1887

Abstract

The NS3 protein of hepatitis C virus (HCV) has a serine protease activity in its N-terminal region, which plays a crucial role in virus replication. This region has also been reported to interact not only with its viral cofactor NS4A, but also with a number of host-cell proteins, which suggests a multifunctional feature of NS3. By means of yeast two-hybrid screening using an N-terminal region of NS3 as bait, a human cDNA encoding a region of ELKS-, a member of a novel family of proteins involved in intracellular transport and secretory pathways, was molecularly cloned. Using co-immunoprecipitation, GST pull-down and confocal and immunoelectron microscopic analyses, it was shown that full-length NS3 interacted physically with full-length ELKS- and its splice variant, ELKS-, both in the absence and presence of NS4A, in cultured human cells, including Huh-7 cells harbouring an HCV subgenomic RNA replicon. The degree of binding to ELKS- varied with different sequences of the N-terminal 180 residues of NS3. Interestingly, NS3, either full-length or N-terminal fragments, enhanced secretion of secreted alkaline phosphatase (SEAP) from the cells, and the increase in SEAP secretion correlated well with the degree of binding between NS3 and ELKS-. Taken together, these results suggest the possibility that NS3 plays a role in modulating host-cell functions such as intracellular transport and secretion through its binding to ELKS- and ELKS-, which may facilitate the virus life cycle and/or mediate the pathogenesis of HCV.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.80862-0
2005-08-01
2021-03-05
Loading full text...

Full text loading...

/deliver/fulltext/jgv/86/8/vir862197.html?itemId=/content/journal/jgv/10.1099/vir.0.80862-0&mimeType=html&fmt=ahah

