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Tuning a cellular lipid kinase activity adapts hepatitis C virus to replication in cell culture

Nature Microbiology volume 2, Article number: 16247 (2017) Cite this article

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Abstract

With a single exception, all isolates of hepatitis C virus (HCV) require adaptive mutations to replicate efficiently in cell culture. Here, we show that a major class of adaptive mutations regulates the activity of a cellular lipid kinase, phosphatidylinositol 4-kinase IIIα (PI4KA). HCV needs to stimulate PI4KA to create a permissive phosphatidylinositol 4-phosphate-enriched membrane microenvironment in the liver and in primary human hepatocytes (PHHs). In contrast, in Huh7 hepatoma cells, the virus must acquire loss-of-function mutations that prevent PI4KA overactivation. This adaptive mechanism is necessitated by increased PI4KA levels in Huh7 cells compared with PHHs, and is conserved across HCV genotypes. PI4KA-specific inhibitors promote replication of unadapted viral isolates and allow efficient replication of patient-derived virus in cell culture. In summary, this study has uncovered a long-sought mechanism of HCV cell-culture adaptation and demonstrates how a virus can adapt to changes in a cellular environment associated with tumorigenesis.

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Figure 1: PI4KA knockdown and inhibition have differing impacts on non-adapted and adapted HCV variants.
Figure 2: Cell-culture adaptive mutations in NS5A and NS5B eliminate PI4P accumulation and alter the morphology of the membranous web.
Figure 3: PI4KA activation by WT HCV and loss of activation for adapted variants is conserved among different genotypes.
Figure 4: Replication-enhancing effects of adaptive mutations in NS5A correlate with elimination of PI4P induction.
Figure 5: Combined PI4KA and CKIa inhibition allows highly efficient RNA replication of unadapted HCV in cell culture.
Figure 6: HCV WT requires low levels of PI4KA and adapts to high PI4KA expression levels in Huh7-derived hepatoma cell lines.

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  • 14 July 2017

    In the PDF version of this article previously published, the year of publication provided in the footer of each page and in the 'How to cite' section was erroneously given as 2017, it should have been 2016. This error has now been corrected. The HTML version of the article was not affected.

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Acknowledgements

The authors would especially like to thank U. Herian, R. Klein, S. Kallis and M. Bartenschlager for excellent technical assistance. We are grateful to T. Wakita for the JFH-1 isolate, to S. Breitfelder for compounds PI4KA-A and PI4KA-B, J. Botyanszki for compounds PI4KA-F1 and PI4KA-G1 and M. Harris for the polyclonal sheep serum recognizing NS5A. We are grateful to the Electron Microscopy Core Facility at the Bioquant (Heidelberg University) for providing access to their equipment and for excellent support. This project was funded by grants from the Deutsche Forschungsgemeinschaft (LO 1556/1-2, LO 1556/4-1 and TRR77, TPA1 to V.L., and TRR83, TP13 and TRR77, TPA1 to R.B.), as well as an HBIGS Postdoc stipend to C.H. This work was supported in part by the National Institutes of Health National Cancer Institute grant R01CA057973 and National Institute of Allergy and Infectious Diseases grants R01AI072613 and R01AI099284 (to C.M.R.) and by a Helmsley Postdoctoral Fellowship for Basic and Translational Research on Disorders of the Digestive System at The Rockefeller University (to M.S.). The Greenberg Medical Research Institute, Starr Foundation, Ronald A. Shellow, M.D. Memorial Fund and anonymous donors provided additional funding (to C.M.R.).

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Author notes

  1. Christian Harak, Max Meyrath & Katharina Esser-Nobis

    Present address: †Present address: Abbott GmbH & Co. KG, Wiesbaden, Germany (C.H.); Infectious Diseases Research Unit, Department of Infection and Immunity, Luxembourg Institute of Health, Luxembourg (M.M.); Department of Immunology, University of Washington, Seattle, Washington, USA (K.E.-N.),

Authors and Affiliations

  1. Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany

    Christian Harak, Max Meyrath, Inés Romero-Brey, Christian Schenk, Philipp Schult, Katharina Esser-Nobis, Ralf Bartenschlager & Volker Lohmann

  2. INSERM U1183, Montpellier, France

    Claire Gondeau

  3. Department of Hepato-gastroenterology A, Hospital Saint Eloi, CHRU, Montpellier, France

    Claire Gondeau

  4. Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, USA

    Mohsan Saeed & Charles M. Rice

  5. Istituto Nazionale Genetica Molecolare (INGM) ‘Romeo ed Enrica Invernizzi’, Milan, Italy

    Petra Neddermann & Raffaele De Francesco

  6. Department of Infectious Diseases, Virology, Heidelberg University, Heidelberg, Germany

    Paul Schnitzler

  7. Internal Medicine, University Hospital Heidelberg, Heidelberg, Germany

    Daniel Gotthardt

  8. Liver Unit, Hospital Clínic, IDIBAPS, CIBERehd, Barcelona, Spain

    Sofia Perez-del-Pulgar

  9. Internal Medicine, University Hospital Freiburg, Freiburg im Breisgau, Germany

    Christoph Neumann-Haefelin & Robert Thimme

  10. Department of Clinical Chemistry, Microbiology and Immunology, Ghent University, Gent, Belgium

    Philip Meuleman

  11. Department of General, Regenerative Medicine and Experimental Surgery (ReMediES), Visceral and Transplantation Surgery, Hannover Medical School, Hannover, Germany

    Florian W. R. Vondran

  12. German Center for Infection Research (DZIF), partner site Hannover-Braunschweig, Hannover, Germany

    Florian W. R. Vondran

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Contributions

C.H. conceived and carried out experiments, interpreted the results and wrote the manuscript. I.R.-B. performed the EM experiments. M.M., C.S., P.S. and K.E.-N. did some of the cell-culture experiments. C.G. did some of the PHH experiments and F.W.R.V. provided PHHs. P.S., D.G., S.P.-d.-P., C.N.-H., R.T. and P.M. provided patient sera and contributed with helpful discussions. P.N. and R.D.F. provided H479 and contributed to the conception of the project. M.S. and C.M.R. provided the gt3–5 replicons and Con1 WT replicon cell lines and contributed to the interpretation of the results. R.B. contributed to the concept of the study and to critical discussions. V.L. designed the concept of the project, interpreted the results and wrote the manuscript. All authors contributed to the writing of the manuscript.

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Correspondence to Volker Lohmann.

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Harak, C., Meyrath, M., Romero-Brey, I. et al. Tuning a cellular lipid kinase activity adapts hepatitis C virus to replication in cell culture. Nat Microbiol 2, 16247 (2017). https://doi.org/10.1038/nmicrobiol.2016.247

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