J Bacteriol Virol.  2011 Jun;41(2):99-108. 10.4167/jbv.2011.41.2.99.

HMG CoA Reductase Inhibitors Inhibit HCV RNA Replication of HCV Genotype 1b but Not 2a

• Chang KS
• Jo HJ

• KMID: 2168627
• DOI: http://doi.org/10.4167/jbv.2011.41.2.99

Abstract

Replication of hepatitis C virus (HCV) is regulated by statin, one of 3-hydroxy-3-methylglutaryl CoA reducatase (HMG CoA reductase) inhibitors that block mevalonate pathway and cholesterol biosyntheis, which has been used usefully for health improvement and disease control in clinic. In order to know which statin can be used to inhibit HCV replication, we examined the effects of HCV genotype 1b replication by 6 kinds of statins with different structure. We treated six statins to HCV genotype 1b replicon cell. Atorvastatin, simvastatin, fluvastatin, mevastatin, and lovastatin inhibited HCV RNA replication and HCV protein expression in HCV genotype 1b replicon cells, though pravastatin did not affect HCV replication. In order to know whether inhibition of HCV replication by statin is depended on HCV genotype, we treated the statins to HCV genotype 2a producing cells, and investigated HCV RNA replication and HCV protein expression. HCV RNA replication and protein expression was not affected in HCV genotype 2a producing cells by treatment of statins and cholesterol inhibitor. These results suggest that HMG-CoA reductase and cholesterol inhibitors might be used depending on HCV genotype. In addition, inhibition of HCV genotype 1b replication by statins has been depended on structure of various statins which should be seriously selected for HCV clinic. In future, we will study on inhibition of another HCV genotype replication by HMG-CoA reductase and cholesterol inhibitors.

Keyword

Hepatitis C virus; HMG CoA reductase inhibitor; Statin; Mavalonate pathway; Genotypes

MeSH Terms

Acyl Coenzyme A
Anticholesteremic Agents
Atorvastatin Calcium
Cholesterol
Fatty Acids, Monounsaturated
Genotype
Hepacivirus
Heptanoic Acids
Hydroxymethylglutaryl CoA Reductases
Hydroxymethylglutaryl-CoA Reductase Inhibitors
Indoles
Lovastatin
Mevalonic Acid
Oxidoreductases
Pravastatin
Pyrroles
Replicon
RNA
Simvastatin
Acyl Coenzyme A
Anticholesteremic Agents
Cholesterol
Fatty Acids, Monounsaturated
Heptanoic Acids
Hydroxymethylglutaryl CoA Reductases
Hydroxymethylglutaryl-CoA Reductase Inhibitors
Indoles
Lovastatin
Mevalonic Acid
Oxidoreductases
Pravastatin
Pyrroles
RNA
Simvastatin

Figure

• 

  Figure 1. Suppression of HCV genotype 1b RNA replication and HCV protein expression by lovastatin (50 μM), one of HMG-CoA reductase inhibitor, in HCV genotype 1b replicon S1179I #NS3-3 depending on time. Hour is numbered as indicated on the top. Total protein from a clarified lysate (25 μg) was separated by SDS-PAGE and subjected to immunoblot analysis using antibodies directed against NS5A and β-actin. –, not treated; +, treated.

• 

  Figure 2. Inhibition of lovastatin to HCV RNA replication and in HCV genotype 1b replicon S1179I #NS3-3 at 72 hrs with increasing concentration. (A) Total protein from a clarified lysate (25 μg) was separated by SDS-PAGE and subjected to immunoblot analysis using antibodies directed against NS5A and β-actin, (B) HCV genotype 1b replicon cell is treated with various concentrations (0, 0.4, 2, 10, and 50 μM) of lovastatin and incubated for 3 days (Lane 2~6, respectively). RNA was extracted using Trizol solution and chloroform. Positive-stranded HCV RNA in each fraction was determined using a negative-sense 3'UTR RNA probe. RNA products were analyzed on a 6% polyacrylamide/7.7 M urea gel and visualized by autoradiography. H, Huh7.5 cell; P, probe.

