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Single-cell resolution imaging of membrane-anchored hepatitis C virus NS3/4A protease activity

  • Morgan M. Martin and François Jean
Published/Copyright: August 9, 2006
Biological Chemistry
From the journal Volume 387 Issue 8

Abstract

The study of host and viral membrane-associated proteases has been hampered due to a lack of in vivo assays. We report here the development of a cell-based fluorescence assay for detecting hepatitis C virus (HCV) NS3/4A juxtamembrane protease activity. Intracellular membrane-anchored protein substrates were engineered comprising: (1) an endoplasmic reticulum targeting domain, the HCV NS5A N-terminal amphipathic α-helix; (2) a NS3/4A-specific cleavage site; and (3) a red fluorescent reporter group, DsRed. The results of our immunofluorescence and Western blotting studies demonstrate that our membrane-bound fluorescent probe was cleaved specifically and efficiently by NS3/4A expressed in human cells.

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References

Aizaki, H., Lee, K.J., Sung, V.M., Ishiko H., and Lai, M.M. (2004). Characterization of the hepatitis C virus RNA replication complex associated with lipid rafts. Virology324, 450–461.10.1016/j.virol.2004.03.034Search in Google Scholar PubMed

Baruch, A., Jeffery, D.A., and Bogyo, M. (2004). Enzyme activity – it's all about image. Trends Cell Biol.14, 29–35.10.1016/j.tcb.2003.11.002Search in Google Scholar PubMed

Bauvois, B. (2004). Transmembrane proteases in cell growth and invasion: new contributors to angiogenesis?Oncogene23, 317–329.10.1038/sj.onc.1207124Search in Google Scholar PubMed

Egger, D., Wolk, B., Gosert, R., Bianchi, L., Blum, H.E., Moradpour, D., and Bienz, K. (2002). Expression of hepatitis C virus proteins induces distinct membrane alterations including a candidate viral replication complex. J. Virol.76, 5974–5984.10.1128/JVI.76.12.5974-5984.2002Search in Google Scholar PubMed PubMed Central

Ehrmann, M. and Clausen, T. (2004). Proteolysis as a regulatory mechanism. Annu. Rev. Genet.38, 709–724.10.1146/annurev.genet.38.072902.093416Search in Google Scholar PubMed

Elazar, M., Cheong, K.H., Liu, P., Greenberg, H.B., Rice, C.M., and Glenn, J.S. (2003). Amphipathic helix-dependent localization of NS5A mediates hepatitis C virus RNA replication. J. Virol.77, 6055–6061.10.1128/JVI.77.10.6055-6061.2003Search in Google Scholar PubMed PubMed Central

Ferreon, J.C., Ferreon, A.C., Li, K., and Lemon, S.M. (2005). Molecular determinants of TRIF proteolysis mediated by the hepatitis C virus NS3/4A protease. J. Biol. Chem.280, 20483–20492.10.1074/jbc.M500422200Search in Google Scholar PubMed

Golde, T.E. and Eckman, C.B. (2003). Physiologic and pathologic events mediated by intramembranous and juxtamembranous proteolysis. Sci. STKE172, RE4.Search in Google Scholar

Goudreau, N. and Llinas-Brunet, M. (2005). The therapeutic potential of NS3 protease inhibitors in HCV infection. Expert. Opin. Invest. Drugs14, 1129–1144.10.1517/13543784.14.9.1129Search in Google Scholar PubMed

Hamill, P. and Jean, F. (2005). Enzymatic characterization of membrane-associated hepatitis C virus NS3-4A heterocomplex serine protease activity expressed in human cells. Biochemistry44, 6586–6596.10.1021/bi047408jSearch in Google Scholar PubMed

Jean, F., Basak, A., DiMaio, J., Seidah, N.G., and Lazure, C. (1995a). An internally quenched fluorogenic substrate of prohormone convertase 1 and furin leads to a potent prohormone convertase inhibitor. Biochem. J.307, 689–695.10.1042/bj3070689Search in Google Scholar PubMed PubMed Central

Jean, F., Boudreault, A., Basak, A., Seidah, N.G., and Lazure, C. (1995b). Fluorescent peptidyl substrates as an aid in studying the substrate specificity of human prohormone convertase PC1 and human furin and designing a potent irreversible inhibitor. J. Biol. Chem.270, 19225–19231.10.1074/jbc.270.33.19225Search in Google Scholar PubMed

Jones, J., Heim, R., Hare, E., Stack, J., and Pollok, B.A. (2000). Development and application of a GFP-FRET intracellular caspase assay for drug screening. J. Biomol. Screen.5, 307–318.10.1177/108705710000500502Search in Google Scholar PubMed

Kim, S.Y., Park, K.W., Lee, Y.J., Back, S.H., Goo, J.H., Park, O.K., Jang, S.K., and Park, W.J. (2000). In vivo determination of substrate specificity of hepatitis C virus NS3 protease: genetic assay for site-specific proteolysis. Anal. Biochem.284, 42–48.10.1006/abio.2000.4662Search in Google Scholar PubMed

