLV), pravastatin (PRV), simvastatin (SMV), and LOV. We found that ATV, FLV, and SMV exhibited stronger anti-HCV activity than that previously reported for LOV and that PRV exhibited no anti-HCV activity, although it worked as an inhibitor for HMG-CoA reductase. Because FLV showed the strongest anti-HCV activity, we also examined the effect of the combination of IFN-a and FLV on HCV RNA replication and found a synergistic inhibitory effect of IFN-a and FLV on HCV RNA replication.

      Discussion
      In this study, we found that different statins have different anti-HCV profiles. FLV, ATV, and SMV each exerted a stronger inhibitory effect on HCV RNA replication than did that of LOV reported previously.[11][12] However, PRV exhibited no anti-HCV activity. We also demonstrated that anti-HCV activity was drastically increased when these statins except PRV were used in combination with IFN-a. Because these statins are currently used for the clinical treatment of patients with hypercholesterolemia without inducing severe side effects, our findings suggest that these statins might be useful in combination therapy with IFN-a or IFN-a plus ribavirin.

      That PRV exhibited no anti-HCV activity is interesting. From the information on LOV only[10][11] to date, the mechanism underlying statins' inhibition of HCV RNA replication has not been considered their cholesterol-lowering activity but rather their inhibition of geranylgeranylation of cellular proteins. In other words, statins' inhibition of HMG-CoA reductase leads to the reduction of intracellular mevalonate and consequently to a reduction in geranylgeranyl pyrophosphate. In fact, in OR6 cells we observed that mevalonate and geranylgeraniol restored HCV RNA replication in the FLV- or LOV-treated cells. However, we found unexpectedly that PRV did not inhibit HCV RNA replication, whereas PRV inhibited HMG-CoA reductase as effectively as other statins possessing anti-HCV activity. Although PRV is a water-soluble reagent (others are lipophilic), we confirmed PRV did induce expression of HMG-CoA reductase by a positive feedback mechanism[18] and LST-1 was expressed in our cell culture system. These findings suggest the presence of a mechanism in which PRV's inhibition of HMG-CoA reductase does not cause the depletion of geranylgeranyl pyrophosphate. Interestingly, it has been reported that PRV has a unique effect among statins on the induction of p450.[18] Therefore, further studies are needed to explain why PRV exhibits no anti-HCV activity.

      We minutely examined the effect of FLV, the statin exhibiting the strongest inhibition of HCV replication of those tested in this study, in combination with IFN-a. We found that a combination treatment of IFN-a and FLV had a synergistic inhibitory effect on HCV RNA replication. Although high doses of IFN- are more effective than low doses for eliminating HCV from a patient, the side effects increase in a dose-dependent manner. Because ribavirin enhances the effect of IFN-a slightly in a cotreatment, it is the only reagent currently used with IFN-a to treat patients with CH C. In our previous study of anti-HCV activity using the OR6 assay system, we found the IC50 of ribavirin to be 76 umol/L.[10] This concentration is much higher than the clinically achievable ribavirin concentration (10-14 mol/L) previously reported.[25] Furthermore, when administered in combination with IFN-a (2 IU/mL) and ribavirin (50 umol/L), HCV RNA replication was reduced by only approximately 50%, compared with the effect of treatment with IFN-a alone.[10] It has been reported that the maximum blood concentration of FLV after 40 mg/day being administered orally for 4 weeks is approximately 0.6 umol/L.[26] This concentration is rather low for the inhibition of HCV replication in vivo, because the IC90 of FLV was assigned as 6.7 umol/L in our assay system (Fig. 5B). In addition, our study showed reatment of OR6 cells with 5 umol/L FLV alone was almost equal to the effect of 10 IU/mL IFN-a. Although statins are known to concentrate in the liver, FLV monotherapy will not be effective for patients with CH C. However, we demonstrated that the combination of IFN-a and FLV exhibited synergistic effects on HCV RNA replication. For example, when administered in combination with IFN-a (2-8 IU/mL) and FLV (5 umol/L), HCV RNA replication fell remarkably, to approximately 3%, compared with the effects of treatment with IFN-a alone (Fig. 4A). From these results, we propose that therapy combining FLV with IFN-a may be effective for the treatment of patients with CH C. Furthermore, additional treatment with reagents in combination (e.g., IFN-a, ribavirin, and FLV) will help to improve the SVR rate.

