1887

Abstract

The non-structural protein 5A (NS5A) of hepatitis C virus (HCV) has been implicated in inhibition of antiviral activity of IFN. While previous studies have suggested an interaction between NS5A and the double-stranded RNA-dependent protein kinase (PKR), the possibility still remains that interaction with another molecule(s) is involved in the NS5A-mediated inhibition of IFN. In the present study, we investigated a possible interaction between NS5A and 2′,5′-oligoadenylate synthetase (2-5AS), another key molecule in antiviral activity. We observed that NS5A physically interacted with 2-5AS in cultured cells, with an N-terminal portion of NS5A [aa 1–148; NS5A(1–148)] and two separate portions of 2-5AS (aa 52–104 and 184–275) being involved in the interaction. Single point mutations at residue 37 of NS5A affected the degree of the interaction with 2-5AS, with a Phe-to-Leu mutation (F37L) augmenting and a Phe-to-Asn mutation (F37N) diminishing it. Virus rescue assay revealed that the full-length NS5A (NS5A-F) and NS5A(1–148), the latter of which contains neither the IFN sensitivity-determining region (ISDR) nor the PKR-binding domain, significantly counteracted the antiviral activity of IFN. Introduction of a F37N mutation into NS5A(1–148) impaired the otherwise more significant IFN-inhibitory activity of NS5A(1–148). It was also found that the F37N mutation was highly disadvantageous for the replication of an HCV RNA replicon. Taken together, our results suggest the possibility that NS5A interacts with 2-5AS and inhibits the antiviral activity of IFN in an ISDR-independent manner.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.19513-0
2004-04-01
2019-11-21
Loading full text...

Full text loading...

/deliver/fulltext/jgv/85/4/vir850959.html?itemId=/content/journal/jgv/10.1099/vir.0.19513-0&mimeType=html&fmt=ahah

