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Regulation of the hepatitis C virus RNA replicase by endogenous lipid peroxidation.

Yamane D, McGivern DR, Wauthier E, Yi M, Madden VJ, Welsch C, Antes I, Wen Y, Chugh PE, McGee CE, Widman DG, Misumi I, Bandyopadhyay S, Kim S, Shimakami T, Oikawa T, Whitmire JK, Heise MT, Dittmer DP, Kao CC, Pitson SM, Merrill AH, Reid LM, Lemon SM - Nat. Med. (2014)

Bottom Line: Endogenous oxidative membrane damage lowers the 50% effective concentration of direct-acting antivirals in vitro, suggesting critical regulation of the conformation of the NS3-4A protease and the NS5B polymerase, membrane-bound HCV replicase components.Resistance to lipid peroxidation maps genetically to transmembrane and membrane-proximal residues within these proteins and is essential for robust replication in cell culture, as exemplified by the atypical JFH1 strain of HCV.Thus, the typical, wild-type HCV replicase is uniquely regulated by lipid peroxidation, providing a mechanism for attenuating replication in stressed tissue and possibly facilitating long-term viral persistence.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Medicine, Division of Infectious Diseases, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA. [2] Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

ABSTRACT
Oxidative tissue injury often accompanies viral infection, yet there is little understanding of how it influences virus replication. We show that multiple hepatitis C virus (HCV) genotypes are exquisitely sensitive to oxidative membrane damage, a property distinguishing them from other pathogenic RNA viruses. Lipid peroxidation, regulated in part through sphingosine kinase-2, severely restricts HCV replication in Huh-7 cells and primary human hepatoblasts. Endogenous oxidative membrane damage lowers the 50% effective concentration of direct-acting antivirals in vitro, suggesting critical regulation of the conformation of the NS3-4A protease and the NS5B polymerase, membrane-bound HCV replicase components. Resistance to lipid peroxidation maps genetically to transmembrane and membrane-proximal residues within these proteins and is essential for robust replication in cell culture, as exemplified by the atypical JFH1 strain of HCV. Thus, the typical, wild-type HCV replicase is uniquely regulated by lipid peroxidation, providing a mechanism for attenuating replication in stressed tissue and possibly facilitating long-term viral persistence.