      In conclusion, the results of the present study suggest that statins other than PRV are good reagents for combination therapy with IFN-a in patients with CH C. Although the mechanism by which PRV lacks anti-HCV activity has not been clarified in the present study, a better understanding of this mechanism may lead to the discovery of statin-related anti-HCV reagents possessing no cholesterol-lowering activity. Furthermore, our developed OR6 assay system will be useful for the time-saving screening of new anti-HCV reagents.

      Results

      Inhibition of HCV RNA Replication by Statins.
      We recently developed a genome-length HCV RNA (strain O of genotype 1b) replication system (OR6) with luciferase as a reporter.[9][10] This OR6 reporter assay system has enabled prompt and precise quantification of HCV RNA replication. Therefore, in the present study, we examined whether several types of statins currently used in clinical therapy exhibit anti-HCV activity, as has already been reported for LOV.[11][12] This time, the antiviral activities of five statins - ATV, FLV, LOV, PRV, and SMV - were tested using the OR6 assay system (Fig. 1A). The results revealed that ATV, FLV, and SMV exhibited stronger anti-HCV activity than did LOV and that FLV exhibited the strongest anti-HCV activity among the statins tested (Fig. 1B). Surprisingly, however, PRV had no inhibitory effect on HCV RNA replication (Fig. 1B). Similar results were obtained from the analysis of the expression levels of HCV proteins (Fig. 1C). The anti-HCV activity of 5 umol/L FLV was compatible with that of 10 IU/mL IFN-a (Fig. 1C).

      To exclude the possibility that only PRV was unable to inhibit HMG-CoA reductase, we examined the expression of HMG-CoA reductase in statin-treated OR6c cells by RT-PCR, because HMG-CoA reductase was known to show positive feedback when statins were active in the cells.[18] OR6c cells were treated with statins in the same way as were the OR6 cells used to measure the anti-HCV activity of statins, except that sampling for RT-PCR was performed after 24 hours of statin treatment. All statins, including PRV, enhanced expression of the HMG-CoA reductase gene (Fig. 1D). Although PRV is hydrophilic and does not cross cellular membranes passively, it has been reported that a human liver-specific organic anion transporter, LST-1, mediates the uptake of PRV in human hepatocytes but not in HepG2 cells, which showed very low PRV uptake.[19][20] Therefore, we examined the expression levels of LST-1 in OR6 and OR6c cells using an RT-PCR method. OR6 and OR6c cells expressed LST-1 at levels equivalent to that in normal human liver, confirming that LST-1 was not expressed in the HepG2 cells (Fig. 1E). These findings suggest that PRV is taken up actively in OR6 and OR6c cells. In summary, these results indicate all statins tested inhibit HMG-CoA reductase and suggest the anti-HCV action of statins is not a result of direct inhibition of HMG-CoA reductase.

      Regarding the mechanism underlying the anti-HCV action of statins, it has thus far been reported that the inhibitory effect of LOV can be overcome by the addition of mevalonate (the product of the HMG-CoA reductase reaction) or geranylgeraniol (a donor of prenyl groups for protein geranylgeranyl transferase reaction).[11][12] These observations suggest that some geranylgeranylated proteins are required for HCV RNA replication and that LOV blocks HCV RNA replication by depleting endogenous geranylgeranyl pyrophosphate (the mevalonate-derived donor of protein geranylgeranylation). To evaluate this mechanism, we examined the effects of mevalonate and geranylgeraniol on the anti-HCV activities of the statins used in this study. OR6 cells were treated with 5 umol/L FLV, PRV, or LOV alone or in the presence of mevalonate (10 mmol/L) or geranylgeraniol (10 or 30 umol/L). Mevalonate and geranylgeraniol restored HCV RNA replication in the statin-treated cells, although 10 umol/L geranylgeraniol exhibited partial restoration (Fig. 1F). In addition, we observed that the anti-HCV activities of the statins could be blocked by the addition of geranylgeranyl pyrophosphate (20 umol/L) in the OR6 cells (data not shown), indicating geranylgeranyl pyrophosphate is also taken up in OR6 cells. These findings support two previous reports[11][12] that found the inhibition of HCV RNA replication by statins was not correlated with their cholesterol-lowering activities, although the reason for the lack of anti-HCV activity by PRV remains vague.

