Ethanol and reactive species increase basal sequence heterogeneity of hepatitis C virus and produce variants with reduced susceptibility to antivirals

Abstract

Hepatitis C virus (HCV) exhibits a high level of genetic variability, and variants with reduced susceptibility to antivirals can occur even before treatment begins. In addition, alcohol decreases efficacy of antiviral therapy and increases sequence heterogeneity of HCV RNA but how ethanol affects HCV sequence is unknown. Ethanol metabolism and HCV infection increase the level of reactive species that can alter cell metabolism, modify signaling, and potentially act as mutagen to the viral RNA. Therefore, we investigated whether ethanol and reactive species affected the basal sequence variability of HCV RNA in hepatocytes. Human hepatoma cells supporting a continuous replication of genotype 1b HCV RNA (Con1, AJ242652) were exposed to ethanol, acetaldehyde, hydrogen peroxide, or L-buthionine-S,R-sulfoximine (BSO) that decreases intracellular glutathione as seen in patients. Then, NS5A region was sequenced and compared with genotype 1b HCV sequences in the database. Ethanol and BSO elevated nucleotide and amino acid substitution rates of HCV RNA by 4-18 folds within 48 hrs which were accompanied by oxidative RNA damage. Iron chelator and glutathione ester decreased both RNA damage and mutation rates. Furthermore, infectious HCV and HCV core gene were sufficient to induce oxidative RNA damage even in the absence of ethanol or BSO. Interestingly, the dn/ds ratio and percentage of sites undergoing positive selection increased with ethanol and BSO, resulting in an increased detection of NS5A variants with reduced susceptibility to interferon alpha, cyclosporine, and ribavirin and others implicated in immune tolerance and modulation of viral replication. Therefore, alcohol is likely to synergize with virus-induced oxidative/nitrosative stress to modulate the basal mutation rate of HCV. Positive selection induced by alcohol and reactive species may contribute to antiviral resistance.

Figure 1. Ethanol, BSO, and oxidative RNA damage.

(A) Con1 replicon cells were incubated with 0.5% (v/v) ethanol, 20 µM BSO, or 250 µM NiCl₂ daily for 48 hr and analyzed for 8-OHG by ELISA. (B) Con1 replicon cells were incubated with 0.1% (v/v) ethanol, 20 µM BSO, or 50 µM NiCl₂ daily for 2 weeks and analyzed for 8-OHG by ELISA. Data were expressed as fold increase from the controls (0.134±0.013 ng/ml for panel A and 0.418±0.109 ng/ml for panel B). * Indicates statistically significant difference from controls (n = 4 in panel A, n = 6 in panel B).

Figure 2. Effects of dipyridyl and GSH ester on the oxidative RNA damage and HCV mutation rate.

(A-C) Con1 replicon cells were incubated with 0.5% ethanol or 20 µM BSO daily for 48 hr±10 µM dipyridyl or 2 mM GSH ester and analyzed for 8-OHG by ELISA (A) or sequenced and analyzed for nucleotide (B) and amino acid substitution rates (C). Data were expressed as fold increase from the control where the control values were 0.122±0.027 ng/ml (n = 6) for panel A, 2.88±0.77×10^-4 nt. changes per site for panel B, and 2.80±0.61×10^-4 amino acid changes per site for panel C. Letter a indicates statistically significant difference from no inhibitor control. Letters b and c represent statistically significant change from ethanol (no inhibitor) and BSO (no inhibitor), respectively. (D–E) Huh7 cells were transfected with JFH1 RNA and incubated with (D) 0.5% (v/v) ethanol or 20 µM BSO for 48 hrs, or (E) 0.1% (v/v) ethanol or 20 µM BSO±5 µM dipyridyl or 1 mM GSH ester daily for 2 weeks. Then, the samples were analyzed for 8-OHG by ELISA. Data were expressed as fold increase from the control where the control values were 0.106±0.012 and 0.291±0.054 ng/ml for the 48 hr and 2 week time points, respectively. Letters a and b indicate statistically significant difference from the –HCV control and +HCV control, respectively, and c indicates statistically significant difference from respective no inhibitor controls (control no inhibitor, ethanol no inhibitor, and BSO no inhibitor) (n = 4).

Figure 3. Synonymous and nonsynonymous amino acid substitutions and positive selection in the NS5A coding region.

(A) Percentage of sites undergoing positive selection (dn/ds ratio>1) in control, ethanol, and BSO groups. Letter a indicates statistically significant difference from no inhibitor control; b indicates statistically significant difference from respective no inhibitor controls (control no inhibitor, ethanol no inhibitor, and BSO no inhibitor). (B – D) Cumulative synonymous and non-synonymous mutations in control (B), ethanol (C), and BSO (D) groups were plotted against codons via SNAP. Numbers represent NS5A codon number.

Figure 4. Amino acid substitutions in the NS5A coding region.

(A) Structural and functional domains of HCV NS5A . Numbers represent NS5A condon number. (B) Con1(wt) and Con1(Y321C) replicon cells were treated with 1 mM cyclosporine (CsA) for 48 hrs. Total RNA was extracted and HCV RNA was determined. * Indicates a statistically significant difference. (C-D) Location of amino acid substitutions involving serines (C), threonines (D), and tyrosines (D). Putative basal phosphorylation regions are shaded in (A, C, D).