Lamarre, D., Anderson, P.C., Bailey, M., Beulieu, P., Bolger, G., Bonneau, P., Bos, M., Cameron, D.R., Cartier, M., Cordingley, M.G., et al. (2003). An NS3 protease inhibitor with antiviral effects in humans infected with hepatitis C virus. Nature426, 186–189.10.1038/nature02099Search in Google Scholar PubMed

Li, K., Foy, E., Ferreon, J.C., Nakamura, M., Ferreon, A.C., Ikeda, M., Ray, S.C., Gale, M. Jr., and Lemon, S.M. (2005a). Immune evasion by hepatitis C virus NS3/4A protease-mediated cleavage of the Toll-like receptor 3 adaptor protein TRIF. Proc. Natl. Acad. Sci. USA102, 2992–2997.10.1073/pnas.0408824102Search in Google Scholar PubMed PubMed Central

Li, X.D., Sun, L., Seth, R.B., Pineda, G., and Chen, Z.J. (2005b). Hepatitis C virus protease NS3/4A cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity. Proc. Natl. Acad. Sci. USA102, 17717–17722.10.1073/pnas.0508531102Search in Google Scholar PubMed PubMed Central

Matz, M.V., Fradkov, A.F., Labas, Y.A., Savitsky, A.P., Zaraisky, A.G., Markelov, M.L., and Lukyanov, S.A. (1999). Fluorescent proteins from nonbioluminescent Anthozoa species. Nat. Biotechnol.17, 969–973.10.1038/13657Search in Google Scholar PubMed

Moradpour, D., Evans, M.J., Gosert, R., Yuan, Z., Blum, H.E., Goff, S.P., Lindenbach, B.D., and Rice, C.M. (2004). Insertion of green fluorescent protein into nonstructural protein 5A allows direct visualization of functional hepatitis C virus replication complexes. J. Virol.78, 7400–7409.10.1128/JVI.78.14.7400-7409.2004Search in Google Scholar PubMed PubMed Central

Moradpour, D., Brass, V., and Penin, F. (2005). Function follows form: the structure of the N-terminal domain of HCV NS5A. Hepatology42, 732–735.10.1002/hep.20851Search in Google Scholar PubMed

Mottola, G., Cardinali, G., Ceccacci, A., Trozzi, C., Bartholomew, L., Torrisi, M.R., Pedrazzini, E., Bonatti, S., and Migliaccio, G. (2002). Hepatitis C virus nonstructural proteins are localized in a modified endoplasmic reticulum of cells expressing viral subgenomic replicons. Virology293, 31–43.10.1006/viro.2001.1229Search in Google Scholar PubMed

Netzel-Arnett, S., Hooper, J.D., Szabo, R., Madison, E.L., Quigley, J.P., Bugge, T.H., and Antalis, T.M. (2003). Membrane anchored serine proteases: a rapidly expanding group of cell surface proteolytic enzymes with potential roles in cancer. Cancer Metastasis Rev.22, 237–258.10.1023/A:1023003616848Search in Google Scholar

Richer, M.J., Juliano, L., Hashimoto, C., and Jean, F. (2004). Serpin mechanism of hepatitis C virus nonstructural 3 (NS3) protease inhibition: induced fit as a mechanism for narrow specificity. J. Biol. Chem.279, 10222–10227.10.1074/jbc.M313852200Search in Google Scholar

Salonen, A., Ahola, T., and Kaariainen, L. (2005). Viral RNA replication in association with cellular membranes. Curr. Top. Microbiol. Immunol.285, 139–173.Search in Google Scholar

Schechter, I. and Berger, A. (1967). On the size of the active site in proteases. I. Papain. Biochem. Biophys. Res. Commun.27, 157–162.10.1016/S0006-291X(67)80055-XSearch in Google Scholar

Tawa, P., Tam, J., Cassady, R., Nicholson, D.W., and Xanthoudakis, S. (2001). Quantitative analysis of fluorescent caspase substrate cleavage in intact cells and identification of novel inhibitors of apoptosis. Cell Death Differ.8, 30–37.10.1038/sj.cdd.4400769Search in Google Scholar PubMed

Wolfe, M.S. and Kopan, R. (2004). Intramembrane proteolysis: theme and variations. Science305, 1119–1123.10.1126/science.1096187Search in Google Scholar PubMed

Wolk, B., Sansonno, D., Krausslich, H.G., Dammacco, F., Rice, C.M., Blum, H.E., and Moradpour, D. (2000). Subcellular localization, stability, and trans-cleavage competence of the hepatitis C virus NS3-NS4A expressed in tetracycline-regulated cell lines. J. Virol.74, 2293–2304.10.1128/JVI.74.5.2293-2304.2000Search in Google Scholar PubMed PubMed Central

Zhang, R., Durkin, J., Windsor, W.T., McNemar, C., Ramanathan, L., and Le, H.V. (1997). Probing the substrate specificity of hepatitis C virus NS3 serine protease by using synthetic peptides. J. Virol.71, 6208–6213.10.1128/jvi.71.8.6208-6213.1997Search in Google Scholar PubMed PubMed Central

Published Online: 2006-08-09
Published in Print: 2006-08-01

©2006 by Walter de Gruyter Berlin New York

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