Skip to main content
NCBI home page
Journal of Virology logoLink to Journal of Virology
. 1995 Mar;69(3):1769–1777. doi: 10.1128/jvi.69.3.1769-1777.1995

An amino-terminal domain of the hepatitis C virus NS3 protease is essential for interaction with NS4A.

C Failla 1, L Tomei 1, R De Francesco 1
PMCID: PMC188783  PMID: 7853516

Abstract

Hepatitis C virus (HCV) genomic RNA is translated into a large polyprotein that is processed into structural and nonstructural proteins. Processing at the N termini of several nonstructural proteins requires sequences contained in both NS3 and NS4A. NS3 contains a serine protease, whereas the function of NS4A in proteolysis is yet to be determined. By using the vaccinia virus-T7 hybrid expression system to transiently express HCV polypeptides in HeLa cells, we studied the effect of several N-terminal and C-terminal deletions of HCV NS3 on the processing activity at all the downstream cleavage sites. In this way, we have delineated the minimal domain of NS3 required for the serine protease activity associated with this protein. In addition, we demonstrate the formation of a stable complex between NS3 and NS4A: analysis of the deletion mutants reveals a region at the N terminus of NS3 that is necessary for both complex formation and modulation of the proteolytic activity by NS4A but not for the NS4A-independent serine protease activity of NS3.