      Anti-HCV Activity of FLV Significantly Stronger Than Those of Other Statins.
      From the dose-response curves after 72 hours of treatment with the statins, the concentrations of FLV, ATV, SMV, and LOV required for a 50% reduction in RL activity (IC50) were calculated to be 0.90, 1.39, 1.57, and 2.16 umol/L, respectively (Fig. 2). Consistent with the results shown in Fig. 1, the anti-HCV activity of FLV (P < .01 at 0.625-5 umol/L), ATV (P < .05 at 1.25 umol/L; P < .01 at 2.5 and 5 umol/L), and SMV (P < .05 at 0.625 and 1.25 umol/L; P < .01 at 2.5 and 5 umol/L) was significantly stronger than that previously reported for LOV. In addition, the anti-HCV activity of FLV was significantly stronger than those of SMV (P < .01 at 1.25-5 umol/L) and ATV (P < .05 at 1.25 umol/L; P < .01 at 2.5 and 5 umol/L).

      Anti-HCV Activity of Statins Not Due to Their Cytotoxicity.
      Since it has been reported that the proliferation of the HCV replicon is dependent on host-cell growth,[21] it remained to be clarified whether the inhibitory effects of statins on HCV RNA replication were caused by their cytotoxicity. To examine this possibility, we investigated the cytotoxic effects of statins on OR6 cells. A comparison of cell viability in the untreated cells with that in the cells treated with each statin (5 umol/L each) showed no significant decrease in the number of cells following treatment with statins (Fig. 3A). However, it was recently reported that statins inhibited the proliferation of hepatocellular carcinoma cell lines (HuH-7 and HepG2) by inducing apoptosis and G1/S cell-cycle arrest.[22] Because that study found the IC50 of FLV in HuH-7 cells to be 10 ± 3 umol/L, we further examined the effects of FLV on the proliferation of OR6 cells by varying the concentration (up to 10 umol/L) of FLV. FLV (at least at concentrations </=10 umol/L) did not inhibit the proliferation of OR6 cells (Fig. 3B), suggesting that FLV does not induce apoptosis or G1/S cell-cycle arrest in OR6 cells, although the OR6 cell line is a HuH-7-derived cell line.[10] In summary, these results indicate that none of the statins showed any cytotoxicity to the OR6 cells at the concentrations used in our assay system. This suggests the statins possess the ability to inhibit replication of HCV RNA via specific antiviral mechanism(s).

      Anti-HCV Activity of Statins Not Due to Inhibition of RL Activity.
      We clearly showed that the anti-HCV activities of statins were not due to their cytotoxicity. However, it remained to be clarified whether the statins used in this study would directly inhibit RL activity, because we recently found that two antifungal compounds, amphotericin B (AMPH-B) and nystatin, drastically inhibited RL activity (Ikeda et al., unpublished data). To examine this possibility, we investigated the effects of statins on RL activity. The addition of the statins to cell lysates prepared from OR6 cells revealed that none of the statins (up to 10 umol/L) tested exhibited any inhibitory effect on RL activity, although AMPH-B extensively inhibited RL activity (Fig. 3C). This result excludes the possibility that the statins directly inhibit RL activity.

      Anti-HCV Activity of Statins Not Due to Inhibition of EMCV IRES Activity.
      To further exclude the possibility that the anti-HCV activities of statins were a result of the artificial assay system, we next tested the possibility that the statins inhibit EMCV IRES activity, because Core-NS5B is translated in an EMCV-IRES-dependent manner in OR6 cells. The plasmid encoding RL driven by EMCV IRES was transfected into the OR6c cells, and 24 hours after transfection the cells were treated with each statin (5 umol/L each) for 72 hours. The results revealed none of the statins exhibited any inhibitory effect, although AMPH-B drastically inhibited RL activity again (Fig. 3D). These data suggest the statins had no effect on exogenous genes EMCV IRES and RL introduced into HCV RNA.

