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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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Resistance to lipid peroxidation is tightly linked to robust replication in cell culture. (a) (upper panel) Cell culture-adaptive mutations in TNcc34 (yellow arrowheads) confer resistance to lipid peroxidation when introduced into H77S.3/GLucIS (H77S.3/GLuc in which the adaptive mutation S2204I has been removed (black arrowhead), see Supplementary Fig. 10b for details). Huh-7.5 cells were treated with DMSO, 1 μM SKI, 1 μM VE, 10 μM CuOH, CuOH plus VE, or 30 μM sofosbuvir (DAA) beginning 6 h following RNA electroporation. GLuc secretion was measured between 48–72 h. (lower panel) TNcc mutations in NS3 (helicase) and NS4B are not required for lipid peroxidation resistance. Combinations of TNcc substitutions were introduced into H77S.3/GLucIS/GS (NS proteins shown only) that contains the compensatory mutation G1909S (GS) in NS4B (red arrowhead, see Supplementary Fig. 11). Data shown represent mean GLuc activity ± s.e.m. from two independent experiments. L.O.D. = limit of detection. (b) (top) H77D genome containing the I2204S substitution (black), 8 TNcc-derived mutations (yellow) and 3 novel compensatory mutations (red) in the H77S.3 background. (bottom) Huh-7.5 cells transfected with the indicated RNAs encoding GLuc (left) or lacking GLuc (right) were treated with DMSO or 1 μM VE and secreted GLuc activity (left) or released infectious virus (right) measured between 48–72 h. Data shown represent means ± s.d. from triplicate cultures in a representative experiment. (c) Structural models of (left) NS4A and (right) NS5B membrane interactions showing key residues that determine sensitivity to lipid peroxidation. (d) EC50 of direct- vs. indirect-acting antivirals against H77D in the presence of SKI or VE (each 1 μM). Assays were carried out as in Fig. 5g. (e) Huh-7.5 cells transfected with H77S.3/GLuc or HJ3-5/GLuc RNA, genome-length JFH-2 RNA, or subgenomic RNAs encoding FLuc (S52 and ED43) were treated with DMSO, 1 μM SKI or VE, 10 uM CuOH or CuOH plus VE. Data shown represent percent GLuc (H77S.3 and HJ3-5), RNA copies (JFH-2) or FLuc activities (S52 and ED43) at 72 h relative to DMSO controls. Data shown represent mean ± s.e.m. from three independent experiments. (f) The HCV replicase is uniquely regulated by lipid peroxidation. The impact of SKI or VE (each 1 μM) or LA (50 μM) on the abundance of viral RNA (left) or yields of infectious virus (right) was determined for a panel of RNA viruses following infection of Huh-7.5 cells. In addition to H77S.3, viruses studied included the hepatotropic picornavirus, hepatitis A virus (HAV), representative flaviviruses (dengue virus, DENV; West Nile virus, WNV; yellow fever virus, YFV), alphaviruses (Sindbis virus, SINV; Ross River virus, RRV; Chikungunya virus, CHIKV), and lymphocytic choriomeningitis virus (LCMV). Data shown are mean ± s.e.m. from three replicate cultures. See Supplementary Fig. 12 for additional details.
