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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. c DNA 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-NS5 A, SOD-1, SOD-2, catalase, thioredoxin-1, MAT1 A, MAT2 A, 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/MAT2 A, since MAT1 A expression was increased(2.5 fold-times at 48 h) and MAT2 A 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.
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. C DNA 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- using antibodies vs HCV-NS5 A, SOD-1, SOD-2, catalase, thioredoxin-1, MAT1 A, MAT2 A, 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 dith Results: SAM exposition decreased HCV-RNA levels 50% -70% compared to non-treated controls (24-72 h). SAM induced a synergic antiviral (PDTC) was tested as an antioxidant control and H2O2 as a positive oxidant agent. 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 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 the exposed cells. There was a turnover from MAT1A / MAT2 A, since MAT1 A expression was increased (2.5 fold-times at 48 h) and MAT2 A was diminished (from 24 h) upon SAM treatment aCONCLUSION: 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.