References

  1. 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]
  2. Brass, V., Bieck, E., Montserret, R., Wolk, 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.[CrossRef]
    [Google Scholar]
  3. Bruno, S., Silini, E., Crosignani, A. & 9 other authors ( 1997; ). Hepatitis C virus genotypes and risk of hepatocellular carcinoma in cirrhosis: a prospective study. Hepatology 25, 754–758.[CrossRef]
    [Google Scholar]
  4. Dan, K., Seto, Y., Fujita, T., Asaba, Y., Takei, I., Fujita, H. & Kato, R. ( 1995; ). Characterization of insulin-dependent diabetes mellitus induced by a new variant (DK-27) of encephalomyocarditis virus in DBA/2 mice. Exp Anim 44, 211–218.[CrossRef]
    [Google Scholar]
  5. Doi, H., Apichartpiyakul, C., Ohba, K., Mizokami, M. & Hotta, H. ( 1996; ). Hepatitis C virus (HCV) subtype prevalence in Chiang Mai, Thailand, and identification of novel subtypes of HCV major type 6. J Clin Microbiol 34, 569–574.
    [Google Scholar]
  6. Duverlie, G., Khorsi, H., Castelain, S., Jaillon, O., Izopet, J., Lunel, F., Eb, F., Penin, F. & Wychowski, C. ( 1998; ). Sequence analysis of the NS5A protein of European hepatitis C virus 1b isolates and relation to interferon sensitivity. J Gen Virol 79, 1373–1381.
    [Google Scholar]
  7. Enomoto, N., Sakuma, I., Asahina, Y. & 7 other authors ( 1996; ). Mutations in the nonstructural protein 5A gene and response to interferon in patients with chronic hepatitis C virus 1b infection. N Engl J Med 334, 77–81.[CrossRef]
    [Google Scholar]
  8. Francois, 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]
  9. Frese, M., Pietschmann, T., Moradpour, D., Haller, O. & Bartenschlager, R. ( 2001; ). Interferon-alpha inhibits hepatitis C virus subgenomic RNA replication by an MxA-independent pathway. J Gen Virol 82, 723–733.
    [Google Scholar]
  10. Gale, M. J., Jr, Korth, M. J., Tang, N. M., Tan, S.-L., Hopkins, D. A., Dever, T. E., Polyak, S. J., Gretch, D. R. & Katze, M. G. ( 1997; ). Evidence that hepatitis C virus resistance to interferon is mediated through repression of the PKR protein kinase by the nonstructural 5A protein. Virology 230, 217–227.[CrossRef]
    [Google Scholar]
  11. Gale, M., Jr, Blakely, C. M., Kwieciszewski, B. & 7 other authors ( 1998; ). Control of PKR protein kinase by hepatitis C virus nonstructural 5A protein: molecular mechanisms of kinase regulation. Mol Cell Biol 18, 5208–5218.
    [Google Scholar]
  12. Ghosh, A., Sarkar, S. N. & Sen, G. C. ( 2000; ). Cell growth regulatory and antiviral effects of the p69 isozyme of 2-5 (A) synthetase. Virology 266, 319–328.[CrossRef]
    [Google Scholar]
  13. Girard, S., Shalhoub, P., Lescure, P., Sabile, A., Misek, D. E., Hanash, S., Brechot, C. & Beretta, L. ( 2002; ). An altered cellular response to interferon and up-regulation of interleukin-8 induced by the hepatitis C viral protein NS5A uncovered by microarray analysis. Virology 295, 272–283.[CrossRef]
    [Google Scholar]
  14. Goh, P.-Y., Tan, Y.-J., Lim, S.-P., Lim, S.-G., Tan, Y.-H. & Hong, W.-J. ( 2001; ). The hepatitis C virus core protein interacts with NS5A and activates its caspase-mediated proteolytic cleavage. Virology 290, 224–236.[CrossRef]
    [Google Scholar]
  15. Grander, D., Hultcrantz, R., Weiland, O. & 10 other authors ( 1996; ). Factors influencing the response to interferon therapy in chronic hepatitis C. Studies on viral genotype and induction of 2′,5′-oligoadenylate synthetase in the liver and peripheral blood cells. Scand J Gastroenterol 31, 604–611.[CrossRef]
    [Google Scholar]
  16. 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]
  17. Han, J.-Q. & Barton, D. J. ( 2002; ). Activation and evasion of the antiviral 2′-5′ oligoadenylate synthetase/ribonuclease L pathway by hepatitis C virus mRNA. RNA 8, 512–525.[CrossRef]
    [Google Scholar]
  18. Hassel, B. A., Zhou, A., Sotomayor, C., Maran, A. & Silverman, R. H. ( 1993; ). A dominant negative mutant of 2-5A-dependent RNase suppresses antiproliferative and antiviral effects of interferon. EMBO J 12, 3297–3304.
    [Google Scholar]