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Figure 6: Resistance to lipid peroxidation is tightly linked to robust replication in cell culture. (a) (upper panel) Cell culture-adaptive mutations in TNcc34 (yellow arrowheads) confer resistance to lipid peroxidation when introduced into H77S.3/GLucIS (H77S.3/GLuc in which the adaptive mutation S2204I has been removed (black arrowhead), see Supplementary Fig. 10b for details). Huh-7.5 cells were treated with DMSO, 1 μM SKI, 1 μM VE, 10 μM CuOH, CuOH plus VE, or 30 μM sofosbuvir (DAA) beginning 6 h following RNA electroporation. GLuc secretion was measured between 48–72 h. (lower panel) TNcc mutations in NS3 (helicase) and NS4B are not required for lipid peroxidation resistance. Combinations of TNcc substitutions were introduced into H77S.3/GLucIS/GS (NS proteins shown only) that contains the compensatory mutation G1909S (GS) in NS4B (red arrowhead, see Supplementary Fig. 11). Data shown represent mean GLuc activity ± s.e.m. from two independent experiments. L.O.D. = limit of detection. (b) (top) H77D genome containing the I2204S substitution (black), 8 TNcc-derived mutations (yellow) and 3 novel compensatory mutations (red) in the H77S.3 background. (bottom) Huh-7.5 cells transfected with the indicated RNAs encoding GLuc (left) or lacking GLuc (right) were treated with DMSO or 1 μM VE and secreted GLuc activity (left) or released infectious virus (right) measured between 48–72 h. Data shown represent means ± s.d. from triplicate cultures in a representative experiment. (c) Structural models of (left) NS4A and (right) NS5B membrane interactions showing key residues that determine sensitivity to lipid peroxidation. (d) EC50 of direct- vs. indirect-acting antivirals against H77D in the presence of SKI or VE (each 1 μM). Assays were carried out as in Fig. 5g. (e) Huh-7.5 cells transfected with H77S.3/GLuc or HJ3-5/GLuc RNA, genome-length JFH-2 RNA, or subgenomic RNAs encoding FLuc (S52 and ED43) were treated with DMSO, 1 μM SKI or VE, 10 uM CuOH or CuOH plus VE. Data shown represent percent GLuc (H77S.3 and HJ3-5), RNA copies (JFH-2) or FLuc activities (S52 and ED43) at 72 h relative to DMSO controls. Data shown represent mean ± s.e.m. from three independent experiments. (f) The HCV replicase is uniquely regulated by lipid peroxidation. The impact of SKI or VE (each 1 μM) or LA (50 μM) on the abundance of viral RNA (left) or yields of infectious virus (right) was determined for a panel of RNA viruses following infection of Huh-7.5 cells. In addition to H77S.3, viruses studied included the hepatotropic picornavirus, hepatitis A virus (HAV), representative flaviviruses (dengue virus, DENV; West Nile virus, WNV; yellow fever virus, YFV), alphaviruses (Sindbis virus, SINV; Ross River virus, RRV; Chikungunya virus, CHIKV), and lymphocytic choriomeningitis virus (LCMV). Data shown are mean ± s.e.m. from three replicate cultures. See Supplementary Fig. 12 for additional details.

Mentions: TNcc is a recently described genotype 1a virus with 8 cell culture-adaptive mutations that replicates almost as well as JFH1 in Huh-7.5 cells34. Surprisingly, we found it completely resistant to lipid peroxidation (Fig. 6a and Supplementary Fig. 10a). We introduced all 8 TNcc mutations into H77S.3 to determine whether they would confer peroxidation resistance. This RNA (H77S.3/GLuc8mt) failed to replicate, but removal of a key H77S adaptive mutation (S2204I in NS5A)12 restored low-level replication, and this virus, H77S.3/GLucIS/8mt, was resistant to lipid peroxidation (Fig. 6a and Supplementary Fig. 10b). Continued passage of cells infected with H77S.3IS/8mt resulted in additional mutations in NS4B (G1909S), NS5A (D2416G) and NS5B (G2963D) that together enhanced replication by 850-fold (see Supplementary Results and Supplementary Fig. 11). NS4B G1909S compensates for negative effects of TNcc mutations placed into the H77S.3 background, but does not confer peroxidation resistance (Fig. 6a). When all 3 mutations were introduced into H77S.3/GLucIS/8mt (designated H77D), infectious virus yields were comparable to HJ3-5 or JFH1-QL, and not increased by VE supplementation (Fig. 6b). Importantly, the EC50 of DAAs against H77D virus was not altered by SKI or VE (Fig. 6d and Supplementary Fig. 9h).


