1887

Journal of General Virology header logo

Volume 95, Issue 2

Direct visualization of hepatitis C virus-infected Huh7.5 cells with a high titre of infectious chimeric JFH1-EGFP reporter virus in three-dimensional Matrigel cell cultures Free

Abstract

Identification of the hepatitis C virus (HCV) JFH1 isolate enabled the development of infectious HCV cell culture systems. However, the relatively low virus titres and instability of some chimeric JFH1 reporter viruses restricts some uses of this system. We describe a higher-titre JFH1-EGFP reporter virus where the NS5A V3 region was replaced with the EGFP gene and adapted by serial passage in Huh7.5 cells. Six adaptive mutants were identified: one each in E2, P7 and NS4B, plus three in the NS5A region. These adaptive mutants increased the reporter virus titres to 1×10 immunofluorescent focus-forming units ml, which is the highest titre of JFH1-EGFP reporter virus reported to our knowledge. This chimeric virus did not lose EGFP expression following 40 days of passage and it can be used to test the activity of HCV antivirals by measuring EGFP fluorescence in 96-well plates. Moreover, this reporter virus allows living infected Huh7.5 cells in Matrigel three-dimensional (3D) cultures to be visualized and produces infectious viral particles in these 3D cultures. The chimeric NS5A-EGFP infectious JFH1 reporter virus described should enable new studies of the HCV life cycle in 3D cell cultures and will be useful in identifying antivirals that interfere with HCV release or entry.

  • Received:
  • Accepted:
  • Published Online:
© Microbiology Society
Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.055772-0
2014-02-01
2024-04-19
Loading full text...

Full text loading...

/deliver/fulltext/jgv/95/2/423.html?itemId=/content/journal/jgv/10.1099/vir.0.055772-0&mimeType=html&fmt=ahah