  19. Ide, Y., Zhang, L., Chen, M., Inchauspe, G., Bahl, C., Sasaguri, Y. & Padmanabhan, R. ( 1996; ). Characterization of the nuclear localization signal and subcellular distribution of hepatitis C virus nonstructural protein NS5A. Gene 182, 203–211.[CrossRef]
    [Google Scholar]
  20. Kaneko, T., Tanji, Y., Satoh, S., Hijikata, M., Asabe, S., Kimura, K. & Shimotohno, K. ( 1994; ). Production of two phosphoproteins from the NS5A region of the hepatitis C viral genome. Biochem Biophys Res Commun 205, 320–326.[CrossRef]
    [Google Scholar]
  21. Katze, M. G., Kwieciszewski, B., Goodlett, D. R., Blakely, C. M., Neddermann, P., Tan, S.-L. & Aebersold, R. ( 2000; ). Ser2194 is a highly conserved major phosphorylation site of the hepatitis C virus nonstructural protein NS5A. Virology 278, 501–513.[CrossRef]
    [Google Scholar]
  22. Krieger, N., Lohmann, V. & Bartenschlager, R. ( 2001; ). Enhancement of hepatitis C virus RNA replication by cell culture-adaptive mutations. J Virol 75, 4614–4624.[CrossRef]
    [Google Scholar]
  23. Lauer, G. M. & Walker, B. D. ( 2001; ). Hepatitis C virus infection. N Engl J Med 345, 41–52.[CrossRef]
    [Google Scholar]
  24. Li, X.-L., Blackford, J. A. & Hassel, B. A. ( 1998; ). RNase L mediates the antiviral effect of interferon through a selective reduction in viral RNA during encephalomyocarditis virus infection. J Virol 72, 2752–2759.
    [Google Scholar]
  25. Lohmann, V., Korner, F., Dobierzewska, A. & Bartenschlager, R. ( 2001; ). Mutations in hepatitis C virus RNAs conferring cell culture adaptation. J Virol 75, 1437–1449.[CrossRef]
    [Google Scholar]
  26. Lusida, M. I., Nagano-Fujii, M., Nidom, C. A., Soetjipto, Handajani, R., Fujita, T., Oka, K. & Hotta, H. ( 2001; ). Correlation between mutations in the interferon sensitivity-determining region of NS5A protein and viral load of hepatitis C virus subtypes 1b, 1c, and 2a. J Clin Microbiol 39, 3858–3864.[CrossRef]
    [Google Scholar]
  27. McHutchison, J. G., Gordon, S. C., Schiff, E. R. & 7 other authors ( 1998; ). Interferon alfa-2b alone or in combination with ribavirin as initial treatment for chronic hepatitis C. N Engl J Med 339, 1485–1492.[CrossRef]
    [Google Scholar]
  28. Mellor, J., Holmes, E. C., Jarvis, L. M., Yap, P. L., Simmonds, P. & The International HCV Collaborative Study Group ( 1995; ). Investigation of the pattern of hepatitis C virus sequence diversity in different geographical regions: implications for virus classification. J Gen Virol 76, 2493–2507.[CrossRef]
    [Google Scholar]
  29. Muramatsu, S., Ishido, S., Fujita, T., Itoh, M. & Hotta, H. ( 1997; ). Nuclear localization of the NS3 protein of hepatitis C virus and factors affecting the localization. J Virol 71, 4954–4961.
    [Google Scholar]
  30. Murashima, S., Kumashiro, R., Ide, T. & 7 other authors ( 2000; ). Effect of interferon treatment on serum 2′,5′-oligoadenylate synthetase levels in hepatitis C-infected patients. J Med Virol 62, 185–190.[CrossRef]
    [Google Scholar]
  31. Naganuma, A., Nozaki, A., Tanaka, T., Sugiyama, K., Takagi, H., Mori, M., Shimotohno, K. & Kato, N. ( 2000; ). Activation of the interferon-inducible 2′-5′-oligoadenylate synthetase gene by hepatitis C virus core protein. J Virol 74, 8744–8750.[CrossRef]
    [Google Scholar]
  32. Niwa, H., Yamamura, K. & Miyazaki, J. ( 1991; ). Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108, 193–199.[CrossRef]
    [Google Scholar]
  33. Nousbaum, J. B., Pol, S., Nalpas, B., Landais, P., Berthelot, P. & Brechot, C. ( 1995; ). Hepatitis C virus type 1b (II) infection in France and Italy. Ann Intern Med 122, 161–168.[CrossRef]
    [Google Scholar]
  34. Ogata, S., Nagano-Fujii, M., Ku, Y., Yoon, S. & Hotta, H. ( 2002; ). Comparative sequence analysis of the core protein and its frameshift product, the F protein, of hepatitis C virus subtype 1b strains obtained from patients with and without hepatocellular carcinoma. J Clin Microbiol 40, 3625–3630.[CrossRef]
    [Google Scholar]
  35. Ogata, S., Florese, R. H., Nagano-Fujii, M. & 7 other authors ( 2003; ). Identification of hepatitis C virus (HCV) subtype 1b strains that are highly, or only weakly, associated with hepatocellular carcinoma on the basis of secondary structure of an amino-terminal portion of the HCV NS3 protein. J Clin Microbiol 41, 2835–2841.[CrossRef]