      Statins Prevent Replication of Authentic HCV RNA.
      To obtain further evidence that the statins prevent HCV RNA replication, we prepared authentic HCV-O-derived genome-length HCV RNA possessing two adaptive mutations (HCV-O/KE/EG). One adaptive mutation, K1609E in NS3, was previously described,[9] and the other, E1202G in NS3, was found in OA cells harboring genome-length HCV-O RNA (Abe et al., in preparation). The combination of these two mutations markedly enhanced the efficiency of HCV RNA replication, and HCV proteins were continuously detected for at least 8 weeks (Abe et al., in preparation). HCV-O/dGDD, from which the GDD motif in NS5B polymerase was deleted, was used as a control. HCV-O/KE/EG and HCV-O/dGDD RNAs were transfected into OR6c cells, and the production of HCV proteins was monitored for 96 hours. The Core and NS3 in the OR6c cells transfected with HCV-O/dGDD RNA were not detected even 96 hours after transfection. However, the Core and NS3 in the OR6c cells transfected with HCV-O/KE/EG RNA were detected 24 hours after transfection, and their expression increased with time (Fig. 3E). This observation suggests that HCV-O/KE/EG RNA, without exogenous genetic factors such as EMCV IRES and RL, efficiently replicates in OR6c cells. Using such a transient HCV RNA replication system, we demonstrated that the production of Core and NS3 was markedly prevented when the OR6c cells transfected with HCV-O/KE/EG RNA were treated with FLV (5 umol/L) at 24 hours after transfection (Fig. 3E). In summary, our results indicate that the statins prevent HCV RNA replication and that their inhibitory effects are not a result of the inhibitory effect toward the exogenous genes introduced into ORN/C-5B RNA replicating in OR6 cells.

      Combination of a Statin with IFN Efficiently Enhances Anti-HCV Activity of IFN.
      IFN is the world standard of therapy for CH C, and currently its best partner is ribavirin. Because we found the statins efficiently inhibited HCV activity, we expected the statins to be candidates for use in combination with IFN. To address this point, we examined the inhibitory effects of combinations of IFN-a (0, 2, 4, and 8 IU/mL) and the statins (5 umol/L each) using the OR6 assay system. As expected, ATV, SMV, FLV, and LOV markedly enhanced the anti-HCV effect of IFN-a, although PRV did not (Fig. 4A). In combination with IFN-, FLV again was the statin that had the strongest effect. The results indicated that cotreatment was more effective than treatment with IFN- alone. We thought the mechanism underlying this phenomenon might be statin-induced enhancement of the type I IFN signaling pathway. To examine this possibility, we investigated the phosphorylation status of STAT1 after statin treatment. The results revealed that FLV did not cause phosphorylation of STAT1 in OR6c or OR6 cells, although IFN-a did so efficiently in both types of cells (Fig. 4B). In addition, we confirmed that phosphorylation of STAT2 and STAT3 in both cell types was also not induced by FLV treatment (data not shown). Furthermore, we confirmed that FLV did not affect expression of 2-5-OAS1 mRNA and that the expression level induced by IFN-a treatment was not affected by treatment with FLV (Fig. 4C). PRV, which showed no anti-HCV activity, also had no effect on the type I IFN signaling pathway. In summary, these results indicate the statin-induced enhancement of the anti-HCV action of IFN-a is not a result of induction of the type I IFN signaling pathway.

      Cotreatment of IFN- and FLV Exhibits Synergistic Inhibitory Effects on HCV RNA Replication.
      We showed that FLV was the statin tested that exhibited the strongest anti-HCV activity, not only alone, but also in combination with IFN-a. Therefore, we focused on the anti-HCV activity of FLV, minutely examining the inhibitory effects of the combination of IFN-a and FLV on genome-length HCV RNA replication. A dose-response curve of FLV was obtained for fixed concentrations of IFN- of 0, 4, 8, 16, 32, and 64 IU/mL. The results revealed the curves shifted to shift markedly to the bottom as the concentration of IFN- increased (Fig. 5A), indicating that cotreatment was drastically more effective than treatment with IFN-a alone. Furthermore, we observed that RL activity decreased to almost the background level in the OR6 reporter assay when OR6 cells were cotreated with 64 IU/mL of IFN-a and FLV at concentrations above 1.25 umol/L (Fig. 5A). Because the data in Fig. 5A indicate the possibility of a synergistic effect of the combination of IFN- and FLV, we examined whether the effect of this combination is synergistic or additive effect using an isobologram method.[23][24] The anti-HCV activities of IFN-a and FLV in combination were evaluated by the OR6 reporter assay. Dose-response inhibition of HCV RNA replication was evaluated for varying IFN-a concentrations (0-8 IU/mL) in the presence of various doses of FLV (0-7.5 mol/L). The IC90 values of IFN-a and FLV were 4.0 IU/mL and 6.7 mol/L, respectively. These data were used to generate isoboles, which demonstrated 90% inhibition of HCV RNA replication, and the synergistic anti-HCV action of IFN- and FLV was revealed by the curvilinear plots of the 90% isoboles (Fig. 5B). In conclusion, we clearly demonstrated that combination treatment of IFN-a and FLV was an overwhelmingly more effective treatment, compared with the previous results for the combination treatment of IFN-a with ribavirin.[10]