References

  1. Aizaki H., Morikawa K., Fukasawa M., Hara H., Inoue Y., Tani H., Saito K., Nishijima M., Hanada K.other authors 2008; Critical role of virion-associated cholesterol and sphingolipid in hepatitis C virus infection. J Virol 82:5715–5724 [View Article][PubMed]
     [Google Scholar]
  2. Alter H. J., Seeff L. B. 2000; Recovery, persistence, and sequelae in hepatitis C virus infection: a perspective on long-term outcome. Semin Liver Dis 20:17–35 [View Article][PubMed]
     [Google Scholar]
  3. Appel N., Pietschmann T., Bartenschlager R. 2005; Mutational analysis of hepatitis C virus nonstructural protein 5A: potential role of differential phosphorylation in RNA replication and identification of a genetically flexible domain. J Virol 79:3187–3194 [View Article][PubMed]
     [Google Scholar]
  4. Bialek S. R., Terrault N. A. 2006; The changing epidemiology and natural history of hepatitis C virus infection. Clin Liver Dis 10:697–715 [View Article][PubMed]
     [Google Scholar]
  5. Blight K. J., McKeating J. A., Rice C. M. 2002; Highly permissive cell lines for subgenomic and genomic hepatitis C virus RNA replication. J Virol 76:13001–13014 [View Article][PubMed]
     [Google Scholar]
  6. Bowen D. G., Walker C. M. 2005; Adaptive immune responses in acute and chronic hepatitis C virus infection. Nature 436:946–952 [View Article][PubMed]
     [Google Scholar]
  7. Brass V., Bieck E., Montserret R., Wölk B., Hellings J. A., Blum H. E., Penin F., Moradpour D. 2002; An amino-terminal amphipathic alpha-helix mediates membrane association of the hepatitis C virus nonstructural protein 5A. J Biol Chem 277:8130–8139 [View Article][PubMed]
     [Google Scholar]
  8. Cai Z., Zhang C., Chang K. S., Jiang J., Ahn B. C., Wakita T., Liang T. J., Luo G. 2005; Robust production of infectious hepatitis C virus (HCV) from stably HCV cDNA-transfected human hepatoma cells. J Virol 79:13963–13973 [View Article][PubMed]
     [Google Scholar]
  9. Eldrup A. B., Allerson C. R., Bennett C. F., Bera S., Bhat B., Bhat N., Bosserman M. R., Brooks J., Burlein C.other authors 2004; Structure-activity relationship of purine ribonucleosides for inhibition of hepatitis C virus RNA-dependent RNA polymerase. J Med Chem 47:2283–2295 [View Article][PubMed]
     [Google Scholar]
  10. Evans M. J., Rice C. M., Goff S. P. 2004; Phosphorylation of hepatitis C virus nonstructural protein 5A modulates its protein interactions and viral RNA replication. Proc Natl Acad Sci U S A 101:13038–13043 [View Article][PubMed]
     [Google Scholar]
  11. Fried M. W., Shiffman M. L., Reddy K. R., Smith C., Marinos G., Gonçales F. L. Jr, Häussinger D., Diago M., Carosi G.other authors 2002; Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection. N Engl J Med 347:975–982 [View Article][PubMed]
     [Google Scholar]
  12. Guo J. T., Bichko V. V., Seeger C. 2001; Effect of alpha interferon on the hepatitis C virus replicon. J Virol 75:8516–8523 [View Article][PubMed]
     [Google Scholar]
  13. Härmä V., Virtanen J., Mäkelä R., Happonen A., Mpindi J. P., Knuuttila M., Kohonen P., Lötjönen J., Kallioniemi O., Nees M. 2010; A comprehensive panel of three-dimensional models for studies of prostate cancer growth, invasion and drug responses. PLoS ONE 5:e10431 [View Article][PubMed]
     [Google Scholar]
  14. Houghton M., Abrignani S. 2005; Prospects for a vaccine against the hepatitis C virus. Nature 436:961–966 [View Article][PubMed]
     [Google Scholar]
  15. Inchauspe G., Zebedee S., Lee D. H., Sugitani M., Nasoff M., Prince A. M. 1991; Genomic structure of the human prototype strain H of hepatitis C virus: comparison with American and Japanese isolates. Proc Natl Acad Sci U S A 88:10292–10296 [View Article][PubMed]
     [Google Scholar]
  16. Jiang J., Luo G. 2012; Cell culture-adaptive mutations promote viral protein-protein interactions and morphogenesis of infectious hepatitis C virus. J Virol 86:8987–8997 [View Article][PubMed]
     [Google Scholar]
  17. Jones C. T., Murray C. L., Eastman D. K., Tassello J., Rice C. M. 2007; Hepatitis C virus p7 and NS2 proteins are essential for production of infectious virus. J Virol 81:8374–8383 [View Article][PubMed]
     [Google Scholar]
  18. Kato T., Furusaka A., Miyamoto M., Date T., Yasui K., Hiramoto J., Nagayama K., Tanaka T., Wakita T. 2001; Sequence analysis of hepatitis C virus isolated from a fulminant hepatitis patient. J Med Virol 64:334–339 [View Article][PubMed]
     [Google Scholar]
  19. Kaul A., Woerz I., Meuleman P., Leroux-Roels G., Bartenschlager R. 2007; Cell culture adaptation of hepatitis C virus and in vivo viability of an adapted variant. J Virol 81:13168–13179 [View Article][PubMed]
     [Google Scholar]
  20. Kim C. S., Jung J. H., Wakita T., Yoon S. K., Jang S. K. 2007; Monitoring the antiviral effect of alpha interferon on individual cells. J Virol 81:8814–8820 [View Article][PubMed]
     [Google Scholar]
  21. Kim C. S., Keum S. J., Jang S. K. 2011; Generation of a cell culture-adapted hepatitis C virus with longer half life at physiological temperature. PLoS ONE 6:e22808 [View Article][PubMed]
     [Google Scholar]
  22. Kleinman H. K., Martin G. R. 2005; Matrigel: basement membrane matrix with biological activity. Semin Cancer Biol 15:378–386 [View Article][PubMed]
     [Google Scholar]
