1887

Abstract

The hepatitis C virus (HCV) non-structural NS5A protein has been shown to associate with a variety of cellular signalling proteins. Of particular interest is the observation that a highly conserved C-terminal polyproline motif in NS5A was able to interact with the Src-homology 3 (SH3) domains of the adaptor protein Grb2. As it has previously been shown that specific polyproline motifs can interact with a range of SH3 domains, we investigated whether NS5A was capable of interacting with other SH3 domain-containing proteins. We show here that NS5A interacts with the SH3 domains of members of the Src family of tyrosine kinases: a combination of binding assays and co-immunoprecipitation experiments revealed an interaction between NS5A and Hck, Lck, Lyn and Fyn, but interestingly not Src itself. Mutational analysis confirmed that the polyproline motif responsible for binding to Grb2 also bound to the SH3 domains of Hck, Lck, Lyn and Fyn. Furthermore, a previously unidentified polyproline motif, adjacent to the first motif, was also able to mediate binding to the SH3 domain of Lyn. Using transient transfections and Huh-7 cells harbouring a persistently replicating subgenomic HCV replicon we demonstrate that NS5A bound to native Src-family kinases and differentially modulated kinase activity, inhibiting Hck, Lck and Lyn but activating Fyn. Lastly, we show that signalling pathways controlled by Src-family kinases are modulated in replicon cells. We conclude that the interactions between NS5A and Src-family kinases are physiologically relevant and may play a role in either virus replication or pathogenesis.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.19691-0
2004-03-01
2019-11-14
Loading full text...

Full text loading...

/deliver/fulltext/jgv/85/3/vir850721.html?itemId=/content/journal/jgv/10.1099/vir.0.19691-0&mimeType=html&fmt=ahah

