1887

Abstract

The N-terminal 198 residues of NS3 (NS3-N) of (HCV) subtype 1b obtained from 29 patients, as well as full-length NS3 (NS3-Full), were analysed for their subcellular localization, interaction with the tumour suppressor p53 and serine protease activity in the presence and absence of the viral cofactor NS4A. Based on the subcellular-localization patterns in the absence of NS4A, NS3-N sequences were classified into three groups, with each group exhibiting either dot-like, diffuse or a mixed type of localization. Chimeric NS3-Full sequences, each consisting of an individual NS3-N and a shared C-terminal sequence, showed the same localization patterns as those of the respective NS3-N. Site-directed mutagenesis experiments revealed that a single or a few amino acid substitutions at a particular position(s) of NS3-N altered the localization pattern. Interestingly, NS3 of the dot-like type, either NS3-N or NS3-Full, interacted with p53 more strongly than that of the diffuse type, in both the presence and the absence of NS4A. Moreover, NS3-N of the dot-like type suppressed -activating activity of p53 more strongly than that of the diffuse type. Serine protease activity did not differ significantly between the two types of NS3. In HCV RNA replicon-harbouring cells, physical interaction between NS3 and p53 was observed consistently and p53-mediated transcriptional activation was suppressed significantly compared with HCV RNA-negative control cells. Our results collectively suggest the possibility that NS3 plays an important role in the hepatocarcinogenesis of HCV by interacting differentially with p53 in an NS3 sequence-dependent manner.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.81735-0
2006-06-01
2019-10-17
Loading full text...

Full text loading...

/deliver/fulltext/jgv/87/6/1703.html?itemId=/content/journal/jgv/10.1099/vir.0.81735-0&mimeType=html&fmt=ahah