  23. Koutsoudakis G., Kaul A., Steinmann E., Kallis S., Lohmann V., Pietschmann T., Bartenschlager R. 2006; Characterization of the early steps of hepatitis C virus infection by using luciferase reporter viruses. J Virol 80:5308–5320 [View Article][PubMed]
     [Google Scholar]
  24. Krieger N., Lohmann V., Bartenschlager R. 2001; Enhancement of hepatitis C virus RNA replication by cell culture-adaptive mutations. J Virol 75:4614–4624 [View Article][PubMed]
     [Google Scholar]
  25. Li Y. P., Ramirez S., Jensen S. B., Purcell R. H., Gottwein J. M., Bukh J. 2012; Highly efficient full-length hepatitis C virus genotype 1 (strain TN) infectious culture system. Proc Natl Acad Sci U S A 109:19757–19762 [View Article][PubMed]
     [Google Scholar]
  26. Lindenbach B. D., Evans M. J., Syder A. J., Wölk B., Tellinghuisen T. L., Liu C. C., Maruyama T., Hynes R. O., Burton D. R.other authors 2005; Complete replication of hepatitis C virus in cell culture. Science 309:623–626 [View Article][PubMed]
     [Google Scholar]
  27. Liu S., Nelson C. A., Xiao L., Lu L., Seth P. P., Davis D. R., Hagedorn C. H. 2011; Measuring antiviral activity of benzimidazole molecules that alter IRES RNA structure with an infectious hepatitis C virus chimera expressing Renilla luciferase . Antiviral Res 89:54–63 [View Article][PubMed]
     [Google Scholar]
  28. Liu S., Xiao L., Nelson C., Hagedorn C. H. 2012; A cell culture adapted HCV JFH1 variant that increases viral titers and permits the production of high titer infectious chimeric reporter viruses. PLoS ONE 7:e44965 [View Article][PubMed]
     [Google Scholar]
  29. Miyanari Y., Atsuzawa K., Usuda N., Watashi K., Hishiki T., Zayas M., Bartenschlager R., Wakita T., Hijikata M., Shimotohno K. 2007; The lipid droplet is an important organelle for hepatitis C virus production. Nat Cell Biol 9:1089–1097 [View Article][PubMed]
     [Google Scholar]
  30. Molina-Jimenez F., Benedicto I., Dao Thi V. L., Gondar V., Lavillette D., Marin J. J., Briz O., Moreno-Otero R., Aldabe R.other authors 2012; Matrigel-embedded 3D culture of Huh-7 cells as a hepatocyte-like polarized system to study hepatitis C virus cycle. Virology 425:31–39 [View Article][PubMed]
     [Google Scholar]
  31. Moradpour D., Penin F., Rice C. M. 2007; Replication of hepatitis C virus. Nat Rev Microbiol 5:453–463 [View Article][PubMed]
     [Google Scholar]
  32. Papic N., Maxwell C. I., Delker D. A., Liu S., Heale B. S., Hagedorn C. H. 2012; RNA-sequencing analysis of 5′ capped RNAs identifies many new differentially expressed genes in acute hepatitis C virus infection. Viruses 4:581–612 [View Article][PubMed]
     [Google Scholar]
  33. Penin F., Dubuisson J., Rey F. A., Moradpour D., Pawlotsky J. M. 2004; Structural biology of hepatitis C virus. Hepatology 39:5–19 [View Article][PubMed]
     [Google Scholar]
  34. Phan T., Beran R. K., Peters C., Lorenz I. C., Lindenbach B. D. 2009; Hepatitis C virus NS2 protein contributes to virus particle assembly via opposing epistatic interactions with the E1-E2 glycoprotein and NS3-NS4A enzyme complexes. J Virol 83:8379–8395 [View Article][PubMed]
     [Google Scholar]
  35. Reed K. E., Rice C. M. 2000; Overview of hepatitis C virus genome structure, polyprotein processing, and protein properties. Curr Top Microbiol Immunol 242:55–84 [View Article][PubMed]
     [Google Scholar]
  36. Sainz B. Jr, TenCate V., Uprichard S. L. 2009; Three-dimensional Huh7 cell culture system for the study of Hepatitis C virus infection. Virol J 6:103 [View Article][PubMed]
     [Google Scholar]
  37. Tellinghuisen T. L., Marcotrigiano J., Gorbalenya A. E., Rice C. M. 2004; The NS5A protein of hepatitis C virus is a zinc metalloprotein. J Biol Chem 279:48576–48587 [View Article][PubMed]
     [Google Scholar]
  38. Tellinghuisen T. L., Marcotrigiano J., Rice C. M. 2005; Structure of the zinc-binding domain of an essential component of the hepatitis C virus replicase. Nature 435:374–379 [View Article][PubMed]
     [Google Scholar]
  39. Tscherne D. M., Jones C. T., Evans M. J., Lindenbach B. D., McKeating J. A., Rice C. M. 2006; Time- and temperature-dependent activation of hepatitis C virus for low-pH-triggered entry. J Virol 80:1734–1741 [View Article][PubMed]
     [Google Scholar]
  40. Wakita T., Pietschmann T., Kato T., Date T., Miyamoto M., Zhao Z., Murthy K., Habermann A., Kräusslich H. G.other authors 2005; Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat Med 11:791–796 [View Article][PubMed]
     [Google Scholar]
  41. Yi M., Ma Y., Yates J., Lemon S. M. 2007; Compensatory mutations in E1, p7, NS2, and NS3 enhance yields of cell culture-infectious intergenotypic chimeric hepatitis C virus. J Virol 81:629–638 [View Article][PubMed]
     [Google Scholar]
  42. Zhong J., Gastaminza P., Cheng G., Kapadia S., Kato T., Burton D. R., Wieland S. F., Uprichard S. L., Wakita T., Chisari F. V. 2005; Robust hepatitis C virus infection in vitro. Proc Natl Acad Sci U S A 102:9294–9299 [View Article][PubMed]
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.055772-0
Loading
Direct visualization of hepatitis C virus-infected Huh7.5 cells with a high titre of infectious chimeric JFH1-EGFP reporter virus in three-dimensional Matrigel cell cultures
J. Gen. Virol. 95, 423 (2014); https://doi.org/10.1099/vir.0.055772-0
/content/journal/jgv/10.1099/vir.0.055772-0
/content/journal/jgv/10.1099/vir.0.055772-0
Loading

Data & Media loading...

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error