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Differential regulation of HCV strains by SPHK2-mediated lipid peroxidation. (a) Dose-dependent effects of PUFAs on H77S.3/GLuc and HJ3-5/GLuc RNAs in Huh-7.5 cells. Data shown represent percent GLuc activity secreted between 48–72 h relative to DMSO control. (b) Growth kinetics of H77S.3/GLuc and HJ3-5/GLuc RNAs in the presence of 50 μM PUFAs. Data shown are mean ± s.e.m. of GLuc activity in supernatant fluids of two replicate cultures. (c) Cells transfected with HCV RNAs encoding GLuc were treated with DMSO, 100 μM LA or 100 μM LA plus 1 μM SKI. Data shown represent percent GLuc activity secreted between 48–72 h relative to DMSO control. L.O.D. = limit of detection. (d) Effect of 1 μM SKI, 1 μM VE, 100 μM CoQ10 or 50 μM ARA or DHA, on intracellular malondialdehyde (MDA) abundance in cells transfected with the indicated HCV/GLuc RNAs at 72 h. MDA was significantly increased by PUFAs and reduced by SKI or lipophilic antioxidants (P < 0.01). (e) Analysis of 8-isoprostane abundance in cells electroporated with the indicated HCV RNAs and grown in the presence of 1 μM SKI or VE, or 50 μM LA with or without 1 μM SKI and VE for 48 h. (f) Effect of siRNA targeting SPHK isoforms (see Fig. 1f) on MDA accumulation after treatment with increasing concentrations of LA (6.25, 12.5, 25, 50, 100 μM) for 24 h (left panel). MDA levels in Huh-7.5 cells treated with increasing concentrations of LA in the presence of DMSO or 1 μM SKI (right panel). (g) Effects of increasing concentrations of VE (left), 1 μM VE alone, or 1 μM VE plus 1 μM SKI (right) on replication of H77S.3/GLuc and HJ3-5/GLuc RNAs. Data shown represent GLuc secreted between 48–72 h relative to DMSO control. (h) GLuc secretion from Huh-7.5 cells transfected as in a and treated with 10 μM CuOH with or without 10 μM VE. (i) Influence of SKI or VE (each 1 μM) on replication of H77S.3 and HJ3-5 viruses expressing GLuc in cells cultured in the presence or absence of 10% FBS. Medium containing 10% FBS was replaced with FBS-free or 10% FBS media containing SKI, VE or DMSO 6 h after RNA transfection. Data shown represent mean GLuc activity ± s.e.m. from two (a–f,i) or three (g,h) independent experiments. *P < 0.05, **P < 0.01.
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Figure 2: Differential regulation of HCV strains by SPHK2-mediated lipid peroxidation. (a) Dose-dependent effects of PUFAs on H77S.3/GLuc and HJ3-5/GLuc RNAs in Huh-7.5 cells. Data shown represent percent GLuc activity secreted between 48–72 h relative to DMSO control. (b) Growth kinetics of H77S.3/GLuc and HJ3-5/GLuc RNAs in the presence of 50 μM PUFAs. Data shown are mean ± s.e.m. of GLuc activity in supernatant fluids of two replicate cultures. (c) Cells transfected with HCV RNAs encoding GLuc were treated with DMSO, 100 μM LA or 100 μM LA plus 1 μM SKI. Data shown represent percent GLuc activity secreted between 48–72 h relative to DMSO control. L.O.D. = limit of detection. (d) Effect of 1 μM SKI, 1 μM VE, 100 μM CoQ10 or 50 μM ARA or DHA, on intracellular malondialdehyde (MDA) abundance in cells transfected with the indicated HCV/GLuc RNAs at 72 h. MDA was significantly increased by PUFAs and reduced by SKI or lipophilic antioxidants (P < 0.01). (e) Analysis of 8-isoprostane abundance in cells electroporated with the indicated HCV RNAs and grown in the presence of 1 μM SKI or VE, or 50 μM LA with or without 1 μM SKI and VE for 48 h. (f) Effect of siRNA targeting SPHK isoforms (see Fig. 1f) on MDA accumulation after treatment with increasing concentrations of LA (6.25, 12.5, 25, 50, 100 μM) for 24 h (left panel). MDA levels in Huh-7.5 cells treated with increasing concentrations of LA in the presence of DMSO or 1 μM SKI (right panel). (g) Effects of increasing concentrations of VE (left), 1 μM VE alone, or 1 μM VE plus 1 μM SKI (right) on replication of H77S.3/GLuc and HJ3-5/GLuc RNAs. Data shown represent GLuc secreted between 48–72 h relative to DMSO control. (h) GLuc secretion from Huh-7.5 cells transfected as in a and treated with 10 μM CuOH with or without 10 μM VE. (i) Influence of SKI or VE (each 1 μM) on replication of H77S.3 and HJ3-5 viruses expressing GLuc in cells cultured in the presence or absence of 10% FBS. Medium containing 10% FBS was replaced with FBS-free or 10% FBS media containing SKI, VE or DMSO 6 h after RNA transfection. Data shown represent mean GLuc activity ± s.e.m. from two (a–f,i) or three (g,h) independent experiments. *P < 0.05, **P < 0.01.

Mentions: Polyunsaturated fatty acids (PUFAs) inhibit replication of genotype 1b HCV replicons by inducing lipid peroxidation8,29. Surprisingly, although PUFAs such as arachidonic acid (ARA), docosahexaenoic acid (DHA) or linoleic acid (LA) potently suppressed H77S.3/GLuc replication without affecting cell viability, HJ3-5/GLuc was highly resistant (Fig. 2a,b and Supplementary Fig. 5a,b). Thus PUFAs appear to phenocopy the effect of SPHK2 on HCV replication, suggesting that SPHK2 promotes lipid peroxidation. Consistent with this hypothesis, SKI completely abolished the inhibitory effects of PUFAs on H77S.3/GLuc and N.2/GLuc (Fig. 2c). SKI also lowered the abundance of malondialdehyde (MDA), a secondary product of peroxidative degradation (Fig. 2d). SKI was as effective as the lipophilic antioxidants vitamin E (VE) and coenzyme Q10 (CoQ10) in reducing MDA abundance in HCV-infected cells, and like VE and CoQ10 prevented large increases in lipid peroxidation induced by PUFAs (Fig. 2d,e). SKI also reduced both endogenous and PUFA-induced 8-isoprostane, an alternative biomarker of lipid peroxidation (Fig. 2e). Conversely, RNAi-mediated depletion of SPHK1 increased MDA abundance, while SPHK2 knockdown, like SKI, reduced it (Fig. 2f). Thus, the contrasting effects of SPHK1 and SPHK2 on HCV replication can be explained by opposing actions on peroxidation of endogenous PUFA. These results identify SPHK2 as an important mediator of lipid peroxidation.


