S-adenosyl-L-methionine modifies antioxidant-enzymes, glutathione-biosynthesis and methionine adenosyltransferases-1/2 in hepatitis C virus-expressing cells

Abstract

AIM: To elucidate the mechanism(s) by which S-adenosyl-L-methionine (SAM) decreases hepatitis C virus (HCV) expression.

METHODS: We examined the effects of SAM on viral expression using an HCV subgenomic replicon cell culture system. Huh7 HCV-replicon cells were treated with 1 mmol/L SAM for different times (24-72 h), then total RNA and proteins were isolated. cDNA was synthesized and real time-PCR was achieved to quantify HCV-RNA, superoxide dismutase 1 and 2 (SOD-1, SOD-2) catalase, thioredoxin 1, methionine adenosyltransferase 1A and 2A (MAT1A, MAT2A) expression, and GAPDH and RPS18 as endogenous genes. Expression of cellular and viral protein was evaluated by western-blot analysis using antibodies vs HCV-NS5A, SOD-1, SOD-2, catalase, thioredoxin-1, MAT1A, MAT2A, GAPDH and actin. Total glutathione levels were measured at different times by Ellman’s recycling method (0-24 h). Reactive oxidative species (ROS) levels were quantified by the dichlorofluorescein assay (0-48 h); Pyrrolidin dithiocarbamate (PDTC) was tested as an antioxidant control and H₂O₂ as a positive oxidant agent.

RESULTS: SAM exposition decreased HCV-RNA levels 50%-70% compared to non-treated controls (24-72 h). SAM induced a synergic antiviral effect with standard IFN treatment but it was independent of IFN signaling. In addition, 1 mmol/L SAM exposition did not modify viral RNA stability, but it needs cellular translation machinery in order to decrease HCV expression. Total glutathione levels increased upon SAM treatment in HCV-replicon cells. Transcriptional antioxidant enzyme expression (SOD-1, SOD-2 and thioredoxin-1) was increased at different times but interestingly, there was no significant change in ROS levels upon SAM treatment, contrary to what was detected with PDTC treatment, where an average 40% reduction was observed in exposed cells. There was a turnover from MAT1A/MAT2A, since MAT1A expression was increased (2.5 fold-times at 48 h) and MAT2A was diminished (from 24 h) upon SAM treatment at both the transcriptional and translational level.

CONCLUSION: A likely mechanism(s) by which SAM diminish HCV expression could involve modulating antioxidant enzymes, restoring biosynthesis of glutathione and switching MAT1/MAT2 turnover in HCV expressing cells.

Keywords: Hepatitis C virus, S-adenosyl-L-methionine, Superoxide dismutase 1, Superoxide dismutase 2, Replication, Hepatitis C virus-RNA, NS5A, Oxidative stress, Antioxidants, Viral proteins, Reactive oxygen species, Pyrrolidine dithiocarbamate

Core tip: S-adenosyl-L-methionine (SAM) downregulates hepatitis C virus (HCV) expression by unknown mechanisms. We evaluated the effects of SAM on viral expression using an HCV subgenomic replicon cell culture system. We observed that SAM induces a synergic antiviral effect with standard interferon (IFN) treatment independently of IFN signaling pathways; it does not modify viral RNA stability, but it needs cellular translation machinery in order to decrease HCV expression. In addition, results demonstrated that a likely mechanism(s) by which SAM decreases HCV expression could involve modulating antioxidant enzyme systems, restoring biosynthesis of glutathione and switching methionine adenosyltransferase 1 (MAT1)/MAT2 turnover in HCV expressing cells.