References

  1. Appella, E. & Anderson, C. W. ( 2001; ). Post-translational modifications and activation of p53 by genotoxic stresses. Eur J Biochem 268, 2764–2772.[CrossRef]
    [Google Scholar]
  2. 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 IKKε. J Virol 79, 3969–3978.[CrossRef]
    [Google Scholar]
  3. Cheng, P.-L., Chang, M.-H., Chao, C.-H. & Wu Lee, Y.-H. ( 2004; ). Hepatitis C viral proteins interact with Smad3 and differentially regulate TGF-β/Smad3-mediated transcriptional activation. Oncogene 23, 7821–7838.[CrossRef]
    [Google Scholar]
  4. Fernandez-Fernandez, M. R., Veprintsev, D. B. & Fersht, A. R. ( 2005; ). Proteins of the S100 family regulate the oligomerization of p53 tumor suppressor. Proc Natl Acad Sci U S A 102, 4735–4740.[CrossRef]
    [Google Scholar]
  5. Florese, R. H., Nagano-Fujii, M., Iwanaga, Y., Hidajat, R. & Hotta, H. ( 2002; ). Inhibition of protein synthesis by the nonstructural proteins NS4A and NS4B of hepatitis C virus. Virus Res 90, 119–131.[CrossRef]
    [Google Scholar]
  6. Foy, E., Li, K., Wang, C., Sumpter, R., Jr, Ikeda, M., Lemon, S. M. & Gale, M., Jr ( 2003; ). Regulation of interferon regulatory factor-3 by the hepatitis C virus serine protease. Science 300, 1145–1148.[CrossRef]
    [Google Scholar]
  7. 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]
  8. Fuerst, T. R., Niles, E. G., Studier, F. W. & Moss, B. ( 1986; ). Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc Natl Acad Sci U S A 83, 8122–8126.[CrossRef]
    [Google Scholar]
  9. Fujita, T., Ishido, S., Muramatsu, S., Itoh, M. & Hotta, H. ( 1996; ). Suppression of actinomycin D-induced apoptosis by the NS3 protein of hepatitis C virus. Biochem Biophys Res Commun 229, 825–831.[CrossRef]
    [Google Scholar]
  10. Hidajat, R., Nagano-Fujii, M., Deng, L., Tanaka, M., Takigawa, Y., Kitazawa, S. & Hotta, H. ( 2005; ). Hepatitis C virus NS3 protein interacts with ELKS-δ and ELKS-α, members of a novel protein family involved in intracellular transport and secretory pathways. J Gen Virol 86, 2197–2208.[CrossRef]
    [Google Scholar]
  11. Ikeda, M., Abe, K., Dansako, H., Nakamura, T., Naka, K. & Kato, N. ( 2005; ). Efficient replication of a full-length hepatitis C virus genome, strain O, in cell culture, and development of a luciferase reporter system. Biochem Biophys Res Commun 329, 1350–1359.[CrossRef]
    [Google Scholar]
  12. Ishido, S. & Hotta, H. ( 1998; ). Complex formation of the nonstructural protein 3 of hepatitis C virus with the p53 tumor suppressor. FEBS Lett 438, 258–262.[CrossRef]
    [Google Scholar]
  13. Ishido, S., Muramatsu, S., Fujita, T., Iwanaga, Y., Tong, W.-Y., Katayama, Y., Itoh, M. & Hotta, H. ( 1997; ). Wild-type, but not mutant-type, p53 enhances nuclear accumulation of the NS3 protein of hepatitis C virus. Biochem Biophys Res Commun 230, 431–436.[CrossRef]
    [Google Scholar]
  14. Kadoya, H., Nagano-Fujii, M., Deng, L., Nakazono, N. & Hotta, H. ( 2005; ). Nonstructural proteins 4A and 4B of hepatitis C virus transactivate the interleukin 8 promoter. Microbiol Immunol 49, 265–273.[CrossRef]
    [Google Scholar]
  15. Kao, C.-F., Chen, S.-Y., Chen, J.-Y. & Wu Lee, Y.-H. ( 2004; ). Modulation of p53 transcription regulatory activity and post-translational modification by hepatitis C virus core protein. Oncogene 23, 2472–2483.[CrossRef]
    [Google Scholar]
  16. Kim, D. W., Gwack, Y., Han, J. H. & Choe, J. ( 1995; ). C-terminal domain of the hepatitis C virus NS3 protein contains an RNA helicase activity. Biochem Biophys Res Commun 215, 160–166.[CrossRef]
    [Google Scholar]
  17. Kim, J.-E., Song, W. K., Chung, K. M., Back, S. H. & Jang, S. K. ( 1999; ). Subcellular localization of hepatitis C viral proteins in mammalian cells. Arch Virol 144, 329–343.[CrossRef]
    [Google Scholar]
  18. Kwun, H. J., Jung, E. Y., Ahn, J. Y., Lee, M. N. & Jang, K. L. ( 2001; ). p53-dependent transcriptional repression of p21waf1 by hepatitis C virus NS3. J Gen Virol 82, 2235–2241.
    [Google Scholar]
  19. Lan, K.-H., Sheu, M.-L., Hwang, S.-J. & 8 other authors ( 2002; ). HCV NS5A interacts with p53 and inhibits p53-mediated apoptosis. Oncogene 21, 4801–4811.[CrossRef]
    [Google Scholar]
  20. Levine, A. J. ( 1997; ). p53, the cellular gatekeeper for growth and division. Cell 88, 323–331.[CrossRef]
    [Google Scholar]
  21. Lin, C., Prágai, B. M., Grakoui, A., Xu, J. & Rice, C. M. ( 1994; ). Hepatitis C virus NS3 serine proteinase: trans-cleavage requirements and processing kinetics. J Virol 68, 8147–8157.
    [Google Scholar]
  22. Loguercio, C., Cuomo, A., Tuccillo, C., Gazzerro, P., Cioffi, M., Molinari, A. M. & Del Vecchio Blanco, C. ( 2003; ). Liver p53 expression in patients with HCV-related chronic hepatitis. J Viral Hepat 10, 266–270.[CrossRef]
    [Google Scholar]
  23. Lohmann, V., Körner, F., Dobierzewska, A. & Bartenschlager, R. ( 2001; ). Mutations in hepatitis C virus RNAs conferring cell culture adaptation. J Virol 75, 1437–1449.[CrossRef]