      Materials and Methods

      Cell Cultures.
      OR6 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, penicillin, streptomycin, and G418 (300 ug/mL; Geneticin, Invitrogen) and passaged twice a week at a 5:1 split ratio. OR6c cells are cured OR6 cells from which genome-length HCV RNA was eliminated by IFN-a treatment (500 IU/mL for 2 weeks) without G418, as previously described.[9] HepG2 and PH5CH8 (human immortalized hepatocytes) cells were cultured as previously described.[14][15]

      Luciferase Reporter Assay.
      For the renilla luciferase (RL) assay, 2 Ã- 10[4] OR6 cells were plated in 24-well plates at least in triplicate for each assay and were cultured for 24 hours. The cells were treated with statins for 72 hours and were harvested with Renilla lysis reagent (Promega) and subjected to the RL assay according to the manufacturer's protocol.

      Reagents.
      FLV, LOV, and PRV were purchased from Calbiochem. ATV and SMV were purchased from Astellas Pharma Inc. and Banyu Pharmaceutical Co. Ltd., respectively. Mevalonate, geranylgeraniol, and geranylgeranyl pyrophosphate were purchased from Sigma.

      Reverse-Transcriptase Polymerase Chain Reaction.
      Total RNA from the cultured cells was extracted with the RNeasy Mini Kit (Qiagen). Reverse-transcriptase polymerase chain reaction (RT-PCR) for HMG-CoA reductase, human liver-specific organic anion transporter (LST-1), 2-5-oligoadenylate synthetase 1 (2-5-OAS1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was performed by a method described previously.[16] Briefly, using cellular total RNA (2 ug), cDNA was synthesized using Superscript II with oligo dT primer. One-tenth of the synthesized cDNA was subjected to polymerase chain reaction (PCR) with the following primer pairs: HMG-CoA reductase, 5-ATGCCATCCCTGTTGGAGTG-3 and 5-TGTTCATCCCCATGGCATCCC-3; LST-1, 5-TGGCACACGTGGGTCATGTAGG-3 and 5-CACTATCTGCCCCAGCAGAAGG-3, 2-5-OAS1, 5-AGTACCTGAGAAGGCAGCTCACGA-3 and 5-ACTGGCATTCAGAGGATGGTGCAG-3; and GAPDH, 5-GACTCATGACCACAGTCCATGC-3, and 5-GAGGAGACCACCTGGTGCTCAG-3.

      Western Blot Analysis.
      Preparation of the cell lysate, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunoblotting were performed as previously described.[17] The antibodies used in this study were those against Core (Institute of Immunology, Tokyo), NS3 (Novocastra Laboratories, Newcastle upon Tyne, UK), NS5B (a generous gift from Dr. M. Kohara, Tokyo Metropolitan Institute of Medical Science), b-actin (Sigma, St. Louis, MO), STAT1 (BD Transduction Laboratories, San Diego, CA), and phospho-STAT1 (Y701; Cell Signaling Technology, Beverly, MA). Immunocomplexes on the membranes were detected by the enhanced chemiluminescence assay (Renaissance; Perkin Elmer Life Science, Wellesley, MA).

      Plasmid Construction.
      The plasmids pORN/C-5B/KE (Fig. 1A) and pHCV-O were described previously.[9] To construct the pEMCV-RL, two fragments, the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) and the RL gene, were amplified by PCR from pORN/C-5B/KE using the following primer pairs: EMCV IRES, 5-CGGGATCCGCGGGACTCGG- GGGTTCG-3 and 5-CCGCTCGAGGGTATTAT- CGTGTTTTTCAAAGG-3; and RL, 5-CCGCTCGAGATGGCTTCCAAGGTGTACGACC-3, and 5-GCTCTAGACTAGACGTTGATCCTGGCGC-3. The two fragments were ligated into the BamHI and XbaI sites in pcDNA 3.1/Zeo (Invitrogen).



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