Regulation of the hepatitis C virus RNA replicase by endogenous lipid peroxidation.

Yamane D, McGivern DR, Wauthier E, Yi M, Madden VJ, Welsch C, Antes I, Wen Y, Chugh PE, McGee CE, Widman DG, Misumi I, Bandyopadhyay S, Kim S, Shimakami T, Oikawa T, Whitmire JK, Heise MT, Dittmer DP, Kao CC, Pitson SM, Merrill AH, Reid LM, Lemon SM - Nat. Med. (2014)

Differential regulation of HCV strains by SPHK2-mediated lipid peroxidation. (a) Dose-dependent effects of PUFAs on H77S.3/GLuc and HJ3-5/GLuc RNAs in Huh-7.5 cells. Data shown represent percent GLuc activity secreted between 48–72 h relative to DMSO control. (b) Growth kinetics of H77S.3/GLuc and HJ3-5/GLuc RNAs in the presence of 50 μM PUFAs. Data shown are mean ± s.e.m. of GLuc activity in supernatant fluids of two replicate cultures. (c) Cells transfected with HCV RNAs encoding GLuc were treated with DMSO, 100 μM LA or 100 μM LA plus 1 μM SKI. Data shown represent percent GLuc activity secreted between 48–72 h relative to DMSO control. L.O.D. = limit of detection. (d) Effect of 1 μM SKI, 1 μM VE, 100 μM CoQ10 or 50 μM ARA or DHA, on intracellular malondialdehyde (MDA) abundance in cells transfected with the indicated HCV/GLuc RNAs at 72 h. MDA was significantly increased by PUFAs and reduced by SKI or lipophilic antioxidants (P < 0.01). (e) Analysis of 8-isoprostane abundance in cells electroporated with the indicated HCV RNAs and grown in the presence of 1 μM SKI or VE, or 50 μM LA with or without 1 μM SKI and VE for 48 h. (f) Effect of siRNA targeting SPHK isoforms (see Fig. 1f) on MDA accumulation after treatment with increasing concentrations of LA (6.25, 12.5, 25, 50, 100 μM) for 24 h (left panel). MDA levels in Huh-7.5 cells treated with increasing concentrations of LA in the presence of DMSO or 1 μM SKI (right panel). (g) Effects of increasing concentrations of VE (left), 1 μM VE alone, or 1 μM VE plus 1 μM SKI (right) on replication of H77S.3/GLuc and HJ3-5/GLuc RNAs. Data shown represent GLuc secreted between 48–72 h relative to DMSO control. (h) GLuc secretion from Huh-7.5 cells transfected as in a and treated with 10 μM CuOH with or without 10 μM VE. (i) Influence of SKI or VE (each 1 μM) on replication of H77S.3 and HJ3-5 viruses expressing GLuc in cells cultured in the presence or absence of 10% FBS. Medium containing 10% FBS was replaced with FBS-free or 10% FBS media containing SKI, VE or DMSO 6 h after RNA transfection. Data shown represent mean GLuc activity ± s.e.m. from two (a–f,i) or three (g,h) independent experiments. *P < 0.05, **P < 0.01.
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Figure 2: Differential regulation of HCV strains by SPHK2-mediated lipid peroxidation. (a) Dose-dependent effects of PUFAs on H77S.3/GLuc and HJ3-5/GLuc RNAs in Huh-7.5 cells. Data shown represent percent GLuc activity secreted between 48–72 h relative to DMSO control. (b) Growth kinetics of H77S.3/GLuc and HJ3-5/GLuc RNAs in the presence of 50 μM PUFAs. Data shown are mean ± s.e.m. of GLuc activity in supernatant fluids of two replicate cultures. (c) Cells transfected with HCV RNAs encoding GLuc were treated with DMSO, 100 μM LA or 100 μM LA plus 1 μM SKI. Data shown represent percent GLuc activity secreted between 48–72 h relative to DMSO control. L.O.D. = limit of detection. (d) Effect of 1 μM SKI, 1 μM VE, 100 μM CoQ10 or 50 μM ARA or DHA, on intracellular malondialdehyde (MDA) abundance in cells transfected with the indicated HCV/GLuc RNAs at 72 h. MDA was significantly increased by PUFAs and reduced by SKI or lipophilic antioxidants (P < 0.01). (e) Analysis of 8-isoprostane abundance in cells electroporated with the indicated HCV RNAs and grown in the presence of 1 μM SKI or VE, or 50 μM LA with or without 1 μM SKI and VE for 48 h. (f) Effect of siRNA targeting SPHK isoforms (see Fig. 1f) on MDA accumulation after treatment with increasing concentrations of LA (6.25, 12.5, 25, 50, 100 μM) for 24 h (left panel). MDA levels in Huh-7.5 cells treated with increasing concentrations of LA in the presence of DMSO or 1 μM SKI (right panel). (g) Effects of increasing concentrations of VE (left), 1 μM VE alone, or 1 μM VE plus 1 μM SKI (right) on replication of H77S.3/GLuc and HJ3-5/GLuc RNAs. Data shown represent GLuc secreted between 48–72 h relative to DMSO control. (h) GLuc secretion from Huh-7.5 cells transfected as in a and treated with 10 μM CuOH with or without 10 μM VE. (i) Influence of SKI or VE (each 1 μM) on replication of H77S.3 and HJ3-5 viruses expressing GLuc in cells cultured in the presence or absence of 10% FBS. Medium containing 10% FBS was replaced with FBS-free or 10% FBS media containing SKI, VE or DMSO 6 h after RNA transfection. Data shown represent mean GLuc activity ± s.e.m. from two (a–f,i) or three (g,h) independent experiments. *P < 0.05, **P < 0.01.
Mentions: Polyunsaturated fatty acids (PUFAs) inhibit replication of genotype 1b HCV replicons by inducing lipid peroxidation8,29. Surprisingly, although PUFAs such as arachidonic acid (ARA), docosahexaenoic acid (DHA) or linoleic acid (LA) potently suppressed H77S.3/GLuc replication without affecting cell viability, HJ3-5/GLuc was highly resistant (Fig. 2a,b and Supplementary Fig. 5a,b). Thus PUFAs appear to phenocopy the effect of SPHK2 on HCV replication, suggesting that SPHK2 promotes lipid peroxidation. Consistent with this hypothesis, SKI completely abolished the inhibitory effects of PUFAs on H77S.3/GLuc and N.2/GLuc (Fig. 2c). SKI also lowered the abundance of malondialdehyde (MDA), a secondary product of peroxidative degradation (Fig. 2d). SKI was as effective as the lipophilic antioxidants vitamin E (VE) and coenzyme Q10 (CoQ10) in reducing MDA abundance in HCV-infected cells, and like VE and CoQ10 prevented large increases in lipid peroxidation induced by PUFAs (Fig. 2d,e). SKI also reduced both endogenous and PUFA-induced 8-isoprostane, an alternative biomarker of lipid peroxidation (Fig. 2e). Conversely, RNAi-mediated depletion of SPHK1 increased MDA abundance, while SPHK2 knockdown, like SKI, reduced it (Fig. 2f). Thus, the contrasting effects of SPHK1 and SPHK2 on HCV replication can be explained by opposing actions on peroxidation of endogenous PUFA. These results identify SPHK2 as an important mediator of lipid peroxidation.

