1887

Journal of General Virology header logo

Volume 87, Issue 12

A hepatitis C virus (HCV) NS3/4A protease-dependent strategy for the identification and purification of HCV-infected cells Free

Abstract

As a tool for the identification and/or purification of hepatitis C virus (HCV)-infected cells, a chimeric form of the Gal4VP16 transcription factor was engineered to be activated only in the presence of the HCV NS3/4A protease and to induce different reporter genes [choramphenical acetyltransferase (CAT), green fluorescent protein (GFP) and the cell-surface marker H-2K] through the (Gal4)-E1b promoter. For this, the NS5A/5B -cleavage motif of HCV of genotype 1a was inserted between Gal4VP16 and the N terminus of the endoplasmic reticulum (ER)-resident protein PERK, and it was demonstrated that it could be cleaved specifically by NS3/4A. Accordingly, transient transfection in tetracycline-inducible UHCV-11 cells expressing the HCV polyprotein of genotype 1a revealed the migration of the Gal4VP16 moiety of the chimera from the ER to the nucleus upon HCV expression. Activation of the chimera provoked specific gene induction, as shown by CAT assay, first in UHCV-11 cells and then in Huh-7 cells expressing an HCV replicon of genotype 1b (Huh-7 Rep). In addition, the GFP reporter gene allowed rapid fluorescence monitoring of HCV expression in the Huh-7 Rep cells. Finally, the chimera was introduced into Huh-7.5 cells infected with cell culture-generated HCV JFH1 (genotype 2a), allowing the purification of the HCV-infected cells by immunomagnetic cell sorting using H-2K as gene reporter. In conclusion, the Gal4VP16 chimera activation system can be used for the rapid identification and purification of HCV-infected cells.

  • Received:
  • Accepted:
  • Published Online:
SGM
Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.82214-0
2006-12-01
2024-04-24
Loading full text...

Full text loading...

/deliver/fulltext/jgv/87/12/3587.html?itemId=/content/journal/jgv/10.1099/vir.0.82214-0&mimeType=html&fmt=ahah

References

  1. Bartenschlager B. 1999; The NS3/4A proteinase of the hepatitis C virus: unravelling structure and function of an unusual enzyme and a prime target for antiviral therapy. J Viral Hepat 6:165–181 [CrossRef]
     [Google Scholar]
  2. Bartosch B., Vitelli A., Granier C. & 7 other authors 2003; Cell entry of hepatitis C virus requires a set of co-receptors that include the CD81 tetraspanin and the SR-B1 scavenger receptor. J Biol Chem 278:41624–41630 [CrossRef]
     [Google Scholar]
  3. Beyer B. M., Zhang R., Hong Z., Madison V., Malcolm B. A. 2001; Effect of naturally occuring active site mutations on hepatitis C virus NS3 protease specificity. Proteins Struct Funct Genet 43:82–88 [CrossRef]
     [Google Scholar]
  4. Blanchard E., Brand D., Trassard S., Goudeau A., Roingeard P. 2002; Hepatitis C virus-like particle morphogenesis. J Virol 76:4073–4079 [CrossRef]
     [Google Scholar]
  5. Blight K. J., Kolykhalov A. A., Rice C. M. 2000; Efficient initiation of HCV RNA replication in cell culture. Science 290:1972–1974 [CrossRef]
     [Google Scholar]
  6. 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 [CrossRef]
     [Google Scholar]
  7. Blight K. J., McKeating J. A., Marcotrigiano J., Rice C. M. 2003; Efficient replication of hepatitis C virus genotype 1a RNAs in cell culture. J Virol 77:3181–3190 [CrossRef]
     [Google Scholar]
  8. Bonnet M. C., Weil R., Dam E., Hovanessian A. G., Meurs E. F. 2000; PKR stimulates NF- κ B irrespective of its kinase function by interacting with the I κ B kinase complex. Mol Cell Biol 20:4532–4542 [CrossRef]
     [Google Scholar]
  9. Breiman A., Grandvaux N., Lin R., Ottone C., Akira S., Yoneyama M., Fujita T., Hiscott J., Meurs E. F. 2005; Inhibition of RIG-I-dependent signaling to the interferon pathway during hepatitis C virus expression and restoration of signaling by IKK ε . J Virol 79:3969–3978 [CrossRef]
     [Google Scholar]
  10. Bressanelli S., Tomei L., Roussel A., Incitti I., Vitale R. L., Mathieu M., De Francesco R., Rey F. A. 1999; Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus. Proc Natl Acad Sci U S A 96:13034–13039 [CrossRef]
     [Google Scholar]
  11. Bukh J., Purcell R. H. 2006; A milestone for hepatitis C virus research: a virus generated in cell culture is fully viable in vivo . Proc Natl Acad Sci U S A 103:3500–3501 [CrossRef]
     [Google Scholar]
  12. Buonocore L., Blight K. J., Rice C. M., Rose J. K. 2002; Characterization of vesicular stomatitis virus recombinants that express and incorporate high levels of hepatitis C virus glycoproteins. J Virol 76:6865–6872 [CrossRef]
     [Google Scholar]
  13. Castet V., Fournier C., Soulier A., Brillet R., Coste J., Larrey D., Dhumeaux D., Maurel P., Pawlotski J.-M. 2002; Alpha interferon inhibits hepatitis C virus replication in primary human hepatocytes infected in vitro. J Virol 76:8189–8199 [CrossRef]
     [Google Scholar]
  14. De Francesco R., Tomei L., Altamura S., Summa V., Migliaccio G. 2003; Approaching a new era for hepatitis C virus therapy: inhibitors of the NS3-4A serine protease and the NS5B RNA-dependent RNA polymerase. Antiviral Res 58:1–16 [CrossRef]
     [Google Scholar]
  15. Fernandez J., Bode B., Koromilas A., Diehl J. A., Krukovets I., Snider M. D., Hatzoglou M. 2002; Translation mediated by the internal ribosome entry site of the cat-1 mRNA is regulated by glucose availability in a PERK kinase-dependent manner. J Biol Chem 277:11780–11787 [CrossRef]
     [Google Scholar]
  16. Ferrini J. B., Pichard L., Domergue J., Maurel P. 1997; Long-term primary cultures of adult human hepatocytes. Chem Biol Interact 107:31–45 [CrossRef]
     [Google Scholar]
  17. Fournier C., Sureau C., Coste J., Ducos J., Pageaux G., Larrey D., Domergue J., Maurel P. 1998; In vitro infection of adult normal human hepatocytes in primary culture by hepatits C virus. J Gen Virol 79:2367–2374
     [Google Scholar]
  18. Foy E., Li K., Sumpter R. Jr 8 other authors 2005; Control of antiviral defenses through hepatitis C virus disruption of retinoic acid-inducible gene-I signaling. Proc Natl Acad Sci U S A 102:2986–2991 [CrossRef]
     [Google Scholar]
  19. François C., Duverlie G., Rebouillat D. & 7 other authors 2000; Expression of hepatitis C virus proteins interferes with the antiviral action of interferon independently of PKR-mediated control of protein synthesis. J Virol 74:5587–5596 [CrossRef]
     [Google Scholar]
  20. Giannini C., Morosan S., Tralhao J. G. & 7 other authors 2003; A highly efficient, stable, and rapid approach for ex vivo human liver gene therapy via a FLAP lentiviral vector. Hepatology 38:114–122
     [Google Scholar]
  21. Guo J.-T., Bichko V. V., Seeger C. 2001; Effect of alpha interferon on the hepatitis C virus replicon. J Virol 75:8516–8523 [CrossRef]
     [Google Scholar]
  22. Harding H. P., Zhang Y., Ron D. 1999; Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase. Nature 397:271–274 [CrossRef]
     [Google Scholar]
  23. Hirowatari Y., Hijikata M., Shimotohno K. 1995; A novel method for analysis of viral proteinase activity encoded by hepatitis C virus in cultured cells. Anal Biochem 225:113–120 [CrossRef]
     [Google Scholar]
  24. Ikeda M., Yi M., Li K., Lemon S. M. 2002; Selectable subgenomic and genome-length dicistronic RNAs derived from an infectious molecular clone of the HCV-N strain of hepatitis C virus replicate efficiently in cultured Huh7 cells. J Virol 76:2997–3006 [CrossRef]
     [Google Scholar]
  25. Kanazawa N., Kurosaki M., Sakamoto N. & 13 other authors 2004; Regulation of hepatitis C virus replication by interferon regulatory factor 1. J Virol 78:9713–9720 [CrossRef]
     [Google Scholar]
  26. Kato T., Date T., Miyamoto M., Furusaka A., Tokushige K., Mizokami M., Wakita T. 2003; Efficient replication of the genotype 2a hepatitis C virus subgenomic replicon. Gastroenterology 125:1808–1817 [CrossRef]
     [Google Scholar]
  27. Kaufman R. J. 1999; Stress signaling from the lumen of the endoplasmic reticulum: coordination of gene transcriptional and translational controls. Genes Dev 13:1211–1233 [CrossRef]
     [Google Scholar]
  28. Kolykhalov A. A., Agapov E. V., Blight K. J., Mihalik K., Feinstone S. M., Rice C. M. 1997; Transmission of hepatitis C by intrahepatic inoculation with transcribed RNA. Science 277:570–574 [CrossRef]
     [Google Scholar]
  29. Landro J. A., Raybuck S. A., Luong Y. P. C., O'Malley E. T., Harbeson S. L., Morgenstern K. A., Rao G., Livingston D. J. 1997; Mechanistic role of an NS4A peptide cofactor with the truncated NS3 protease of hepatitis C virus: elucidation of the NS4A stimulatory effect via kinetic analysis and inhibitor mapping. Biochemistry 36:9340–9348 [CrossRef]
     [Google Scholar]
  30. Lee J.-C., Shih Y.-F., Hsu S.-P., Chang T.-Y., Chen L.-H., Hsu J. T. A. 2003; Development of a cell-based assay for monitoring specific hepatitis C virus NS3/4A protease activity in mammalian cells. Anal Biochem 316:162–170 [CrossRef]
     [Google Scholar]
  31. Lerat H., Honda M., Beard M. R. & 8 other authors 2002; Steatosis and liver cancer in transgenic mice expressing the structural and nonstructural proteins of hepatitis C virus. Gastroenterology 122:352–365 [CrossRef]
     [Google Scholar]
  32. Lindenbach B. D., Evans M. J., Syder A. J. & 8 other authors 2005; Complete replication of hepatitis C virus in cell culture. Science 309:623–626 [CrossRef]
     [Google Scholar]
  33. Lindenbach B. D., Meuleman P., Ploss A. & 8 other authors 2006; Cell culture-grown hepatitis C virus is infectious in vivo and can be recultured in vitro . Proc Natl Acad Sci U S A 103:3805–3809 [CrossRef]
     [Google Scholar]
  34. Lohmann V., Körner F., Koch J.-O., Herian U., Theilmann L., Bartenschlager R. 1999; Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science 285:110–113 [CrossRef]
     [Google Scholar]
  35. Ma K., Vattem K. M., Wek R. C. 2002; Dimerization and release of molecular chaperone inhibition facilitate activation of eukaryotic initiation factor-2 kinase in response to endoplasmic reticulum stress. J Biol Chem 277:18728–18735 [CrossRef]
     [Google Scholar]
  36. Maillard P., Huby T., Andréo U., Moreau M., Chapman J., Budkowska A. 2006; The interaction of natural hepatitis C virus with human scavenger receptor SR-BI/Cla1 is mediated by ApoB-containing lipoproteins. FASEB J 20:735–737
     [Google Scholar]
  37. Matsuura Y., Tani H., Susuki K. & 8 other authors 2001; Characterization of pseudotype VSV possessing HCV envelope proteins. Virology 286:263–275 [CrossRef]
     [Google Scholar]
  38. Meurs E., Breiman A., Molina S. & 7 other authors 2003; An HCV NS3/4A protease-dependent strategy for the identification and purification of hepatitis C virus-infected primary human hepatocytes. In 10th International Meeting on Hepatitis C Virus and Related Viruses 2–6 December 2003 Kyoto, Japan:
     [Google Scholar]
  39. Moradpour D., Kary P., Rice C. M., Blum H. E. 1998; Continuous human cell lines inducibly expressing hepatitis C virus structural and nonstructural proteins. Hepatology 28:192–201
     [Google Scholar]
  40. Naldini L., Blömer U., Gallay P., Ory D., Mulligan R., Gage F. H., Verma I. M., Trono D. 1996; In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272:263–267 [CrossRef]
     [Google Scholar]
  41. Pacini L., Bartholomew L., Vitelli A., Migliaccio G. 2004; Reporter substrates for assessing the activity of the hepatitis C virus NS3-4A serine protease in living cells. Anal Biochem 331:46–59 [CrossRef]
     [Google Scholar]
  42. Pavio N., Romano P. R., Graczyk T. M., Feinstone S. M., Taylor D. R. 2003; Protein synthesis and endoplasmic reticulum stress can be modulated by the hepatitis C virus envelope protein E2 through the eukaryotic initiation factor 2 α kinase PERK. J Virol 77:3578–3585 [CrossRef]
     [Google Scholar]
  43. Penin F. 2003; Structural biology of hepatitis C virus. Clin Liver Dis 7:1–21 [CrossRef]
     [Google Scholar]
  44. Pietschmann T., Lohmann V., Kaul A., Krieger N., Rinck G., Rutter G., Strand D., Bartenschlager R. 2002; Persistent and transient replication of full-length hepatitis C virus genomes in cell culture. J Virol 76:4008–4021 [CrossRef]
     [Google Scholar]
  45. Rouillé Y., Helle F., Delgrange D. & 10 other authors 2006; Subcellular localization of hepatitis C virus structural proteins in a cell culture system that efficiently replicates the virus. J Virol 80:2832–2841 [CrossRef]
     [Google Scholar]
  46. Rumin S., Berthillon P., Tanaka E. & 8 other authors 1999; Dynamic analysis of hepatitis C virus replication and quasispecies selection in long-term cultures of adult human hepatocytes infected in vitro . J Gen Virol 80:3007–3018
     [Google Scholar]
  47. Shi Y., Vattem K. M., Sood R., An J., Liang J., Stramm L., Wek R. C. 1998; Identification and characterization of pancreatic eukaryotic factor 2 α -subunit kinase, PEK, involved in translational control. Mol Cell Biol 18:7499–7509
     [Google Scholar]
  48. Shi Y., An J., Liang J., Hayes S. E., Sandusky G. E., Stramm L. E., Yang N. N. 1999; Characterization of a mutant pancreatic eIF-2 α kinase, PEK, and co-localization with somatostatin in islet delta cells. J Biol Chem 274:5723–5730 [CrossRef]
     [Google Scholar]
  49. Subkhankulova T., Mitchell S. A., Willis A. E. 2001; Internal ribosome entry segment-mediated initiation of c-Myc protein synthesis following genotoxic stress. Biochem J 359:183–192 [CrossRef]
     [Google Scholar]
  50. Terrault N. A., Dailey P. J., Ferrell L., Collins M. L., Wilber J. C., Urdea M. S., Bhandari B. N., Wright T. L. 1997; Hepatitis C virus: quantitation and distribution in liver. J Med Virol 51:217–224 [CrossRef]
     [Google Scholar]
  51. Wakita T., Pietschmann T., Kato T. & 9 other authors 2005; Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat Med 11:791–796 [CrossRef]
     [Google Scholar]
  52. Yan Y., Li Y., Munshi S. & 8 other authors 1998; Complex of NS3 protease and NS4A peptide of BK strain hepatitis C virus: a 2.2 A resolution structure in a hexagonal crystal form. Protein Sci 7:837–847
     [Google Scholar]
  53. Yanagi M., Purcell R. H., Emerson S. U., Bukh J. 1997; Transcripts from a single full-length cDNA clone of hepatitis C virus are infectious when directly transfected into the liver of a chimpanzee. Proc Natl Acad Sci U S A 94:8738–8743 [CrossRef]
     [Google Scholar]
  54. Yi M., Lemon S. M. 2004; Adaptive mutations producing efficient replication of genotype 1a hepatitis C virus RNA in normal Huh7 cells. J Virol 78:7904–7915 [CrossRef]
     [Google Scholar]
  55. Zennou V., Petit C., Guetard D., Nerhbass U., Montagnier L., Charneau P. 2000; HIV-1 genome nuclear import is mediated by a central DNA flap. Cell 101:173–185 [CrossRef]
     [Google Scholar]
  56. Zhong J., Gastaminza P., Cheng G. & 7 other authors 2005; Robust hepatitis C virus infection in vitro . Proc Natl Acad Sci U S A 102:9294–9299 [CrossRef]
     [Google Scholar]
  57. Zoulim F., Chevallier M., Maynard M., Trepo C. 2003; Clinical consequences of hepatitis C virus infection. Rev Med Virol 13:57–68 [CrossRef]
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.82214-0
Loading
A hepatitis C virus (HCV) NS3/4A protease-dependent strategy for the identification and purification of HCV-infected cells
J. Gen. Virol. 87, 3587 (2006); https://doi.org/10.1099/vir.0.82214-0
/content/journal/jgv/10.1099/vir.0.82214-0
/content/journal/jgv/10.1099/vir.0.82214-0
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF

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