Letter | Published:

SEC14L2 enables pan-genotype HCV replication in cell culture

Nature volume 524, pages 471475 (27 August 2015) | Download Citation

Abstract

Since its discovery in 1989, efforts to grow clinical isolates of the hepatitis C virus (HCV) in cell culture have met with limited success. Only the JFH-1 isolate has the capacity to replicate efficiently in cultured hepatoma cells without cell culture-adaptive mutations1,2,3. We hypothesized that cultured cells lack one or more factors required for the replication of clinical isolates. To identify the missing factors, we transduced Huh-7.5 human hepatoma cells with a pooled lentivirus-based human complementary DNA (cDNA) library, transfected the cells with HCV subgenomic replicons lacking adaptive mutations, and selected for stable replicon colonies. This led to the identification of a single cDNA, SEC14L2, that enabled RNA replication of diverse HCV genotypes in several hepatoma cell lines. This effect was dose-dependent, and required the continuous presence of SEC14L2. Full-length HCV genomes also replicated and produced low levels of infectious virus. Remarkably, SEC14L2-expressing Huh-7.5 cells also supported HCV replication following inoculation with patient sera. Mechanistic studies suggest that SEC14L2 promotes HCV infection by enhancing vitamin E-mediated protection against lipid peroxidation. This provides a foundation for development of in vitro replication systems for all HCV isolates, creating a useful platform to dissect the mechanisms by which cell culture-adaptive mutations act.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    , & Efficient initiation of HCV RNA replication in cell culture. Science 290, 1972–1974 (2000)

  2. 2.

    et al. Efficient replication of genotype 3a and 4a hepatitis C virus replicons in human hepatoma cells. Antimicrob. Agents Chemother. 56, 5365–5373 (2012)

  3. 3.

    . et al. Efficient replication of the genotype 2a hepatitis C virus subgenomic replicon. Gastroenterology 125, 1808–1817 (2003)

  4. 4.

    , , & Global epidemiology of hepatitis C virus infection: new estimates of age-specific antibody to HCV seroprevalence. Hepatology 57, 1333–1342 (2013)

  5. 5.

    et al. Novel infectious cDNA clones of hepatitis C virus genotype 3a (strain S52) and 4a (strain ED43): genetic analyses and in vivo pathogenesis studies. J. Virol. 84, 5277–5293 (2010)

  6. 6.

    et al. The NS3 helicase and NS5B-to-3′X regions are important for efficient hepatitis C virus strain JFH-1 replication in Huh7 cells. J. Virol. 81, 8030–8040 (2007)

  7. 7.

    et al. RNA polymerase activity and specific RNA structure are required for efficient HCV replication in cultured cells. PLoS Pathog. 6, e1000885 (2010)

  8. 8.

    et al. Mutations that permit efficient replication of hepatitis C virus RNA in Huh-7 cells prevent productive replication in chimpanzees. Proc. Natl Acad. Sci. USA 99, 14416–14421 (2002)

  9. 9.

    , & Current thoughts on the phosphatidylinositol transfer protein family. FEBS Lett. 531, 74–80 (2002)

  10. 10.

    , & Sec14p-like domains in NF1 and Dbl-like proteins indicate lipid regulation of Ras and Rho signaling. Curr. Biol. 9, R195–R197 (1999)

  11. 11.

    et al. Cloning of novel human SEC14p-like proteins: ligand binding and functional properties. Free Radic. Biol. Med. 34, 1458–1472 (2003)

  12. 12.

    et al. Real-time imaging of hepatitis C virus infection using a fluorescent cell-based reporter system. Nature Biotechnol. 28, 167–171 (2010)

  13. 13.

    et al. Prevention of hepatitis C virus infection in chimpanzees by hyperimmune serum against the hypervariable region 1 of the envelope 2 protein. Proc. Natl Acad. Sci. USA 93, 15394–15399 (1996)

  14. 14.

    et al. A humanized mouse model to study hepatitis C virus infection, immune response, and liver disease. Gastroenterology 140, 1334–1344 (2011)

  15. 15.

    et al. Ligand specificity in the CRAL-TRIO protein family. Biochemistry 42, 6467–6474 (2003)

  16. 16.

    et al. Tocopherol-associated protein suppresses prostate cancer cell growth by inhibition of the phosphoinositide 3-kinase pathway. Cancer Res. 65, 9807–9816 (2005)

  17. 17.