    [Google Scholar]
  36. Podevin, P., Guechot, J., Serfaty, L., Monrand-Joubert, L., Veyrunes, C., Bonnefis, M. T. & Poupon, R. ( 1997; ). Evidence for a deficiency of interferon response in mononuclear cells from hepatitis C viremic patients. J Hepatol 27, 265–271.[CrossRef]
    [Google Scholar]
  37. Podevin, P., Sabile, A., Gajardo, R., Delhem, N., Abadie, A., Lozach, P.-Y., Beretta, L. & Brechot, C. ( 2001; ). Expression of hepatitis C virus NS5A natural mutants in a hepatocytic cell line inhibits the antiviral effect of interferon in a PKR-independent manner. Hepatology 33, 1503–1511.[CrossRef]
    [Google Scholar]
  38. Polyak, S. J., Paschal, D. M., McArdle, S., Gale, M. J., Jr, Moradpour, D. & Gretch, D. R. ( 1999; ). Characterization of the effects of hepatitis C virus nonstructural 5A protein expression in human cell lines and on interferon-sensitive virus replication. Hepatology 29, 1262–1271.[CrossRef]
    [Google Scholar]
  39. Polyak, S. J., Khabar, K. S., Paschal, D. M., Ezelle, H. J., Duverlie, G., Barber, G. N., Levy, D. E., Mukaida, N. & Gretch, D. R. ( 2001; ). Hepatitis C virus nonstructural 5A protein induces interleukin-8, leading to partial inhibition of the interferon-induced antiviral response. J Virol 75, 6095–6106.[CrossRef]
    [Google Scholar]
  40. Reed, K. E. & Rice, C. M. ( 1999; ). Identification of the major phosphorylation site of the hepatitis C virus H strain NS5A protein as serine 2321. J Biol Chem 274, 28011–28018.[CrossRef]
    [Google Scholar]
  41. 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.
    [Google Scholar]
  42. Robertson, B., Myers, G., Howard, C. & 14 other authors ( 1998; ). Classification, nomenclature, and database development for hepatitis C virus (HCV) and related virus: proposals for standardization. Arch Virol 143, 2493–2503.[CrossRef]
    [Google Scholar]
  43. Satoh, S., Hirota, M., Noguchi, T., Hijikata, M., Handa, H. & Shimotohno, K. ( 2000; ). Cleavage of hepatitis C virus nonstructural protein 5A by a caspase-like protease(s) in mammalian cells. Virology 270, 476–487.[CrossRef]
    [Google Scholar]
  44. Sen, G. C. & Ransohoff, R. M. ( 1993; ). Interferon-induced antiviral actions and their regulation. Adv Virus Res 42, 57–102.
    [Google Scholar]
  45. Shi, S. T., Polyak, S. J., Tu, H., Taylor, D. R., Gretch, D. R. & Lai, M. M. C. ( 2002; ). Hepatitis C virus NS5A colocalizes with the core protein on lipid droplets and interacts with apolipoproteins. Virology 292, 198–210.[CrossRef]
    [Google Scholar]
  46. Shirota, Y., Luo, H., Qin, W., Kaneko, S., Yamashita, T., Kobayashi, K. & Murakami, S. ( 2002; ). Hepatitis C virus (HCV) NS5A binds RNA-dependent RNA polymerase (RdRP) NS5B and modulates RNA-dependent RNA polymerase activity. J Biol Chem 277, 11149–11155.[CrossRef]
    [Google Scholar]
  47. Song, J., Fujii, M., Wang, F., Itoh, M. & Hotta, H. ( 1999; ). The NS5A protein of hepatitis C virus partially inhibits the antiviral activity of interferon. J Gen Virol 80, 879–886.
    [Google Scholar]
  48. Song, J., Nagano-Fujii, M., Wang, F., Florese, R., Fujita, T., Ishido, S. & Hotta, H. ( 2000; ). Nuclear localization and intramolecular cleavage of N-terminally deleted NS5A protein of hepatitis C virus. Virus Res 69, 109–117.[CrossRef]
    [Google Scholar]
  49. Staeheli, P. & Pavlovic, J. ( 1991; ). Inhibition of vesicular stomatitis virus mRNA synthesis by human MxA protein. J Virol 65, 4498–4501.
    [Google Scholar]
  50. Tanji, Y., Kaneko, T., Satoh, S. & Shimotohno, K. ( 1995; ). Phosphorylation of hepatitis C virus-encoded nonstructural protein NS5A. J Virol 69, 3980–3986.
    [Google Scholar]
  51. Yamamoto, Y., Sono, D. & Sokawa, Y. ( 2000; ). Effects of specific mutations in active site motifs of 2′,5′-oligoadenylate synthetase on enzymatic activity. J Interferon Cytokine Res 20, 337–344.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.19513-0
Loading
/content/journal/jgv/10.1099/vir.0.19513-0
Loading

Data & Media loading...

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