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 reduces HCV-induced membranous web abundance and alters the EC50 of DAAs. (a) Transmission electron microscopic images of the membranous web in Huh-7.5 cells electroporated with H77S.3 or HJ3-5 RNA and treated with DMSO, SKI (1 μM), VE (1 μM), LA (50 μM), or CuOH (10 μM). Scale bar = 500 nm. (b) Quantitation of area occupied by double-membrane vesicles (DMV) within individual cells infected with H77S.3 virus and treated with SKI, VE, or LA as in a. *P ≤ 0.002 vs. DMSO by two-sided Mann-Whitney test. (c) SKI and VE mask the antiviral effect of PSI-6130 against H77S.3/GLuc replication. (left) Huh-7.5 cells electroporated with H77S.3/GLuc or HJ3-5/GLuc RNA were cultured for 7 d, then treated with DMSO, 10 μM PSI-6130, 1 μM VE or both PSI-6130 and VE. Culture supernatant fluids were replaced every 24 h and assayed for GLuc activity. (right) Huh-7.5 cells were electroporated with H77S.3 RNA, cultured for 5 d and then treated with DMSO, PSI-6130, SKI or VE, or PSI-6130 plus SKI or VE. Cell-associated HCV RNA was quantified by qRT-PCR. Results represent mean ± s.e.m from two (left) or three (right) replicate cultures. (d) Inhibition of H77S.3 (left) and HJ3-5 (right) replication by the NS5B inhibitor PSI-6130 in the presence of SKI or VE (each 1 μM) or DMSO vehicle. Inhibition was assessed by quantifying GLuc secreted 48–72 h after drug addition. Results represent mean ± s.e.m. of two replicate cultures. (e,f) Inhibition of H77S.3 replication by (e) MK-7009, an NS3/4A inhibitor, and (f) SCY-635, a host-targeting cyclophilin inhibitor. (g) EC50 values of representative direct- versus indirect-acting antivirals against H77S.3 (left) and HJ3-5 (right) viruses in the presence of SKI or VE (each 1 μM). Assays were carried out as in panels d–f. Colored bars represent limits of the 95% c.i. of EC50 values calculated from Hill plots. ‘NA’ = not measureable due to poor antiviral activity. See Supplementary Fig. 9 for additional details.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Lipid peroxidation reduces HCV-induced membranous web abundance and alters the EC50 of DAAs. (a) Transmission electron microscopic images of the membranous web in Huh-7.5 cells electroporated with H77S.3 or HJ3-5 RNA and treated with DMSO, SKI (1 μM), VE (1 μM), LA (50 μM), or CuOH (10 μM). Scale bar = 500 nm. (b) Quantitation of area occupied by double-membrane vesicles (DMV) within individual cells infected with H77S.3 virus and treated with SKI, VE, or LA as in a. *P ≤ 0.002 vs. DMSO by two-sided Mann-Whitney test. (c) SKI and VE mask the antiviral effect of PSI-6130 against H77S.3/GLuc replication. (left) Huh-7.5 cells electroporated with H77S.3/GLuc or HJ3-5/GLuc RNA were cultured for 7 d, then treated with DMSO, 10 μM PSI-6130, 1 μM VE or both PSI-6130 and VE. Culture supernatant fluids were replaced every 24 h and assayed for GLuc activity. (right) Huh-7.5 cells were electroporated with H77S.3 RNA, cultured for 5 d and then treated with DMSO, PSI-6130, SKI or VE, or PSI-6130 plus SKI or VE. Cell-associated HCV RNA was quantified by qRT-PCR. Results represent mean ± s.e.m from two (left) or three (right) replicate cultures. (d) Inhibition of H77S.3 (left) and HJ3-5 (right) replication by the NS5B inhibitor PSI-6130 in the presence of SKI or VE (each 1 μM) or DMSO vehicle. Inhibition was assessed by quantifying GLuc secreted 48–72 h after drug addition. Results represent mean ± s.e.m. of two replicate cultures. (e,f) Inhibition of H77S.3 replication by (e) MK-7009, an NS3/4A inhibitor, and (f) SCY-635, a host-targeting cyclophilin inhibitor. (g) EC50 values of representative direct- versus indirect-acting antivirals against H77S.3 (left) and HJ3-5 (right) viruses in the presence of SKI or VE (each 1 μM). Assays were carried out as in panels d–f. Colored bars represent limits of the 95% c.i. of EC50 values calculated from Hill plots. ‘NA’ = not measureable due to poor antiviral activity. See Supplementary Fig. 9 for additional details.