    , & Supernatant protein factor stimulates HMG-CoA reductase in cell culture and in vitro. Arch. Biochem. Biophys. 433, 474–480 (2005)

  18. 18.

    & Supernatant protein factor requires phosphorylation and interaction with Golgi to stimulate cholesterol synthesis in hepatoma cells. Arch. Biochem. Biophys. 435, 175–181 (2005)

  19. 19.

    , , & Tocopherol-associated protein-1 accelerates apoptosis induced by alpha-tocopheryl succinate in mesothelioma cells. Biochem. Biophys. Res. Commun. 343, 1113–1117 (2006)

  20. 20.

    et al. Regulation of the hepatitis C virus RNA replicase by endogenous lipid peroxidation. Nature Med. 20, 927–935 (2014)

  21. 21.

    et al. Positive modulation of IL-12 signaling by sphingosine kinase 2 associating with the IL-12 receptor beta 1 cytoplasmic region. J. Immunol. 171, 1352–1359 (2003)

  22. 22.

    et al. Update on hepatitis C virus resistance to direct-acting antiviral agents. Antiviral Res. 108, 181–191 (2014)

  23. 23.

    et al. Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science 285, 110–113 (1999)

  24. 24.

    et al. Hepatitis C virus genotype 5a subgenomic replicons for evaluation of direct-acting antiviral agents. Antimicrob. Agents Chemother. 58, 5386–5394 (2014)

  25. 25.

    et al. Host-cell sensors for Plasmodium activate innate immunity against liver-stage infection. Nature Med. 20, 47–53 (2014)

  26. 26.

    , , & Hepatitis C virus: an infectious molecular clone of a second major genotype (2a) and lack of viability of intertypic 1a and 2a chimeras. Virology 262, 250–263 (1999)

  27. 27.

    , , & Mutations in hepatitis C virus RNAs conferring cell culture adaptation. J. Virol. 75, 1437–1449 (2001)

  28. 28.

    , , & Efficient replication of hepatitis C virus genotype 1a RNAs in cell culture. J. Virol. 77, 3181–3190 (2003)

  29. 29.

    et al. Protease inhibitor-resistant hepatitis C virus mutants with reduced fitness from impaired production of infectious virus. Gastroenterology 140, 667–675 (2011)

  30. 30.

    et al. Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nature Med. 11, 791–796 (2005)

  31. 31.

    et al. Complete replication of hepatitis C virus in cell culture. Science 309, 623–626 (2005)

  32. 32.

    et al. Cell culture-produced hepatitis C virus does not infect peripheral blood mononuclear cells. Hepatology 48, 1843–1850 (2008)

  33. 33.

    et al. Expression of microRNA miR-122 facilitates an efficient replication in nonhepatic cells upon infection with hepatitis C virus. J. Virol. 86, 7918–7933 (2012)

  34. 34.

    , & Pooled shRNA screenings: experimental approach. Methods Mol. Biol. 980, 353–370 (2013)

  35. 35.

    et al. Identification and characterization of the host protein DNAJC14 as a broadly active flavivirus replication modulator. PLoS Pathog. 7, e1001255 (2011)

  36. 36.

    et al. Dengue reporter viruses reveal viral dynamics in interferon receptor-deficient mice and sensitivity to interferon effectors in vitro. Proc. Natl Acad. Sci. USA 109, 14610–14615 (2012)

  37. 37.

