Limits...
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.

Show MeSH

Related in: MedlinePlus

Lipid peroxidation regulates wild-type HCV replication and represses cell culture-adapted virus in primary human liver cultures. (a) Effects of SKI, VE (each 1 μM), LA (20 μM), LA + SKI, LA + VE, or a DAA (MK-0608, 10 μM) on replication of wild-type H77c/GLuc or HCV-N/GLuc RNAs, or a replication-defective control (H77c/GLuc-AAG) in Huh-7.5 cells. (b) Wild-type JFH1/GLuc RNA was electroporated and treated with drugs as in (a) with PSI-6130 (10 μM) as the DAA control. (c) Phase contrast microscopy of fetal hepatoblasts at 3 d. Scale bar, 50 μm. (d) Human fetal hepatoblasts (HFH) were infected with H77S.3/GLuc or HJ3-5/GLuc viruses in HFH media containing SKI or VE (each 1 μM), LA (50 μM), LA + VE, or a DAA, MK-0608 or PSI-6130 (each 10 μM) and assayed for GLuc. Results represent mean ± s.e.m. from three replicate cultures with cells from two donors. (e) HFH were infected with H77S.3 or HJ3-5 (MOI = 0.01) and treated as in d. Cell-associated viral RNA was quantified by qRT-PCR at 5 d (*P < 0.05, **P < 0.01). (f) Infectious virus released from H77S.3- or HJ3-5 virus-inoculated HFH (MOI = 0.01). Virus was quantified by FFU assay. Results represent mean ± s.e.m. from three replicate cultures.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4126843&req=5

Figure 4: Lipid peroxidation regulates wild-type HCV replication and represses cell culture-adapted virus in primary human liver cultures. (a) Effects of SKI, VE (each 1 μM), LA (20 μM), LA + SKI, LA + VE, or a DAA (MK-0608, 10 μM) on replication of wild-type H77c/GLuc or HCV-N/GLuc RNAs, or a replication-defective control (H77c/GLuc-AAG) in Huh-7.5 cells. (b) Wild-type JFH1/GLuc RNA was electroporated and treated with drugs as in (a) with PSI-6130 (10 μM) as the DAA control. (c) Phase contrast microscopy of fetal hepatoblasts at 3 d. Scale bar, 50 μm. (d) Human fetal hepatoblasts (HFH) were infected with H77S.3/GLuc or HJ3-5/GLuc viruses in HFH media containing SKI or VE (each 1 μM), LA (50 μM), LA + VE, or a DAA, MK-0608 or PSI-6130 (each 10 μM) and assayed for GLuc. Results represent mean ± s.e.m. from three replicate cultures with cells from two donors. (e) HFH were infected with H77S.3 or HJ3-5 (MOI = 0.01) and treated as in d. Cell-associated viral RNA was quantified by qRT-PCR at 5 d (*P < 0.05, **P < 0.01). (f) Infectious virus released from H77S.3- or HJ3-5 virus-inoculated HFH (MOI = 0.01). Virus was quantified by FFU assay. Results represent mean ± s.e.m. from three replicate cultures.

Mentions: To assess replication of wild-type HCV genomes possessing no cell culture-adaptive mutations, we inserted GLuc sequence into infectious molecular clones of H77c and N31,32. Both H77c/GLuc and N/GLuc RNAs produced more GLuc in electroporated cells than RNA with a lethal mutation in NS5B, H77c/GLuc-AAG (Fig. 4a). This was eliminated by direct-acting antivirals (DAA) targeting NS5B, confirming it represents replication. In contrast, treatment with either SKI or VE markedly increased GLuc, while LA reduced it to background (Fig. 4a). Moreover, the inhibitory effect of LA was reversed by co-treatment with SKI or VE. In contrast, the wild-type JFH1/GLuc was not affected by SKI, VE, or LA (Fig. 4b). Thus, endogenous lipid peroxidation is a critical restriction factor for H77c and N viruses, but not wild-type JFH1.