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)

Resistance to lipid peroxidation is tightly linked to robust replication in cell culture. (a) (upper panel) Cell culture-adaptive mutations in TNcc34 (yellow arrowheads) confer resistance to lipid peroxidation when introduced into H77S.3/GLucIS (H77S.3/GLuc in which the adaptive mutation S2204I has been removed (black arrowhead), see Supplementary Fig. 10b for details). Huh-7.5 cells were treated with DMSO, 1 μM SKI, 1 μM VE, 10 μM CuOH, CuOH plus VE, or 30 μM sofosbuvir (DAA) beginning 6 h following RNA electroporation. GLuc secretion was measured between 48–72 h. (lower panel) TNcc mutations in NS3 (helicase) and NS4B are not required for lipid peroxidation resistance. Combinations of TNcc substitutions were introduced into H77S.3/GLucIS/GS (NS proteins shown only) that contains the compensatory mutation G1909S (GS) in NS4B (red arrowhead, see Supplementary Fig. 11). Data shown represent mean GLuc activity ± s.e.m. from two independent experiments. L.O.D. = limit of detection. (b) (top) H77D genome containing the I2204S substitution (black), 8 TNcc-derived mutations (yellow) and 3 novel compensatory mutations (red) in the H77S.3 background. (bottom) Huh-7.5 cells transfected with the indicated RNAs encoding GLuc (left) or lacking GLuc (right) were treated with DMSO or 1 μM VE and secreted GLuc activity (left) or released infectious virus (right) measured between 48–72 h. Data shown represent means ± s.d. from triplicate cultures in a representative experiment. (c) Structural models of (left) NS4A and (right) NS5B membrane interactions showing key residues that determine sensitivity to lipid peroxidation. (d) EC50 of direct- vs. indirect-acting antivirals against H77D in the presence of SKI or VE (each 1 μM). Assays were carried out as in Fig. 5g. (e) Huh-7.5 cells transfected with H77S.3/GLuc or HJ3-5/GLuc RNA, genome-length JFH-2 RNA, or subgenomic RNAs encoding FLuc (S52 and ED43) were treated with DMSO, 1 μM SKI or VE, 10 uM CuOH or CuOH plus VE. Data shown represent percent GLuc (H77S.3 and HJ3-5), RNA copies (JFH-2) or FLuc activities (S52 and ED43) at 72 h relative to DMSO controls. Data shown represent mean ± s.e.m. from three independent experiments. (f) The HCV replicase is uniquely regulated by lipid peroxidation. The impact of SKI or VE (each 1 μM) or LA (50 μM) on the abundance of viral RNA (left) or yields of infectious virus (right) was determined for a panel of RNA viruses following infection of Huh-7.5 cells. In addition to H77S.3, viruses studied included the hepatotropic picornavirus, hepatitis A virus (HAV), representative flaviviruses (dengue virus, DENV; West Nile virus, WNV; yellow fever virus, YFV), alphaviruses (Sindbis virus, SINV; Ross River virus, RRV; Chikungunya virus, CHIKV), and lymphocytic choriomeningitis virus (LCMV). Data shown are mean ± s.e.m. from three replicate cultures. See Supplementary Fig. 12 for additional details.