• 

  Figure 3. Effects of mevalonate and geranylgeranyl to replication in HCV genotype 1b replicon S1179I #NS3-3 at 72 hrs treated with 50 μM of lovastatin. Total protein from a clarified lysate (25 μg) was separated by SDS-PAGE and subjected to immunoblot analysis using antibodies directed against NS5A and β-actin.

• 

  Figure 4. Effects of various statins to HCV replication in HCV genotype 1b replicon S1179I #NS3-3 at 72 hrs by Western blot assay. Total protein from a clarified lysate (25 μg) was separated by SDS-PAGE and subjected to immunoblot analysis using antibodies directed against NS5A and β-actin. ∗cytotoxic effects.

• 

  Figure 5. Effects of various statins in HCV genotype 1b replicon S1179I #NS3-3 at 72 hrs by RNase protection assay. HCV genotype 1b replicon cell is treated with various doses (0, 2, 4, 6, 8 and 10 μM) of lovastatin and incubated for 3 days (Lane 2~6, respectively). RNA was extracted using Trizol solution and chloroform. Positive-stranded HCV RNA in each fraction was determined using a negative-sense 3'UTR RNA probe. RNA products were analyzed on a 6% polyacrylamide/7.7 M urea gel and visualized by autoradiography. (D) The intensities of images on gel were quantified using Phosphor imager (Molecular Device). Each experiment was repeated at least three times, and a representative result is shown here. R, RNA marker; P, probe; C, positive control.

• 

  Figure 6. Effects of various statins to HCV RNA replication in HCV genotype 2a producing cells at 72 hrs by different dose. (A) Total protein from a clarified lysate (25 μg) was separated by SDS-PAGE and subjected to immunoblot analysis using antibodies directed against NS3 and. β-actin, (B)-(D) HCV genotype 1b replicon cell is treated with different doses (0, 2, 4, 6, 8 and 10 μM) of six statins and incubated for 3 days. RNA was extracted using Trizol solution and chloroform. Positive-stranded HCV RNA in each fraction was determined using a negative-sense 3'UTR RNA probe. RNA products were analyzed on a 6% polyacrylamide/7.7 M urea gel and visualized by autoradiography. R, RNA marker; P, probe; NT, not treated (negative control).

• 

  Figure 7. Effects of various statins to HCV protein expression in HCV genotype 2a producing cell lines at 72 hrs by different concentration. Total protein from a clarified lysate (25 μg) was separated by SDS-PAGE and subjected to immunoblot analysis using antibodies directed against NS3 and β-actin. (A) NS3 protein expression in HCV genetype producing cell treated with 50 μM of six statins, (B) Effects to HCV replication in HCV producing cell treated with 2 and 10 μM of fluvastatin and simvastatin, (C) Effects to HCV infection in Huh7.5 infected with the supernatant from HCV producing cell treated with fluvastatin and simvastatin.

• 

  Figure 8. Effects of HCV replication in HCV genotype 1b replicon S1179I #NS3-3 (A) and HCV genotype 2a replicon (B) treated with different concentration of MβCD at 72 hrs. Total protein from a clarified lysate (25 μg) was separated by SDS-PAGE and subjected to immunoblot analysis using antibodies directed against NS5A (A), NS3 (B) and β-actin.

• 

  Figure 9. Effect of MβCD to HCV RNA replication in HCV genotype 2a producing cell lines at 72 hrs by different concentration. Total protein from a clarified lysate (25 μg) was separated by SDS-PAGE and subjected to immunoblot analysis using antibodies directed against NS3 and β-actin. (A) Effects to HCV replication in HCV producing cell treated with MβCD. (B). Effects to HCV infection in Huh7.5 infected with the sup. AI, treatment after infection; BI, treatment before infectio; NI, not infected; HS, supernatant from Huh7.5 cell. ∗cytotoxic effects.

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