Bottom Line: Endogenous oxidative membrane damage lowers the 50% effective concentration of direct-acting antivirals in vitro, suggesting critical regulation of the conformation of the NS3-4A protease and the NS5B polymerase, membrane-bound HCV replicase components.Resistance to lipid peroxidation maps genetically to transmembrane and membrane-proximal residues within these proteins and is essential for robust replication in cell culture, as exemplified by the atypical JFH1 strain of HCV.Thus, the typical, wild-type HCV replicase is uniquely regulated by lipid peroxidation, providing a mechanism for attenuating replication in stressed tissue and possibly facilitating long-term viral persistence.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Medicine, Division of Infectious Diseases, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA. [2] Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

ABSTRACT
Oxidative tissue injury often accompanies viral infection, yet there is little understanding of how it influences virus replication. We show that multiple hepatitis C virus (HCV) genotypes are exquisitely sensitive to oxidative membrane damage, a property distinguishing them from other pathogenic RNA viruses. Lipid peroxidation, regulated in part through sphingosine kinase-2, severely restricts HCV replication in Huh-7 cells and primary human hepatoblasts. Endogenous oxidative membrane damage lowers the 50% effective concentration of direct-acting antivirals in vitro, suggesting critical regulation of the conformation of the NS3-4A protease and the NS5B polymerase, membrane-bound HCV replicase components. Resistance to lipid peroxidation maps genetically to transmembrane and membrane-proximal residues within these proteins and is essential for robust replication in cell culture, as exemplified by the atypical JFH1 strain of HCV. Thus, the typical, wild-type HCV replicase is uniquely regulated by lipid peroxidation, providing a mechanism for attenuating replication in stressed tissue and possibly facilitating long-term viral persistence.

Show MeSH
Related in: MedlinePlus