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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Acknowledgements
We thank L.F. Ping and W. Lovell for expert technical assistance, R. Purcell (US National Institute of Allergy and Infectious Diseases) and J. Bukh (Copenhagen University Hospital, Denmark) for pCV-H77C and pTNcc plasmids, C.M. Rice and M. Saeed (The Rockefeller University) for Huh-7.5 cells and S52/SG-Feo and ED43/SG-Feo plasmids, T. Wakita (National Institute of Infectious Diseases, Japan) for pJFH1 and pJFH-2 plasmids, M.J. Otto (Pharmasset) for PSI-6130, A. Sluder (SCYNEXIS) for SCY-635, R. De Francesco (Istituto Nazionale di Genetica Molecolare, Italy) for compound 23, A.Y. Howe (Merck Research Laboratory) for boceprevir, HCV-796, MK-0608 and MK-7009 and Z. Feng (University of North Carolina) for hepatitis A virus stocks. We also thank S.A. Weinman for critical reading of the manuscript and D.L. Tyrrell, M. Joyce, R.A. Coleman and T. Masaki for helpful discussions. This work was supported by US National Institutes of Health grants RO1-AI095690, RO1-CA164029 and U19-AI109965 (S.M.L.), R21-CA182322 (L.R.), R01-AI075090 (M.Y.), RO1-AI073335 (C.C.K.), RO1-DE018304 (D.P.D.), F32-AI094941 (D.G.W.) and U54-GM069338 (S.B.), a National Cancer Institute Center Core Support Grant to the Lineberger Comprehensive Cancer Center (P30-CA016086) and the University of North Carolina Cancer Research Fund. C.W. was supported by the Deutsche Forschungsgemeinschaft (WE 4388/3-1 and WE 4388/6-1). I.A. was supported by the CIPSM Cluster of Excellence.
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D.Y. and S.M.L. conceived the study and wrote the paper; D.Y., D.R.M., E.W., V.J.M., Y.W., P.E.C., C.E.M., D.G.W. and I.M. conducted experiments; C.W. and I.A. modeled membrane interactions of proteins; S.B. and A.H.M. Jr. carried out mass spectrometry analysis of sphingolipids; J.K.W., M.T.H., D.P.D. and C.C.K. provided reagents and supervised experiments involving viruses other than HCV; M.Y., S.K., T.S., T.O., S.M.P. and L.M.R. provided research materials; and all authors discussed the results and commented on the manuscript.
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L.M.R. has received research support from Vesta Therapeutics and the Dow Chemical Company related to human hepatic and biliary tree stem cells. S.M.L. and D.Y. are named inventors in a U.S. Provisional Patent Application related to the H77D virus described in this paper.
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Yamane, D., McGivern, D., Wauthier, E. et al. Regulation of the hepatitis C virus RNA replicase by endogenous lipid peroxidation. Nat Med 20, 927–935 (2014). https://doi.org/10.1038/nm.3610
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DOI: https://doi.org/10.1038/nm.3610
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