References

  1. Aizaki H., Lee K.-J., Sung V. M.-H., Ishiko H., Lai M. M. C. 2004; Characterization of the hepatitis C virus RNA replication complex associated with lipid rafts. Virology 324:450–461 [CrossRef]
    [Google Scholar]
  2. Aoubala M., Holt J., Clegg R. A., Rowlands D. J., Harris M. 2001; The inhibition of cAMP-dependent protein kinase by full-length hepatitis C virus NS3/4A complex is due to ATP hydrolysis. J Gen Virol 82:1637–1646
    [Google Scholar]
  3. Borowski P., Heiland M., Feucht H., Laufs R. 1999a; Characterisation of non-structural protein 3 of hepatitis C virus as modulator of protein phosphorylation mediated by PKA and PKC: evidences for action on the level of substrate and enzyme. Arch Virol 144:687–701 [CrossRef]
    [Google Scholar]
  4. Borowski P., Schulze zur Wiesch J., Resch K., Feucht H., Laufs R., Schmitz H. 1999b; Protein kinase C recognizes the protein kinase A-binding motif of nonstructural protein 3 of hepatitis C virus. J Biol Chem 274:30722–30728 [CrossRef]
    [Google Scholar]
  5. Borowski P., Kühl R., Laufs R., Schulze zur Wiesch J., Heiland M. 1999c; Identification and characterization of a histone binding site of the non-structural protein 3 of hepatitis C virus. J Clin Virol 13:61–69 [CrossRef]
    [Google Scholar]
  6. 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]
  7. Cullen B. R., Malim M. H. 1992; Secreted placental alkaline phosphatase as a eukaryotic reporter gene. Methods Enzymol 216:362–368
    [Google Scholar]
  8. Deguchi-Tawarada M., Inoue E., Takao-Rikitsu E., Inoue M., Ohtsuka T., Takai Y. 2004; CAST2: identification and characterization of a protein structurally related to the presynaptic cytomatrix CAST. Genes Cells 9:15–23 [CrossRef]
    [Google Scholar]
  9. Ducut Sigala J. L., Bottero V., Young D. B., Shevchenko A., Mercurio F., Verma I. M. 2004; Activation of transcription factor NF- κ B requires ELKS, and I κ B kinase regulatory subunit. Science 304:1963–1967 [CrossRef]
    [Google Scholar]
  10. Echard A., Opdam F. J., de Leeuw H. J., Jollivet F., Savelkoul P., Hendriks W., Voorberg J., Goud B., Fransen J. A. 2000; Alternative splicing of the human Rab6A gene generates two close but functionally different isoforms. Mol Biol Cell 11:3819–3833 [CrossRef]
    [Google Scholar]
  11. Feldmann G., Durand-Schneider A. M., Goud B. 1995; Behaviour of the small GTP-binding protein rab6 in the liver of normal rats and rats presenting an acute inflammatory reaction. Biol Cell 83:121–125 [CrossRef]
    [Google Scholar]
  12. He Q.-Q., Cheng R.-X., Sun Y., Feng D.-Y., Chen Z.-C., Zeng H. 2003; Hepatocyte transformation and tumor development induced by hepatitis C virus NS3 C-terminal deleted protein. World J Gastroenterol 9:474–478
    [Google Scholar]
  13. Hijikata M., Kato N., Ootsuyama Y., Nakagawa M., Shimotohno K. 1991; Gene mapping of the putative structural region of the hepatitis C virus genome by in vitro processing analysis. Proc Natl Acad Sci U S A 88:5547–5551 [CrossRef]
    [Google Scholar]
  14. Hijikata M., Mizushima H., Tanji Y., Komoda Y., Hirowatari Y., Akagi T., Kato N., Kimura K., Shimotohno K. 1993; Proteolytic processing and membrane association of putative nonstructural proteins of hepatitis C virus. Proc Natl Acad Sci U S A 90:10773–10777 [CrossRef]
    [Google Scholar]
  15. 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]
  16. Iwai A., Hasumura Y., Nojima T., Takegami T. 2003; Hepatitis C virus nonstructural protein NS3 binds to Sm-D1, a small nuclear ribonucleoprotein associated with autoimmune disease. Microbiol Immunol 47:601–611 [CrossRef]
    [Google Scholar]
  17. Khu Y.-L., Tan Y.-J., Lim S.-G., Hong W., Goh P.-Y. 2004; Hepatitis C virus non-structural protein NS3 interacts with LMP7, a component of the immunoproteasome, and affects its proteasome activity. Biochem J 384:401–409 [CrossRef]
    [Google Scholar]
  18. Konan K. V., Giddings T. H. Jr, Ikeda M., Li K., Lemon S. M., Kirkegaard K. 2003; Nonstructural protein precursor NS4A/B from hepatitis C virus alters function and ultrastructure of host secretory apparatus. J Virol 77:7843–7855 [CrossRef]
    [Google Scholar]
  19. 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]
  20. Martinez O., Schmidt A., Salamero J., Hoflack B., Roa M., Goud B. 1994; The small GTP-binding protein rab6 functions in intra-Golgi transport. J Cell Biol 127:1575–1588 [CrossRef]
    [Google Scholar]
  21. Monier S., Jollivet F., Janoueix-Lerosey I., Johannes L., Goud B. 2002; Characterization of novel Rab6-interacting proteins involved in endosome-to-TGN transport. Traffic 3:289–297 [CrossRef]
    [Google Scholar]
  22. 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]
  23. Nakata T., Kitamura Y., Shimizu K., Tanaka S., Fujimori M., Yokoyama S., Ito K., Emi M. 1999; Fusion of a novel gene, ELKS , to RET due to translocation t(10; 12)(q11; p13) in a papillary thyroid carcinoma. Genes Chromosomes Cancer 25:97–103 [CrossRef]
    [Google Scholar]
  24. Nakata T., Yokota T., Emi M., Minami S. 2002; Differential expression of multiple isoforms of the ELKS mRNAs involved in a papillary thyroid carcinoma. Genes Chromosomes Cancer 35:30–37 [CrossRef]
    [Google Scholar]
  25. 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 protein 3 (NS3) of hepatitis C virus and development of hepatocellular carcinoma. Microbiol Immunol 46:549–554 [CrossRef]
    [Google Scholar]
  26. 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]
  27. Ohtsuka T., Takao-Rikitsu E., Inoue E. 8 other authors 2002; CAST: a novel protein of the cytomatrix at the active zone of synapses that forms a ternary complex with RIM1 and Munc13-1. J Cell Biol 158:577–590 [CrossRef]
    [Google Scholar]
  28. 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]
  29. Rho J., Choi S., Seong Y. R., Choi J., Im D.-S. 2001; The arginine-1493 residue in QRRGRTGR1493G motif IV of the hepatitis C virus NS3 helicase domain is essential for NS3 protein methylation by the protein arginine methyltransferase 1. J Virol 75:8031–8044 [CrossRef]
    [Google Scholar]
  30. Sakamuro D., Furukawa T., Takegami T. 1995; Hepatitis C virus nonstructural protein NS3 transforms NIH 3T3 cells. J Virol 69:3893–3896
    [Google Scholar]
  31. 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]
  32. Takigawa Y., Nagano-Fujii M., Deng L., Hidajat R., Tanaka M., Mizuta H., Hotta H. 2004; Suppression of hepatitis C virus replicon by RNA interference directed against the NS3 and NS5B regions of the viral genome. Microbiol Immunol 48:591–598 [CrossRef]
    [Google Scholar]
  33. Tellinghuisen T. L., Rice C. M. 2002; Interaction between hepatitis C virus proteins and host cell factors. Curr Opin Microbiol 5:419–427 [CrossRef]
    [Google Scholar]
  34. Tong W.-Y., Nagano-Fujii M., Hidajat R., Deng L., Takigawa Y., Hotta H. 2002; Physical interaction between hepatitis C virus NS4B protein and CREB-RP/ATF6 β . Biochem Biophys Res Commun 299:366–372 [CrossRef]
    [Google Scholar]
  35. Wang Y., Liu X., Biederer T., Südhof T. C. 2002; A family of RIM-binding proteins regulated by alternative splicing: implications for the genesis of synaptic active zones. Proc Natl Acad Sci U S A 99:14464–14469 [CrossRef]
    [Google Scholar]
  36. WHO 1999; Hepatitis C – global prevalence (update). Wkly Epidemiol Rec 74:425–427
    [Google Scholar]
  37. Wölk B., Sansonno D., Kräusslich H. G., Dammacco F., Rice C. M., Blum H. E., Moradpour D. 2000; Subcellular locacalization, stability, and trans -cleavage competence of the hepatitis C virus NS3–NS4A complex expressed in tetracycline-regulated cell lines. J Virol 74:2293–2304 [CrossRef]
    [Google Scholar]
  38. 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.0.80862-0
Loading
/content/journal/jgv/10.1099/vir.0.80862-0
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF

Most cited this month 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