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Figure 6: Resistance to lipid peroxidation is tightly linked to robust replication in cell culture. (a) (upper panel) Cell culture-adaptive mutations in TNcc34 (yellow arrowheads) confer resistance to lipid peroxidation when introduced into H77S.3/GLucIS (H77S.3/GLuc in which the adaptive mutation S2204I has been removed (black arrowhead), see Supplementary Fig. 10b for details). Huh-7.5 cells were treated with DMSO, 1 μM SKI, 1 μM VE, 10 μM CuOH, CuOH plus VE, or 30 μM sofosbuvir (DAA) beginning 6 h following RNA electroporation. GLuc secretion was measured between 48–72 h. (lower panel) TNcc mutations in NS3 (helicase) and NS4B are not required for lipid peroxidation resistance. Combinations of TNcc substitutions were introduced into H77S.3/GLucIS/GS (NS proteins shown only) that contains the compensatory mutation G1909S (GS) in NS4B (red arrowhead, see Supplementary Fig. 11). Data shown represent mean GLuc activity ± s.e.m. from two independent experiments. L.O.D. = limit of detection. (b) (top) H77D genome containing the I2204S substitution (black), 8 TNcc-derived mutations (yellow) and 3 novel compensatory mutations (red) in the H77S.3 background. (bottom) Huh-7.5 cells transfected with the indicated RNAs encoding GLuc (left) or lacking GLuc (right) were treated with DMSO or 1 μM VE and secreted GLuc activity (left) or released infectious virus (right) measured between 48–72 h. Data shown represent means ± s.d. from triplicate cultures in a representative experiment. (c) Structural models of (left) NS4A and (right) NS5B membrane interactions showing key residues that determine sensitivity to lipid peroxidation. (d) EC50 of direct- vs. indirect-acting antivirals against H77D in the presence of SKI or VE (each 1 μM). Assays were carried out as in Fig. 5g. (e) Huh-7.5 cells transfected with H77S.3/GLuc or HJ3-5/GLuc RNA, genome-length JFH-2 RNA, or subgenomic RNAs encoding FLuc (S52 and ED43) were treated with DMSO, 1 μM SKI or VE, 10 uM CuOH or CuOH plus VE. Data shown represent percent GLuc (H77S.3 and HJ3-5), RNA copies (JFH-2) or FLuc activities (S52 and ED43) at 72 h relative to DMSO controls. Data shown represent mean ± s.e.m. from three independent experiments. (f) The HCV replicase is uniquely regulated by lipid peroxidation. The impact of SKI or VE (each 1 μM) or LA (50 μM) on the abundance of viral RNA (left) or yields of infectious virus (right) was determined for a panel of RNA viruses following infection of Huh-7.5 cells. In addition to H77S.3, viruses studied included the hepatotropic picornavirus, hepatitis A virus (HAV), representative flaviviruses (dengue virus, DENV; West Nile virus, WNV; yellow fever virus, YFV), alphaviruses (Sindbis virus, SINV; Ross River virus, RRV; Chikungunya virus, CHIKV), and lymphocytic choriomeningitis virus (LCMV). Data shown are mean ± s.e.m. from three replicate cultures. See Supplementary Fig. 12 for additional details.
Mentions: TNcc is a recently described genotype 1a virus with 8 cell culture-adaptive mutations that replicates almost as well as JFH1 in Huh-7.5 cells34. Surprisingly, we found it completely resistant to lipid peroxidation (Fig. 6a and Supplementary Fig. 10a). We introduced all 8 TNcc mutations into H77S.3 to determine whether they would confer peroxidation resistance. This RNA (H77S.3/GLuc8mt) failed to replicate, but removal of a key H77S adaptive mutation (S2204I in NS5A)12 restored low-level replication, and this virus, H77S.3/GLucIS/8mt, was resistant to lipid peroxidation (Fig. 6a and Supplementary Fig. 10b). Continued passage of cells infected with H77S.3IS/8mt resulted in additional mutations in NS4B (G1909S), NS5A (D2416G) and NS5B (G2963D) that together enhanced replication by 850-fold (see Supplementary Results and Supplementary Fig. 11). NS4B G1909S compensates for negative effects of TNcc mutations placed into the H77S.3 background, but does not confer peroxidation resistance (Fig. 6a). When all 3 mutations were introduced into H77S.3/GLucIS/8mt (designated H77D), infectious virus yields were comparable to HJ3-5 or JFH1-QL, and not increased by VE supplementation (Fig. 6b). Importantly, the EC50 of DAAs against H77D virus was not altered by SKI or VE (Fig. 6d and Supplementary Fig. 9h).

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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Related in: MedlinePlus