Full Text

The Full Text of this article is available as a PDF (390.8 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Arias C. F., Preugschat F., Strauss J. H. Dengue 2 virus NS2B and NS3 form a stable complex that can cleave NS3 within the helicase domain. Virology. 1993 Apr;193(2):888–899. doi: 10.1006/viro.1993.1198. [DOI] [PubMed] [Google Scholar]
  2. Baker D., Sohl J. L., Agard D. A. A protein-folding reaction under kinetic control. Nature. 1992 Mar 19;356(6366):263–265. doi: 10.1038/356263a0. [DOI] [PubMed] [Google Scholar]
  3. Bartenschlager R., Ahlborn-Laake L., Mous J., Jacobsen H. Kinetic and structural analyses of hepatitis C virus polyprotein processing. J Virol. 1994 Aug;68(8):5045–5055. doi: 10.1128/jvi.68.8.5045-5055.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bartenschlager R., Ahlborn-Laake L., Mous J., Jacobsen H. Nonstructural protein 3 of the hepatitis C virus encodes a serine-type proteinase required for cleavage at the NS3/4 and NS4/5 junctions. J Virol. 1993 Jul;67(7):3835–3844. doi: 10.1128/jvi.67.7.3835-3844.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bazan J. F., Fletterick R. J. Detection of a trypsin-like serine protease domain in flaviviruses and pestiviruses. Virology. 1989 Aug;171(2):637–639. doi: 10.1016/0042-6822(89)90639-9. [DOI] [PubMed] [Google Scholar]
  6. Chambers T. J., Grakoui A., Rice C. M. Processing of the yellow fever virus nonstructural polyprotein: a catalytically active NS3 proteinase domain and NS2B are required for cleavages at dibasic sites. J Virol. 1991 Nov;65(11):6042–6050. doi: 10.1128/jvi.65.11.6042-6050.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chambers T. J., Nestorowicz A., Amberg S. M., Rice C. M. Mutagenesis of the yellow fever virus NS2B protein: effects on proteolytic processing, NS2B-NS3 complex formation, and viral replication. J Virol. 1993 Nov;67(11):6797–6807. doi: 10.1128/jvi.67.11.6797-6807.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chien D. Y., Choo Q. L., Tabrizi A., Kuo C., McFarland J., Berger K., Lee C., Shuster J. R., Nguyen T., Moyer D. L. Diagnosis of hepatitis C virus (HCV) infection using an immunodominant chimeric polyprotein to capture circulating antibodies: reevaluation of the role of HCV in liver disease. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10011–10015. doi: 10.1073/pnas.89.21.10011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Choo Q. L., Kuo G., Weiner A. J., Overby L. R., Bradley D. W., Houghton M. Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science. 1989 Apr 21;244(4902):359–362. doi: 10.1126/science.2523562. [DOI] [PubMed] [Google Scholar]
  10. Choo Q. L., Richman K. H., Han J. H., Berger K., Lee C., Dong C., Gallegos C., Coit D., Medina-Selby R., Barr P. J. Genetic organization and diversity of the hepatitis C virus. Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2451–2455. doi: 10.1073/pnas.88.6.2451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Craig E. A. Chaperones: helpers along the pathways to protein folding. Science. 1993 Jun 25;260(5116):1902–1903. doi: 10.1126/science.8100364. [DOI] [PubMed] [Google Scholar]
  12. Eckart M. R., Selby M., Masiarz F., Lee C., Berger K., Crawford K., Kuo C., Kuo G., Houghton M., Choo Q. L. The hepatitis C virus encodes a serine protease involved in processing of the putative nonstructural proteins from the viral polyprotein precursor. Biochem Biophys Res Commun. 1993 Apr 30;192(2):399–406. doi: 10.1006/bbrc.1993.1429. [DOI] [PubMed] [Google Scholar]
  13. Elroy-Stein O., Fuerst T. R., Moss B. Cap-independent translation of mRNA conferred by encephalomyocarditis virus 5' sequence improves the performance of the vaccinia virus/bacteriophage T7 hybrid expression system. Proc Natl Acad Sci U S A. 1989 Aug;86(16):6126–6130. doi: 10.1073/pnas.86.16.6126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Falgout B., Pethel M., Zhang Y. M., Lai C. J. Both nonstructural proteins NS2B and NS3 are required for the proteolytic processing of dengue virus nonstructural proteins. J Virol. 1991 May;65(5):2467–2475. doi: 10.1128/jvi.65.5.2467-2475.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fuerst T. R., Niles E. G., Studier F. W., Moss B. Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc Natl Acad Sci U S A. 1986 Nov;83(21):8122–8126. doi: 10.1073/pnas.83.21.8122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Grakoui A., McCourt D. W., Wychowski C., Feinstone S. M., Rice C. M. A second hepatitis C virus-encoded proteinase. Proc Natl Acad Sci U S A. 1993 Nov 15;90(22):10583–10587. doi: 10.1073/pnas.90.22.10583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Grakoui A., McCourt D. W., Wychowski C., Feinstone S. M., Rice C. M. Characterization of the hepatitis C virus-encoded serine proteinase: determination of proteinase-dependent polyprotein cleavage sites. J Virol. 1993 May;67(5):2832–2843. doi: 10.1128/jvi.67.5.2832-2843.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Grakoui A., Wychowski C., Lin C., Feinstone S. M., Rice C. M. Expression and identification of hepatitis C virus polyprotein cleavage products. J Virol. 1993 Mar;67(3):1385–1395. doi: 10.1128/jvi.67.3.1385-1395.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hijikata M., Kato N., Ootsuyama Y., Nakagawa M., Shimotohno K. 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. 1991 Jul 1;88(13):5547–5551. doi: 10.1073/pnas.88.13.5547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hijikata M., Mizushima H., Akagi T., Mori S., Kakiuchi N., Kato N., Tanaka T., Kimura K., Shimotohno K. Two distinct proteinase activities required for the processing of a putative nonstructural precursor protein of hepatitis C virus. J Virol. 1993 Aug;67(8):4665–4675. doi: 10.1128/jvi.67.8.4665-4675.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ikemura H., Inouye M. In vitro processing of pro-subtilisin produced in Escherichia coli. J Biol Chem. 1988 Sep 15;263(26):12959–12963. [PubMed] [Google Scholar]
  22. Jang S. K., Davies M. V., Kaufman R. J., Wimmer E. Initiation of protein synthesis by internal entry of ribosomes into the 5' nontranslated region of encephalomyocarditis virus RNA in vivo. J Virol. 1989 Apr;63(4):1651–1660. doi: 10.1128/jvi.63.4.1651-1660.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kato N., Hijikata M., Ootsuyama Y., Nakagawa M., Ohkoshi S., Sugimura T., Shimotohno K. Molecular cloning of the human hepatitis C virus genome from Japanese patients with non-A, non-B hepatitis. Proc Natl Acad Sci U S A. 1990 Dec;87(24):9524–9528. doi: 10.1073/pnas.87.24.9524. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kuo G., Choo Q. L., Alter H. J., Gitnick G. L., Redeker A. G., Purcell R. H., Miyamura T., Dienstag J. L., Alter M. J., Stevens C. E. An assay for circulating antibodies to a major etiologic virus of human non-A, non-B hepatitis. Science. 1989 Apr 21;244(4902):362–364. doi: 10.1126/science.2496467. [DOI] [PubMed] [Google Scholar]
  25. Lin C., Amberg S. M., Chambers T. J., Rice C. M. Cleavage at a novel site in the NS4A region by the yellow fever virus NS2B-3 proteinase is a prerequisite for processing at the downstream 4A/4B signalase site. J Virol. 1993 Apr;67(4):2327–2335. doi: 10.1128/jvi.67.4.2327-2335.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Minton E. J., Tysoe C., Sinclair J. H., Sissons J. G. Human cytomegalovirus infection of the monocyte/macrophage lineage in bone marrow. J Virol. 1994 Jun;68(6):4017–4021. doi: 10.1128/jvi.68.6.4017-4021.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Pizzi E., Tramontano A., Tomei L., La Monica N., Failla C., Sardana M., Wood T., De Francesco R. Molecular model of the specificity pocket of the hepatitis C virus protease: implications for substrate recognition. Proc Natl Acad Sci U S A. 1994 Feb 1;91(3):888–892. doi: 10.1073/pnas.91.3.888. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Preugschat F., Yao C. W., Strauss J. H. In vitro processing of dengue virus type 2 nonstructural proteins NS2A, NS2B, and NS3. J Virol. 1990 Sep;64(9):4364–4374. doi: 10.1128/jvi.64.9.4364-4374.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Silen J. L., Agard D. A. The alpha-lytic protease pro-region does not require a physical linkage to activate the protease domain in vivo. Nature. 1989 Oct 5;341(6241):462–464. doi: 10.1038/341462a0. [DOI] [PubMed] [Google Scholar]
  30. Takamizawa A., Mori C., Fuke I., Manabe S., Murakami S., Fujita J., Onishi E., Andoh T., Yoshida I., Okayama H. Structure and organization of the hepatitis C virus genome isolated from human carriers. J Virol. 1991 Mar;65(3):1105–1113. doi: 10.1128/jvi.65.3.1105-1113.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Tomei L., Failla C., Santolini E., De Francesco R., La Monica N. NS3 is a serine protease required for processing of hepatitis C virus polyprotein. J Virol. 1993 Jul;67(7):4017–4026. doi: 10.1128/jvi.67.7.4017-4026.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Zhu X. L., Ohta Y., Jordan F., Inouye M. Pro-sequence of subtilisin can guide the refolding of denatured subtilisin in an intermolecular process. Nature. 1989 Jun 8;339(6224):483–484. doi: 10.1038/339483a0. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES