1887

Abstract

Hepatitis C virus (HCV) infects over 130 million people causing a worldwide epidemic of liver cirrhosis and hepatocellular-carcinoma. Because current HCV treatments are only partially effective, molecular mechanisms involved in HCV propagation are actively being pursued as possible drug targets. Here, we report on a new macromolecular interaction between the HCV capsid core protein and the helicase portion of HCV non-structural protein 3 (NS3h), confirmed by four different biochemical methods. The protease portion of NS3 is not required. Interaction between the two proteins could be disrupted by two types of specific inhibitors of core dimerization, the small molecule SL201 and core106, a C-terminally truncated core protein. Cross-linking experiments suggest that the physical interaction with NS3h is probably driven by core oligomerization. Moreover, SL201 blocks the production of infectious virus, but not the production of a subgenomic HCV replicon by hepatoma cells. Time-of-addition experiments confirm that SL201 has no effect on entry of the virus. These data underline the essential role of core as a key organizer of HCV particle assembly, confirm the importance of oligomerization, reveal the interaction with viral helicase and support a new molecular understanding of the formation of the viral particle at the level of the lipid droplets, before its migration to the site of release and budding.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.023325-0
2011-01-01
2019-11-19
Loading full text...

Full text loading...

/deliver/fulltext/jgv/92/1/101.html?itemId=/content/journal/jgv/10.1099/vir.0.023325-0&mimeType=html&fmt=ahah

References

  1. Alter, M. J. ( 2007; ). Epidemiology of hepatitis C virus infection. World J Gastroenterol 13, 2436–2441.[CrossRef]
    [Google Scholar]
  2. Angus, A. G., Dalrymple, D., Boulant, S., McGivern, D. R., Clayton, R. F., Scott, M. J., Adair, R., Graham, S., Owsianka, A. M. & other authors ( 2010; ). Requirement of cellular DDX3 for hepatitis C virus replication is unrelated to its interaction with the viral core protein. J Gen Virol 91, 122–132.[CrossRef]
    [Google Scholar]
  3. Appel, N., Zayas, M., Miller, S., Krijnse-Locker, J., Schaller, T., Friebe, P., Kallis, S., Engel, U. & Bartenschlager, R. ( 2008; ). Essential role of domain III of nonstructural protein 5A for hepatitis C virus infectious particle assembly. PLoS Pathog 4, e1000035.[CrossRef]
    [Google Scholar]
  4. Belon, C. A. & Frick, D. N. ( 2008; ). Monitoring helicase activity with molecular beacons. Biotechniques 45, 433–440, 442.[CrossRef]
    [Google Scholar]
  5. Boulant, S., Vanbelle, C., Ebel, C., Penin, F. & Lavergne, J. P. ( 2005; ). Hepatitis C virus core protein is a dimeric alpha-helical protein exhibiting membrane protein features. J Virol 79, 11353–11365.[CrossRef]
    [Google Scholar]
  6. Boulant, S., Montserret, R., Hope, R. G., Ratinier, M., Targett-Adams, P., Lavergne, J. P., Penin, F. & McLauchlan, J. ( 2006; ). Structural determinants that target the hepatitis C virus core protein to lipid droplets. J Biol Chem 281, 22236–22247.[CrossRef]
    [Google Scholar]
  7. Chen, W., Zhang, Z., Chen, J., Zhang, J., Zhang, J., Wu, Y., Huang, Y., Cai, X. & Huang, A. ( 2008; ). HCV core protein interacts with Dicer to antagonize RNA silencing. Virus Res 133, 250–258.[CrossRef]
    [Google Scholar]
  8. Choo, Q. L., Kuo, G., Weiner, A. J., Overby, L. R., Bradley, D. W. & Houghton, M. ( 1989; ). Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 244, 359–362.[CrossRef]
    [Google Scholar]
  9. Frick, D. N., Rypma, R. S., Lam, A. M. & Gu, B. ( 2004; ). The nonstructural protein 3 protease/helicase requires an intact protease domain to unwind duplex RNA efficiently. J Biol Chem 279, 1269–1280.[CrossRef]
    [Google Scholar]
  10. Gallinari, P., Brennan, D., Nardi, C., Brunetti, M., Tomei, L., Steinkuhler, C. & De Francesco, R. ( 1998; ). Multiple enzymatic activities associated with recombinant NS3 protein of hepatitis C virus. J Virol 72, 6758–6769.
    [Google Scholar]
  11. Jirasko, V., Montserret, R., Appel, N., Janvier, A., Eustachi, L., Brohm, C., Steinmann, E., Pietschmann, T., Penin, F. & Bartenschlager, R. ( 2008; ). Structural and functional characterization of nonstructural protein 2 for its role in hepatitis C virus assembly. J Biol Chem 283, 28546–28562.[CrossRef]
    [Google Scholar]
  12. 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.[CrossRef]
    [Google Scholar]
  13. Jones, D. M., Patel, A. H., Targett-Adams, P. & McLauchlan, J. ( 2009; ). The hepatitis C virus NS4B protein can trans-complement viral RNA replication and modulates production of infectious virus. J Virol 83, 2163–2177.[CrossRef]
    [Google Scholar]
  14. Klein, K. C., Polyak, S. J. & Lingappa, J. R. ( 2004; ). Unique features of hepatitis C virus capsid formation revealed by de novo cell-free assembly. J Virol 78, 9257–9269.[CrossRef]
    [Google Scholar]
  15. Kota, S., Coito, C., Mousseau, G., Lavergne, J. P. & Strosberg, A. D. ( 2009; ). Peptide inhibitors of hepatitis C virus core oligomerization and virus production. J Gen Virol 90, 1319–1328.[CrossRef]
    [Google Scholar]
  16. Kota, S., Scampavia, L., Spicer, T., Beeler, A. B., Takahashi, V., Snyder, J. K., Porco, J. A., Hodder, P. & Strosberg, A. D. ( 2010; ). A time-resolved fluorescence-resonance energy transfer assay for identifying inhibitors of hepatitis C virus core dimerization. Assay Drug Dev Technol 8, 96–105.[CrossRef]
    [Google Scholar]
  17. Kunkel, M., Lorinczi, M., Rijnbrand, R., Lemon, S. M. & Watowich, S. J. ( 2001; ). Self-assembly of nucleocapsid-like particles from recombinant hepatitis C virus core protein. J Virol 75, 2119–2129.[CrossRef]
    [Google Scholar]
  18. Lam, A. M., Keeney, D., Eckert, P. Q. & Frick, D. N. ( 2003; ). Hepatitis C virus NS3 ATPases/helicases from different genotypes exhibit variations in enzymatic properties. J Virol 77, 3950–3961.[CrossRef]
    [Google Scholar]
  19. Lavanchy, D. ( 2009; ). The global burden of hepatitis C. Liver Int 29 (Suppl. 1), 74–81.
    [Google Scholar]
  20. Lindenbach, B. D., Evans, M. J., Syder, A. J., Wolk, 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.[CrossRef]
    [Google Scholar]
  21. Ma, Y., Yates, J., Liang, Y., Lemon, S. M. & Yi, M. ( 2008; ). NS3 helicase domains involved in infectious intracellular hepatitis C virus particle assembly. J Virol 82, 7624–7639.[CrossRef]
    [Google Scholar]
  22. Mamiya, N. & Worman, H. J. ( 1999; ). Hepatitis C virus core protein binds to a DEAD box RNA helicase. J Biol Chem 274, 15751–15756.[CrossRef]
    [Google Scholar]
  23. Masaki, T., Suzuki, R., Murakami, K., Aizaki, H., Ishii, K., Murayama, A., Date, T., Matsuura, Y., Miyamura, T. & other authors ( 2008; ). Interaction of hepatitis C virus nonstructural protein 5A with core protein is critical for the production of infectious virus particles. J Virol 82, 7964–7976.[CrossRef]
    [Google Scholar]
  24. Matsumoto, M., Hwang, S. B., Jeng, K. S., Zhu, N. & Lai, M. M. ( 1996; ). Homotypic interaction and multimerization of hepatitis C virus core protein. Virology 218, 43–51.[CrossRef]
    [Google Scholar]
  25. McLauchlan, J. ( 2000; ). Properties of the hepatitis C virus core protein: a structural protein that modulates cellular processes. J Viral Hepat 7, 2–14.[CrossRef]
    [Google Scholar]
  26. McLauchlan, J. ( 2009a; ). Hepatitis C virus: viral proteins on the move. Biochem Soc Trans 37, 986–990.[CrossRef]
    [Google Scholar]
  27. McLauchlan, J. ( 2009b; ). Lipid droplets and hepatitis C virus infection. Biochim Biophys Acta 1791, 552–559.[CrossRef]
    [Google Scholar]
  28. McLauchlan, J., Lemberg, M. K., Hope, G. & Martoglio, B. ( 2002; ). Intramembrane proteolysis promotes trafficking of hepatitis C virus core protein to lipid droplets. EMBO J 21, 3980–3988.[CrossRef]
    [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.[CrossRef]
    [Google Scholar]
  30. Moradpour, D., Penin, F. & Rice, C. M. ( 2007; ). Replication of hepatitis C virus. Nat Rev Microbiol 5, 453–463.[CrossRef]
    [Google Scholar]
  31. Murray, C. L., Jones, C. T., Tassello, J. & Rice, C. M. ( 2007; ). Alanine scanning of the hepatitis C virus core protein reveals numerous residues essential for production of infectious virus. J Virol 81, 10220–10231.[CrossRef]
    [Google Scholar]
  32. Murray, C. L., Jones, C. T. & Rice, C. M. ( 2008; ). Architects of assembly: roles of Flaviviridae non-structural proteins in virion morphogenesis. Nat Rev Microbiol 6, 699–708.[CrossRef]
    [Google Scholar]
  33. Nolandt, O., Kern, V., Muller, H., Pfaff, E., Theilmann, L., Welker, R. & Krausslich, H. G. ( 1997; ). Analysis of hepatitis C virus core protein interaction domains. J Gen Virol 78, 1331–1340.
    [Google Scholar]
  34. Owsianka, A. M. & Patel, A. H. ( 1999; ). Hepatitis C virus core protein interacts with a human DEAD box protein DDX3. Virology 257, 330–340.[CrossRef]
    [Google Scholar]
  35. Patkar, C. G. & Kuhn, R. J. ( 2008; ). Yellow fever virus NS3 plays an essential role in virus assembly independent of its known enzymatic functions. J Virol 82, 3342–3352.[CrossRef]
    [Google Scholar]
  36. Penin, F., Dubuisson, J., Rey, F. A., Moradpour, D. & Pawlotsky, J. M. ( 2004; ). Structural biology of hepatitis C virus. Hepatology 39, 5–19.[CrossRef]
    [Google Scholar]
  37. Pietschmann, T., Kaul, A., Koutsoudakis, G., Shavinskaya, A., Kallis, S., Steinmann, E., Abid, K., Negro, F., Dreux, M. & other authors ( 2006; ). Construction and characterization of infectious intragenotypic and intergenotypic hepatitis C virus chimeras. Proc Natl Acad Sci U S A 103, 7408–7413.[CrossRef]
    [Google Scholar]
  38. Rouille, Y., Helle, F., Delgrange, D., Roingeard, P., Voisset, C., Blanchard, E., Belouzard, S., McKeating, J., Patel, A. H. & 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]
  39. Sakamoto, N. & Watanabe, M. ( 2009; ). New therapeutic approaches to hepatitis C virus. J Gastroenterol 44, 643–649.[CrossRef]
    [Google Scholar]
  40. Shavinskaya, A., Boulant, S., Penin, F., McLauchlan, J. & Bartenschlager, R. ( 2007; ). The lipid droplet binding domain of hepatitis C virus core protein is a major determinant for efficient virus assembly. J Biol Chem 282, 37158–37169.[CrossRef]
    [Google Scholar]
  41. Shi, S. T., Polyak, S. J., Tu, H., Taylor, D. R., Gretch, D. R. & Lai, M. M. ( 2002; ). Hepatitis C virus NS5A colocalizes with the core protein on lipid droplets and interacts with apolipoproteins. Virology 292, 198–210.[CrossRef]
    [Google Scholar]
  42. Steinmann, E., Penin, F., Kallis, S., Patel, A. H., Bartenschlager, R. & Pietschmann, T. ( 2007; ). Hepatitis C virus p7 protein is crucial for assembly and release of infectious virions. PLoS Pathog 3, e103.[CrossRef]
    [Google Scholar]
  43. Strosberg, A. D., Kota, S., Takahashi, V., Snyder, J. K. & Mousseau, G. ( 2010; ). Core as a novel target for hepatitis C drugs. Viruses 2, 1734–1751.[CrossRef]
    [Google Scholar]
  44. Tan, S. L., Pause, A., Shi, Y. & Sonenberg, N. ( 2002; ). Hepatitis C therapeutics: current status and emerging strategies. Nat Rev Drug Discov 1, 867–881.[CrossRef]
    [Google Scholar]
  45. Tellinghuisen, T. L., Foss, K. L. & Treadaway, J. ( 2008; ). Regulation of hepatitis C virion production via phosphorylation of the NS5A protein. PLoS Pathog 4, e1000032.[CrossRef]
    [Google Scholar]
  46. Wakita, T., Pietschmann, T., Kato, T., Date, T., Miyamoto, M., Zhao, Z., Murthy, K., Habermann, A., Krausslich, H. G. & 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]
  47. Wei, W., Cai, C., Kota, S., Takahashi, V., Ni, F., Strosberg, A. D. & Snyder, J. K. ( 2009; ). New small molecule inhibitors of hepatitis C virus. Bioorg Med Chem Lett 19, 6926–6930.[CrossRef]
    [Google Scholar]
  48. Wu, J., Bera, A. K., Kuhn, R. J. & Smith, J. L. ( 2005; ). Structure of the Flavivirus helicase: implications for catalytic activity, protein interactions, and proteolytic processing. J Virol 79, 10268–10277.[CrossRef]
    [Google Scholar]
  49. Yan, X. B., Battaglia, S., Boucreux, D., Chen, Z., Brechot, C. & Pavio, N. ( 2007; ). Mapping of the interacting domains of hepatitis C virus core protein and the double-stranded RNA-activated protein kinase PKR. Virus Res 125, 79–87.[CrossRef]
    [Google Scholar]
  50. 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.[CrossRef]
    [Google Scholar]
  51. Yi, M., Ma, Y., Yates, J. & Lemon, S. M. ( 2009; ). Trans-complementation of an NS2 defect in a late step in hepatitis C virus (HCV) particle assembly and maturation. PLoS Pathog 5, e1000403.[CrossRef]
    [Google Scholar]
  52. You, L. R., Chen, C. M., Yeh, T. S., Tsai, T. Y., Mai, R. T., Lin, C. H. & Lee, Y. H. ( 1999; ). Hepatitis C virus core protein interacts with cellular putative RNA helicase. J Virol 73, 2841–2853.
    [Google Scholar]
  53. 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.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.023325-0
Loading
/content/journal/jgv/10.1099/vir.0.023325-0
Loading

Data & Media loading...

Supplements

vol. , part 1, pp. 101 - 111

Effect of SL201 on HCV infectivity

Effect of SL201 on HCV RNA levels

Effective concentration at 50% (EC ) values for SL201 and SL209 on cells before, during and after HCV infection [Single PDF file](54 KB)

 



PDF

Most Cited This Month

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