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)

Lipid peroxidation regulates wild-type HCV replication and represses cell culture-adapted virus in primary human liver cultures. (a) Effects of SKI, VE (each 1 μM), LA (20 μM), LA + SKI, LA + VE, or a DAA (MK-0608, 10 μM) on replication of wild-type H77c/GLuc or HCV-N/GLuc RNAs, or a replication-defective control (H77c/GLuc-AAG) in Huh-7.5 cells. (b) Wild-type JFH1/GLuc RNA was electroporated and treated with drugs as in (a) with PSI-6130 (10 μM) as the DAA control. (c) Phase contrast microscopy of fetal hepatoblasts at 3 d. Scale bar, 50 μm. (d) Human fetal hepatoblasts (HFH) were infected with H77S.3/GLuc or HJ3-5/GLuc viruses in HFH media containing SKI or VE (each 1 μM), LA (50 μM), LA + VE, or a DAA, MK-0608 or PSI-6130 (each 10 μM) and assayed for GLuc. Results represent mean ± s.e.m. from three replicate cultures with cells from two donors. (e) HFH were infected with H77S.3 or HJ3-5 (MOI = 0.01) and treated as in d. Cell-associated viral RNA was quantified by qRT-PCR at 5 d (*P < 0.05, **P < 0.01). (f) Infectious virus released from H77S.3- or HJ3-5 virus-inoculated HFH (MOI = 0.01). Virus was quantified by FFU assay. Results represent mean ± s.e.m. from three replicate cultures.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4126843&req=5

Figure 4: Lipid peroxidation regulates wild-type HCV replication and represses cell culture-adapted virus in primary human liver cultures. (a) Effects of SKI, VE (each 1 μM), LA (20 μM), LA + SKI, LA + VE, or a DAA (MK-0608, 10 μM) on replication of wild-type H77c/GLuc or HCV-N/GLuc RNAs, or a replication-defective control (H77c/GLuc-AAG) in Huh-7.5 cells. (b) Wild-type JFH1/GLuc RNA was electroporated and treated with drugs as in (a) with PSI-6130 (10 μM) as the DAA control. (c) Phase contrast microscopy of fetal hepatoblasts at 3 d. Scale bar, 50 μm. (d) Human fetal hepatoblasts (HFH) were infected with H77S.3/GLuc or HJ3-5/GLuc viruses in HFH media containing SKI or VE (each 1 μM), LA (50 μM), LA + VE, or a DAA, MK-0608 or PSI-6130 (each 10 μM) and assayed for GLuc. Results represent mean ± s.e.m. from three replicate cultures with cells from two donors. (e) HFH were infected with H77S.3 or HJ3-5 (MOI = 0.01) and treated as in d. Cell-associated viral RNA was quantified by qRT-PCR at 5 d (*P < 0.05, **P < 0.01). (f) Infectious virus released from H77S.3- or HJ3-5 virus-inoculated HFH (MOI = 0.01). Virus was quantified by FFU assay. Results represent mean ± s.e.m. from three replicate cultures.
Mentions: To assess replication of wild-type HCV genomes possessing no cell culture-adaptive mutations, we inserted GLuc sequence into infectious molecular clones of H77c and N31,32. Both H77c/GLuc and N/GLuc RNAs produced more GLuc in electroporated cells than RNA with a lethal mutation in NS5B, H77c/GLuc-AAG (Fig. 4a). This was eliminated by direct-acting antivirals (DAA) targeting NS5B, confirming it represents replication. In contrast, treatment with either SKI or VE markedly increased GLuc, while LA reduced it to background (Fig. 4a). Moreover, the inhibitory effect of LA was reversed by co-treatment with SKI or VE. In contrast, the wild-type JFH1/GLuc was not affected by SKI, VE, or LA (Fig. 4b). Thus, endogenous lipid peroxidation is a critical restriction factor for H77c and N viruses, but not wild-type JFH1.

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