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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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SKI enhances genotype 1 HCV replication while suppressing JFH1-based viruses by inhibiting type 2 sphingosine kinase (SPHK2). (a) HCV RNA genomes that express Gaussia Luciferase (GLuc) fused to foot-and-mouth disease virus 2A autoprotease as part of the HCV polyprotein. Arrowheads indicate cell culture-adaptive mutations. (b) (left) Dose-response effects of SKI on replication of H77S.3/GLuc (red) or HJ3-5/GLuc (blue) RNAs in Huh-7.5 cells. (right) Effect of 1 μM SKI on replication of H77S.3 (red) or HJ3-5 (blue) RNAs. Data shown represent relative amounts of GLuc secreted between 48–72 h (left) or intracellular RNA levels at 72 h (right). (c) Effect of 1 μM SKI on GLuc activities of the indicated viruses are presented as fold-change from baseline (6 h). (d) Flow cytometric analysis of NS5A expression in Huh-7.5 cells electroporated with H77S.3 or HJ3-5 RNA and treated with 1 μM SKI or DMSO. (e–f) Effect of siRNAs targeting SPHK isoforms or non-targeting control siRNA on replication of different HCV RNAs (e) and abundance of each SPHK isoform (f). Results represent the mean ± s.e.m. from two independent (b,c,d) or triplicate (e) experiments. *P < 0.05, **P < 0.01.
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Figure 1: SKI enhances genotype 1 HCV replication while suppressing JFH1-based viruses by inhibiting type 2 sphingosine kinase (SPHK2). (a) HCV RNA genomes that express Gaussia Luciferase (GLuc) fused to foot-and-mouth disease virus 2A autoprotease as part of the HCV polyprotein. Arrowheads indicate cell culture-adaptive mutations. (b) (left) Dose-response effects of SKI on replication of H77S.3/GLuc (red) or HJ3-5/GLuc (blue) RNAs in Huh-7.5 cells. (right) Effect of 1 μM SKI on replication of H77S.3 (red) or HJ3-5 (blue) RNAs. Data shown represent relative amounts of GLuc secreted between 48–72 h (left) or intracellular RNA levels at 72 h (right). (c) Effect of 1 μM SKI on GLuc activities of the indicated viruses are presented as fold-change from baseline (6 h). (d) Flow cytometric analysis of NS5A expression in Huh-7.5 cells electroporated with H77S.3 or HJ3-5 RNA and treated with 1 μM SKI or DMSO. (e–f) Effect of siRNAs targeting SPHK isoforms or non-targeting control siRNA on replication of different HCV RNAs (e) and abundance of each SPHK isoform (f). Results represent the mean ± s.e.m. from two independent (b,c,d) or triplicate (e) experiments. *P < 0.05, **P < 0.01.

Mentions: We determined how inhibitors of sphingolipid converting enzymes influence replication of two cell culture-adapted HCVs: H77S.3/GLuc, a genotype 1a virus, and HJ3-5/GLuc, an inter-genotypic chimera expressing the genotype 2a JFH1 replicase (Fig. 1a). To assess replication, we monitored Gaussia princeps luciferase (GLuc) produced from in-frame insertions in each viral genome after transfecting Huh-7.5 cells with synthetic RNA27. Surprisingly, these viral RNAs demonstrated contrary responses to many inhibitors, including most notably SKI, a sphingosine kinase (SPHK) inhibitor (Fig. 1b and Supplementary Fig. 1a,b). We also observed contrasting responses to sphingolipid supplementation (Supplementary Fig. 1c). SKI (1 μM) enhanced replication of H77S.3/GLuc as well as N.2/GLuc, a cell culture-adapted genotype 1b virus (Fig. 1a), by 3–6 fold, while suppressing replication of HJ3-5 (Fig. 1b,c). These effects were evident within 48 h of exposure. SKI also enhanced H77S.3 protein expression 10-fold, while slightly suppressing HJ3-5 protein expression (Fig. 1d). Thus, changes in the cellular environment induced by SKI favor H77S.3/GLuc and N.2/GLuc replication, while inhibiting HJ3-5/GLuc. These effects are not due to altered cell proliferation or viral RNA translation, and were also observed with autonomously replicating, subgenomic HCV RNAs (“replicons”) in multiple cell types (Supplementary Fig. 2).


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)

SKI enhances genotype 1 HCV replication while suppressing JFH1-based viruses by inhibiting type 2 sphingosine kinase (SPHK2). (a) HCV RNA genomes that express Gaussia Luciferase (GLuc) fused to foot-and-mouth disease virus 2A autoprotease as part of the HCV polyprotein. Arrowheads indicate cell culture-adaptive mutations. (b) (left) Dose-response effects of SKI on replication of H77S.3/GLuc (red) or HJ3-5/GLuc (blue) RNAs in Huh-7.5 cells. (right) Effect of 1 μM SKI on replication of H77S.3 (red) or HJ3-5 (blue) RNAs. Data shown represent relative amounts of GLuc secreted between 48–72 h (left) or intracellular RNA levels at 72 h (right). (c) Effect of 1 μM SKI on GLuc activities of the indicated viruses are presented as fold-change from baseline (6 h). (d) Flow cytometric analysis of NS5A expression in Huh-7.5 cells electroporated with H77S.3 or HJ3-5 RNA and treated with 1 μM SKI or DMSO. (e–f) Effect of siRNAs targeting SPHK isoforms or non-targeting control siRNA on replication of different HCV RNAs (e) and abundance of each SPHK isoform (f). Results represent the mean ± s.e.m. from two independent (b,c,d) or triplicate (e) experiments. *P < 0.05, **P < 0.01.
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Figure 1: SKI enhances genotype 1 HCV replication while suppressing JFH1-based viruses by inhibiting type 2 sphingosine kinase (SPHK2). (a) HCV RNA genomes that express Gaussia Luciferase (GLuc) fused to foot-and-mouth disease virus 2A autoprotease as part of the HCV polyprotein. Arrowheads indicate cell culture-adaptive mutations. (b) (left) Dose-response effects of SKI on replication of H77S.3/GLuc (red) or HJ3-5/GLuc (blue) RNAs in Huh-7.5 cells. (right) Effect of 1 μM SKI on replication of H77S.3 (red) or HJ3-5 (blue) RNAs. Data shown represent relative amounts of GLuc secreted between 48–72 h (left) or intracellular RNA levels at 72 h (right). (c) Effect of 1 μM SKI on GLuc activities of the indicated viruses are presented as fold-change from baseline (6 h). (d) Flow cytometric analysis of NS5A expression in Huh-7.5 cells electroporated with H77S.3 or HJ3-5 RNA and treated with 1 μM SKI or DMSO. (e–f) Effect of siRNAs targeting SPHK isoforms or non-targeting control siRNA on replication of different HCV RNAs (e) and abundance of each SPHK isoform (f). Results represent the mean ± s.e.m. from two independent (b,c,d) or triplicate (e) experiments. *P < 0.05, **P < 0.01.
Mentions: We determined how inhibitors of sphingolipid converting enzymes influence replication of two cell culture-adapted HCVs: H77S.3/GLuc, a genotype 1a virus, and HJ3-5/GLuc, an inter-genotypic chimera expressing the genotype 2a JFH1 replicase (Fig. 1a). To assess replication, we monitored Gaussia princeps luciferase (GLuc) produced from in-frame insertions in each viral genome after transfecting Huh-7.5 cells with synthetic RNA27. Surprisingly, these viral RNAs demonstrated contrary responses to many inhibitors, including most notably SKI, a sphingosine kinase (SPHK) inhibitor (Fig. 1b and Supplementary Fig. 1a,b). We also observed contrasting responses to sphingolipid supplementation (Supplementary Fig. 1c). SKI (1 μM) enhanced replication of H77S.3/GLuc as well as N.2/GLuc, a cell culture-adapted genotype 1b virus (Fig. 1a), by 3–6 fold, while suppressing replication of HJ3-5 (Fig. 1b,c). These effects were evident within 48 h of exposure. SKI also enhanced H77S.3 protein expression 10-fold, while slightly suppressing HJ3-5 protein expression (Fig. 1d). Thus, changes in the cellular environment induced by SKI favor H77S.3/GLuc and N.2/GLuc replication, while inhibiting HJ3-5/GLuc. These effects are not due to altered cell proliferation or viral RNA translation, and were also observed with autonomously replicating, subgenomic HCV RNAs (“replicons”) in multiple cell types (Supplementary Fig. 2).

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