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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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Inhibition of lipid peroxidation by SKI or VE promotes production and spread of infectious genotype 1 HCV. (a) Infectious virus yields from Huh-7.5 cells transfected with the indicated viral RNAs and grown in the presence of 1 μM SKI, 1 μM VE or DMSO vehicle. Culture supernatant fluids were harvested and replaced with fresh media containing compounds every 24 h. Infectivity titers are expressed as focus forming units (FFU) ml−1. Data shown are mean ± s.e.m. from three replicate cultures. (b) SKI promotes spread of H77S.3 but inhibits HJ3-5 virus. Cells electroporated with H77S.3 or HJ3-5 RNAs were mixed with carboxyfluorescein succinimidyl ester (CFSE)-labeled naïve Huh-7.5 cells; CFSE/NS5A double-positive cells (upper right quadrant) are indicative of virus spread. (c) Buoyant density of H77S.3 virus particles released from H77S.3 RNA-transfected Huh-7.5 cells grown in 1 μM SKI (left) or 1 μM VE (right) vs. DMSO control. Fractions from isopycnic iodixanol gradients were assayed for infectious virus (FFU) or HCV RNA (GE = genome equivalents). The data shown are representative of two independent experiments.
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Figure 3: Inhibition of lipid peroxidation by SKI or VE promotes production and spread of infectious genotype 1 HCV. (a) Infectious virus yields from Huh-7.5 cells transfected with the indicated viral RNAs and grown in the presence of 1 μM SKI, 1 μM VE or DMSO vehicle. Culture supernatant fluids were harvested and replaced with fresh media containing compounds every 24 h. Infectivity titers are expressed as focus forming units (FFU) ml−1. Data shown are mean ± s.e.m. from three replicate cultures. (b) SKI promotes spread of H77S.3 but inhibits HJ3-5 virus. Cells electroporated with H77S.3 or HJ3-5 RNAs were mixed with carboxyfluorescein succinimidyl ester (CFSE)-labeled naïve Huh-7.5 cells; CFSE/NS5A double-positive cells (upper right quadrant) are indicative of virus spread. (c) Buoyant density of H77S.3 virus particles released from H77S.3 RNA-transfected Huh-7.5 cells grown in 1 μM SKI (left) or 1 μM VE (right) vs. DMSO control. Fractions from isopycnic iodixanol gradients were assayed for infectious virus (FFU) or HCV RNA (GE = genome equivalents). The data shown are representative of two independent experiments.

Mentions: Both SKI and VE induced a 10-fold increase in the yield of infectious virus released by H77S.3 RNA-transfected cells, reaching ~20,000 focus-forming units (FFU) ml−1 (Fig. 3a and Supplementary Fig. 6a). Infectious N.2 virus yields were increased up to 100-fold (Fig. 3a). SKI also enhanced virus spread when H77S.3 RNA-transfected cells were co-cultured with non-transfected CFSE-labeled cells (Fig. 3b). In contrast, neither SKI nor VE enhanced infectious yields of JFH1-QL or HJ3-5 virus (Fig. 3a), while SKI reduced the spread of HJ3-5 virus by >40% (Fig. 3b).


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)

Inhibition of lipid peroxidation by SKI or VE promotes production and spread of infectious genotype 1 HCV. (a) Infectious virus yields from Huh-7.5 cells transfected with the indicated viral RNAs and grown in the presence of 1 μM SKI, 1 μM VE or DMSO vehicle. Culture supernatant fluids were harvested and replaced with fresh media containing compounds every 24 h. Infectivity titers are expressed as focus forming units (FFU) ml−1. Data shown are mean ± s.e.m. from three replicate cultures. (b) SKI promotes spread of H77S.3 but inhibits HJ3-5 virus. Cells electroporated with H77S.3 or HJ3-5 RNAs were mixed with carboxyfluorescein succinimidyl ester (CFSE)-labeled naïve Huh-7.5 cells; CFSE/NS5A double-positive cells (upper right quadrant) are indicative of virus spread. (c) Buoyant density of H77S.3 virus particles released from H77S.3 RNA-transfected Huh-7.5 cells grown in 1 μM SKI (left) or 1 μM VE (right) vs. DMSO control. Fractions from isopycnic iodixanol gradients were assayed for infectious virus (FFU) or HCV RNA (GE = genome equivalents). The data shown are representative of two independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4126843&req=5

Figure 3: Inhibition of lipid peroxidation by SKI or VE promotes production and spread of infectious genotype 1 HCV. (a) Infectious virus yields from Huh-7.5 cells transfected with the indicated viral RNAs and grown in the presence of 1 μM SKI, 1 μM VE or DMSO vehicle. Culture supernatant fluids were harvested and replaced with fresh media containing compounds every 24 h. Infectivity titers are expressed as focus forming units (FFU) ml−1. Data shown are mean ± s.e.m. from three replicate cultures. (b) SKI promotes spread of H77S.3 but inhibits HJ3-5 virus. Cells electroporated with H77S.3 or HJ3-5 RNAs were mixed with carboxyfluorescein succinimidyl ester (CFSE)-labeled naïve Huh-7.5 cells; CFSE/NS5A double-positive cells (upper right quadrant) are indicative of virus spread. (c) Buoyant density of H77S.3 virus particles released from H77S.3 RNA-transfected Huh-7.5 cells grown in 1 μM SKI (left) or 1 μM VE (right) vs. DMSO control. Fractions from isopycnic iodixanol gradients were assayed for infectious virus (FFU) or HCV RNA (GE = genome equivalents). The data shown are representative of two independent experiments.
Mentions: Both SKI and VE induced a 10-fold increase in the yield of infectious virus released by H77S.3 RNA-transfected cells, reaching ~20,000 focus-forming units (FFU) ml−1 (Fig. 3a and Supplementary Fig. 6a). Infectious N.2 virus yields were increased up to 100-fold (Fig. 3a). SKI also enhanced virus spread when H77S.3 RNA-transfected cells were co-cultured with non-transfected CFSE-labeled cells (Fig. 3b). In contrast, neither SKI nor VE enhanced infectious yields of JFH1-QL or HJ3-5 virus (Fig. 3a), while SKI reduced the spread of HJ3-5 virus by >40% (Fig. 3b).

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