    , , , & Identification of adult mouse neurovirulence determinants of the Sindbis virus strain AR86. J. Virol. 79, 4219–4228 (2005)

  38. 38.

    et al. Differential induction of type I interferon responses in myeloid dendritic cells by mosquito and mammalian-cell-derived alphaviruses. J. Virol. 81, 237–247 (2007)

  39. 39.

    et al. A stable full-length yellow fever virus cDNA clone and the role of conserved RNA elements in flavivirus replication. J. Gen. Virol. 84, 1261–1268 (2003)

  40. 40.

    et al. Construction of infectious cDNA clones for dengue 2 virus: strain 16681 and its attenuated vaccine derivative, strain PDK-53. Virology 230, 300–308 (1997)

  41. 41.

    , , , & Seed sequence-matched controls reveal limitations of small interfering RNA knockdown in functional and structural studies of hepatitis C virus NS5A-MOBKL1B interaction. J. Virol. 88, 11022–11033 (2014)

  42. 42.

    , , & Fluorescent proteins as proteomic probes. Mol. Cell. Proteomics 4, 1933–1941 (2005)

  43. 43.

    et al. Lipid peroxidation and oxidant stress regulate hepatic apolipoprotein B degradation and VLDL production. J. Clin. Invest. 113, 1277–1287 (2004)

  44. 44.

    et al. Analysis of functional differences between hepatitis C virus NS5A of genotypes 1-7 in infectious cell culture systems. PLoS Pathog. 8, e1002696 (2012)

  45. 45.

    et al. Regulation of hepatitis C virus replication by nuclear translocation of nonstructural 5A protein and transcriptional activation of host genes. J. Virol. 87, 5523–5539 (2013)

Download references

Acknowledgements

We thank Y. Matsuura for HEP3B/miR-122 cells, J. Bukh for pJ6CF and pTNcc/GLuc, S. M. Lemon and D. Yamane for pH77S.3/GLuc, pH77S.3, and pH77D/GLuc, and D. Manor for [14C]RRR-α-tocopherol. We also thank E. Castillo and A. Webson for laboratory assistance, H. H. Hoffman and L. Andrus for technical input, and W. M. Schneider, M. M. Li and M. R. MacDonald for critical reading of the manuscript. This work was supported in part by the National Institutes of Health, NCI grant R01CA057973, NIAID grants R01AI072613 and R01AI099284 (to C.M.R.), NIDDK grant R01DK090317 and NIDA grant DA031095 (to A.D.B.), by a Helmsley Postdoctoral Fellowship for Basic and Translational Research on Disorders of the Digestive System at The Rockefeller University (to M.S.), and by a Liver Scholar Award from American Association for the Study of Liver Diseases (U.A.). The Greenberg Medical Research Institute, the Starr Foundation, the Ronald A. Shellow, M.D. Memorial Fund and anonymous donors provided the additional funding (to C.M.R.).

Author information

Affiliations

  1. Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, New York 10065, USA

    • Mohsan Saeed
    • , Ursula Andreo
    • , Hyo-Young Chung
    • , Christine Espiritu
    •  & Charles M. Rice
  2. Division of Liver Diseases, Icahn School of Medicine at Mount Sinai, New York, New York 10029, USA

    • Andrea D. Branch
  3. Department of Pathology, Icahn School of Medicine at Mount Sinai, New York, New York 10029, USA

    • Jose M. Silva

Authors

  1. Search for Mohsan Saeed in:

  2. Search for Ursula Andreo in:

  3. Search for Hyo-Young Chung in:

  4. Search for Christine Espiritu in:

  5. Search for Andrea D. Branch in:

  6. Search for Jose M. Silva in:

  7. Search for Charles M. Rice in:

Contributions

M.S. and C.M.R. designed the project, analysed results, and wrote the manuscript. M.S., U.A., H.-Y.C. and C.E. performed the experimental work. A.D.B. and J.M.S. contributed reagents and advice.

Competing interests

C.M.R. has equity in Apath, LLC, which holds commercial licenses for the Huh-7.5 cell line and certain HCV cell culture systems.

Corresponding author

Correspondence to Charles M. Rice.

Extended data

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nature14899

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.