Mentions: Both SKI and VE treatment increased the area occupied by the membranous web in H77S.3-infected cells (Fig. 5a,b), without altering the morphology of double-membrane vesicles which are the likely site of genome replication14. Increased lipid peroxidation had the opposite effect, while the HJ3-5 membranous web was insensitive to changes in 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)

Lipid peroxidation reduces HCV-induced membranous web abundance and alters the EC50 of DAAs. (a) Transmission electron microscopic images of the membranous web in Huh-7.5 cells electroporated with H77S.3 or HJ3-5 RNA and treated with DMSO, SKI (1 μM), VE (1 μM), LA (50 μM), or CuOH (10 μM). Scale bar = 500 nm. (b) Quantitation of area occupied by double-membrane vesicles (DMV) within individual cells infected with H77S.3 virus and treated with SKI, VE, or LA as in a. *P ≤ 0.002 vs. DMSO by two-sided Mann-Whitney test. (c) SKI and VE mask the antiviral effect of PSI-6130 against H77S.3/GLuc replication. (left) Huh-7.5 cells electroporated with H77S.3/GLuc or HJ3-5/GLuc RNA were cultured for 7 d, then treated with DMSO, 10 μM PSI-6130, 1 μM VE or both PSI-6130 and VE. Culture supernatant fluids were replaced every 24 h and assayed for GLuc activity. (right) Huh-7.5 cells were electroporated with H77S.3 RNA, cultured for 5 d and then treated with DMSO, PSI-6130, SKI or VE, or PSI-6130 plus SKI or VE. Cell-associated HCV RNA was quantified by qRT-PCR. Results represent mean ± s.e.m from two (left) or three (right) replicate cultures. (d) Inhibition of H77S.3 (left) and HJ3-5 (right) replication by the NS5B inhibitor PSI-6130 in the presence of SKI or VE (each 1 μM) or DMSO vehicle. Inhibition was assessed by quantifying GLuc secreted 48–72 h after drug addition. Results represent mean ± s.e.m. of two replicate cultures. (e,f) Inhibition of H77S.3 replication by (e) MK-7009, an NS3/4A inhibitor, and (f) SCY-635, a host-targeting cyclophilin inhibitor. (g) EC50 values of representative direct- versus indirect-acting antivirals against H77S.3 (left) and HJ3-5 (right) viruses in the presence of SKI or VE (each 1 μM). Assays were carried out as in panels d–f. Colored bars represent limits of the 95% c.i. of EC50 values calculated from Hill plots. ‘NA’ = not measureable due to poor antiviral activity. See Supplementary Fig. 9 for additional details.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Lipid peroxidation reduces HCV-induced membranous web abundance and alters the EC50 of DAAs. (a) Transmission electron microscopic images of the membranous web in Huh-7.5 cells electroporated with H77S.3 or HJ3-5 RNA and treated with DMSO, SKI (1 μM), VE (1 μM), LA (50 μM), or CuOH (10 μM). Scale bar = 500 nm. (b) Quantitation of area occupied by double-membrane vesicles (DMV) within individual cells infected with H77S.3 virus and treated with SKI, VE, or LA as in a. *P ≤ 0.002 vs. DMSO by two-sided Mann-Whitney test. (c) SKI and VE mask the antiviral effect of PSI-6130 against H77S.3/GLuc replication. (left) Huh-7.5 cells electroporated with H77S.3/GLuc or HJ3-5/GLuc RNA were cultured for 7 d, then treated with DMSO, 10 μM PSI-6130, 1 μM VE or both PSI-6130 and VE. Culture supernatant fluids were replaced every 24 h and assayed for GLuc activity. (right) Huh-7.5 cells were electroporated with H77S.3 RNA, cultured for 5 d and then treated with DMSO, PSI-6130, SKI or VE, or PSI-6130 plus SKI or VE. Cell-associated HCV RNA was quantified by qRT-PCR. Results represent mean ± s.e.m from two (left) or three (right) replicate cultures. (d) Inhibition of H77S.3 (left) and HJ3-5 (right) replication by the NS5B inhibitor PSI-6130 in the presence of SKI or VE (each 1 μM) or DMSO vehicle. Inhibition was assessed by quantifying GLuc secreted 48–72 h after drug addition. Results represent mean ± s.e.m. of two replicate cultures. (e,f) Inhibition of H77S.3 replication by (e) MK-7009, an NS3/4A inhibitor, and (f) SCY-635, a host-targeting cyclophilin inhibitor. (g) EC50 values of representative direct- versus indirect-acting antivirals against H77S.3 (left) and HJ3-5 (right) viruses in the presence of SKI or VE (each 1 μM). Assays were carried out as in panels d–f. Colored bars represent limits of the 95% c.i. of EC50 values calculated from Hill plots. ‘NA’ = not measureable due to poor antiviral activity. See Supplementary Fig. 9 for additional details.
Mentions: Both SKI and VE treatment increased the area occupied by the membranous web in H77S.3-infected cells (Fig. 5a,b), without altering the morphology of double-membrane vesicles which are the likely site of genome replication14. Increased lipid peroxidation had the opposite effect, while the HJ3-5 membranous web was insensitive to changes in 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