References

  1. Bartenschlager, R. & Lohmann, V. ( 2000; ). Replication of hepatitis C virus. J Gen Virol 81, 1631–1648.
    [Google Scholar]
  2. Berchtold, S., Moriggl, R., Gouilleux, F., Silvennoinen, O., Beisenherz, C., Pfitzner, E., Wissler, M., Stocklin, E. & Groner, B. ( 1997; ). Cytokine receptor-independent, constitutively active variants of STAT5. J Biol Chem 272, 30237–30243.[CrossRef]
    [Google Scholar]
  3. 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]
  4. Briggs, S. D., Lerner, E. C. & Smithgall, T. E. ( 2000; ). Affinity of Src family kinase SH3 domains for HIV Nef in vitro does not predict kinase activation by Nef in vivo. Biochemistry 39, 489–495.[CrossRef]
    [Google Scholar]
  5. Collette, Y. & Olive, D. ( 1997; ). Non-receptor protein tyrosine kinases as immune targets of viruses. Immunol Today 18, 393–400.[CrossRef]
    [Google Scholar]
  6. Gale, M. J., Korth, M. J. & Katze, M. G. ( 1998; ). Repression of the PKR protein kinase by the hepatitis C virus NS5A protein: a potential mechanism of interferon resistance. Clin Diagn Virol 10, 157–162.[CrossRef]
    [Google Scholar]
  7. Ghosh, A. K., Steele, R., Meyer, K., Ray, R. & Ray, R. B. ( 1999; ). Hepatitis C virus NS5A protein modulates cell cycle regulatory genes and promotes cell growth. J Gen Virol 80, 1179–1183.
    [Google Scholar]
  8. Ghosh, A. K., Majumder, M., Steele, R., Yaciuk, P., Chrivia, J., Ray, R. & Ray, R. B. ( 2000; ). Hepatitis C virus NS5A protein modulates transcription through a novel cellular transcription factor SRCAP. J Biol Chem 275, 7184–7188.[CrossRef]
    [Google Scholar]
  9. Gong, G. Z., Waris, G., Tanveer, R. & Siddiqui, A. ( 2001; ). Human hepatitis C virus NS5A protein alters intracellular calcium levels, induces oxidative stress, and activates STAT-3 and NF-κB. Proc Natl Acad Sci U S A 98, 9599–9604.[CrossRef]
    [Google Scholar]
  10. Green, S., Issemann, I. & Sheer, E. ( 1988; ). A versatile in vivo and in vitro eukaryotic expression vector for protein engineering. Nucleic Acids Res 16, 369.[CrossRef]
    [Google Scholar]
  11. Higuchi, R. ( 1992; ). Using PCR to engineer DNA. In PCR Technology. Principles and Applications. Edited by H. A. Erlich. New York: Freeman.
  12. Hiipakka, M., Poikonen, K. & Saksela, K. ( 1999; ). SH3 domains with high affinity and engineered ligand specificities targeted to HIV-1 Nef. J Mol Biol 293, 1097–1106.[CrossRef]
    [Google Scholar]
  13. Ichaso, N. & Dilworth, S. M. ( 2001; ). Cell transformation by the middle T-antigen of polyoma virus. Oncogene 20, 7908–7916.[CrossRef]
    [Google Scholar]
  14. Jayaraman, T., Ondrias, K., Ondriasova, E. & Marks, A. R. ( 1996; ). Regulation of the inositol 1,4,5-trisphosphate receptor by tyrosine phosphorylation. Science 272, 1492–1494.[CrossRef]
    [Google Scholar]
  15. 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]
  16. Lavanchy, D. & 31 other authors ( 1999; ). Global surveillance and control of hepatitis C. J Viral Hepat 6, 35–47.[CrossRef]
    [Google Scholar]
  17. Macdonald, A., Crowder, K., Street, A., McCormick, C., Saksela, K. & Harris, M. ( 2003; ). The hepatitis C virus NS5A protein inhibits activating protein-1 (AP1) function by perturbing Ras-ERK pathway signalling. J Biol Chem 278, 17775–17784.[CrossRef]
    [Google Scholar]
  18. Majumder, M., Ghosh, A. K., Steele, R., Ray, R. & Ray, R. B. ( 2001; ). Hepatitis C virus NS5A physically associates with p53 and regulates p21/waf1 gene expression in a p53-dependent manner. J Virol 75, 1401–1407.[CrossRef]
    [Google Scholar]
  19. Mayer, B. J. ( 2001; ). SH3 domains: complexity in moderation. J Cell Sci 114, 1253–1263.
    [Google Scholar]
  20. Meyers, G., Tautz, N., Becher, P., Thiel, H. J. & Kummerer, B. M. ( 1996; ). Recovery of cytopathogenic and noncytopathogenic bovine viral diarrhea viruses from cDNA constructs. J Virol 70, 8606–8613.
    [Google Scholar]
  21. Miller, C. L., Burkhardt, A. L., Lee, J. H., Stealey, B., Longnecker, R., Bolen, J. B. & Kieff, E. ( 1995; ). Integral membrane-protein 2 of Epstein–Barr virus regulates reactivation from latency through dominant-negative effects on protein-tyrosine kinases. Immunity 2, 155–166.[CrossRef]
    [Google Scholar]
  22. Saksela, K., Cheng, G. & Baltimore, D. ( 1995; ). Proline-rich (PxxP) motifs in HIV-1 Nef bind to SH3 domains of a subset of Src kinases and are required for the enhanced growth of Nef+ viruses but not for down-regulation of CD4. EMBO J 14, 484–491.
    [Google Scholar]
  23. Schreiner, S. J., Schiavone, A. P. & Smithgall, T. E. ( 2002; ). Activation of STAT3 by the Src family kinase Hck requires a functional SH3 domain. J Biol Chem 277, 45680–45687.[CrossRef]
    [Google Scholar]
  24. Smith, D. B. & Johnson, K. S. ( 1988; ). Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione-S-transferase. Gene 67, 31–40.[CrossRef]
    [Google Scholar]
  25. Tan, S. L., Nakao, H., He, Y. P., Vijaysri, S., Neddermann, P., Jacobs, B. L., Mayer, B. J. & Katze, M. G. ( 1999; ). NS5A, a nonstructural protein of hepatitis C virus, binds growth factor receptor-bound protein 2 adaptor protein in a Src homology 3 domain/ligand-dependent manner and perturbs mitogenic signaling. Proc Natl Acad Sci U S A 96, 5533–5538.[CrossRef]
    [Google Scholar]
  26. Tatosyan, A. G. & Mizenina, O. A. ( 2000; ). Kinases of the Src family: structure and functions. Biochemistry (Mosc) 65, 49–58.
    [Google Scholar]
  27. 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]
  28. Yanagi, M., St Claire, M., Shapiro, M., Emerson, S. U., Purcell, R. H. & Bukh, J. ( 1998; ). Transcripts of a chimeric cDNA clone of hepatitis C virus genotype 1b are infectious in vivo. Virology 244, 161–172.[CrossRef]
    [Google Scholar]
  29. Yanagi, M., Purcell, R. H., Emerson, S. U. & Bukh, J. ( 1999; ). Hepatitis C virus: an infectious molecular clone of a second major genotype (2a) and lack of viability of intertypic 1a and 2a chimeras. Virology 262, 250–263.[CrossRef]
    [Google Scholar]
  30. Yoshikawa, H., Matsubara, K., Qian, G. S., Jackson, P., Groopman, J. D., Manning, J. E., Harris, C. C. & Herman, J. G. ( 2001; ). SOCS-1, a negative regulator of the JAK/STAT pathway, is silenced by methylation in human hepatocellular carcinoma and shows growth-suppression activity. Nat Genet 28, 29–35.
    [Google Scholar]
  31. 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.0.19691-0
Loading
/content/journal/jgv/10.1099/vir.0.19691-0
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