    [Google Scholar]
  24. Longworth, M. S. & Laimins, L. A. ( 2004; ). Pathogenesis of human papillomaviruses in differentiating epithelia. Microbiol Mol Biol Rev 68, 362–372.[CrossRef]
    [Google Scholar]
  25. Martin, M. E. D. & Berk, A. J. ( 1998; ). Adenovirus E1B 55K represses p53 activation in vitro. J Virol 72, 3146–3154.
    [Google Scholar]
  26. McLure, K. G. & Lee, P. W. K. ( 1998; ). How p53 binds DNA as a tetramer. EMBO J 17, 3342–3350.[CrossRef]
    [Google Scholar]
  27. Moss, B., Elroy-Stein, O., Mizukami, T., Alexander, W. A. & Fuerst, T. R. ( 1990; ). New mammalian expression vectors. Nature 348, 91–92.[CrossRef]
    [Google Scholar]
  28. Mottola, G., Cardinali, G., Ceccacci, A., Trozzi, C., Bartholomew, L., Torrisi, M. R., Pedrazzini, E., Bonatti, S. & Migliaccio, G. ( 2002; ). Hepatitis C virus nonstructural proteins are localized in a modified endoplasmic reticulum of cells expressing viral subgenomic replicons. Virology 293, 31–43.[CrossRef]
    [Google Scholar]
  29. Müller-Tiemann, B. F., Halazonetis, T. D. & Elting, J. J. ( 1998; ). Identification of an additional negative regulatory region for p53 sequence-specific DNA binding. Proc Natl Acad Sci U S A 95, 6079–6084.[CrossRef]
    [Google Scholar]
  30. Münger, K. & Howley, P. M. ( 2002; ). Human papillomavirus immortalization and transformation functions. Virus Res 89, 213–228.[CrossRef]
    [Google Scholar]
  31. 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]
  32. Ogata, S., Ku, Y., Yoon, S., Makino, S., Nagano-Fujii, M. & Hotta, H. ( 2002; ). Correlation between secondary structure of an amino-terminal portion of the nonstructural proteins 3 (NS3) of hepatitis C virus and development of hepatocellular carcinoma. Microbiol Immunol 46, 549–554.[CrossRef]
    [Google Scholar]
  33. 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 the secondary structure of an amino-terminal portion of the HCV NS3 protein. J Clin Microbiol 41, 2835–2841.[CrossRef]
    [Google Scholar]
  34. Qadri, I., Iwahashi, M. & Simon, F. ( 2002; ). Hepatitis C virus NS5A protein binds TBP and p53, inhibiting their DNA binding and p53 interactions with TBP and ERCC3. Biochim Biophys Acta 1592, 193–204.[CrossRef]
    [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.
    [Google Scholar]
  36. Saito, I., Miyamura, T., Ohbayashi, A. & 10 other authors ( 1990; ). Hepatitis C virus infection is associated with the development of hepatocellular carcinoma. Proc Natl Acad Sci U S A 87, 6547–6549.[CrossRef]
    [Google Scholar]
  37. Sakamuro, D., Furukawa, T. & Takegami, T. ( 1995; ). Hepatitis C virus nonstructural protein NS3 transforms NIH3T3 cells. J Virol 69, 3893–3896.
    [Google Scholar]
  38. Sheppard, H. M., Corneillie, S. I., Espiritu, C., Gatti, A. & Liu, X. ( 1999; ). New insights into the mechanism of inhibition of p53 by simian virus 40 large T antigen. Mol Cell Biol 19, 2746–2753.
    [Google Scholar]
  39. Taguchi, T., Nagano-Fujii, M., Akutsu, M., Kadoya, H., Ohgimoto, S., Ishido, S. & Hotta, H. ( 2004; ). Hepatitis C virus NS5A protein interacts with 2′,5′-oligoadenylate synthetase and inhibits antiviral activity of IFN in an IFN sensitivity-determining region-independent manner. J Gen Virol 85, 959–969.[CrossRef]
    [Google Scholar]
  40. Tanaka, M., Nagano-Fujii, M., Deng, L., Ishido, S., Sada, K. & Hotta, H. ( 2006; ). Single-point mutations of hepatitis C virus NS3 that impair p53 interaction and anti-apoptotic activity of NS3. Biochem Biophys Res Commun 340, 792–799.[CrossRef]
    [Google Scholar]
  41. Truant, R., Antunovic, J., Greenblatt, J., Prives, C. & Cromlish, J. A. ( 1995; ). Direct interaction of the hepatitis B virus HBx protein with p53 leads to inhibition by HBx of p53 response element directed-transactivation. J Virol 69, 1851–1859.
    [Google Scholar]
  42. Waterman, M. J. F., Stavridi, E. S., Waterman, J. L. F. & Halazonetis, T. D. ( 1998; ). ATM-dependent activation of p53 involves dephosphorylation and association with 14-3-3 proteins. Nat Genet 19, 175–178.[CrossRef]
    [Google Scholar]
  43. Weinberg, R. L., Veprintsev, D. B. & Fersht, A. R. ( 2004; ). Cooperative binding of tetrameric p53 to DNA. J Mol Biol 341, 1145–1159.[CrossRef]
    [Google Scholar]
  44. Zemel, R., Gerechet, S., Greif, H. & 7 other authors ( 2001; ). Cell transformation induced by hepatitis C virus NS3 serine protease. J Viral Hepat 8, 96–102.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.81735-0
Loading
/content/journal/jgv/10.1099/vir.0.81735-0
Loading

Data & Media loading...

Supplements

vol. , part 6, pp. 1703 – 1713

Distinct subcellular-localization patterns of NS3-N of different HCV-1b isolates

Sequence alignment of all 29 isolates tested

[ Single PDF file] (3.1 MB)



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