This Article
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Pieroni, L.
Right arrow Articles by La Monica, N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Pieroni, L.
Right arrow Articles by La Monica, N.

 Previous Article  |  Next Article 

J. Virol., Sep 1997, 6373-6380, Vol 71, No. 9
Copyright © 1997, American Society for Microbiology

In vitro study of the NS2-3 protease of hepatitis C virus

L Pieroni, E Santolini, C Fipaldini, L Pacini, G Migliaccio and N La Monica
I.R.B.M. Instituto di Ricerche di Biologia Molecolare P. Angeletti, Italy.

Processing at the C terminus of the NS2 protein of hepatitis C virus (HCV) is mediated by a virus-encoded protease which spans most of the NS2 protein and part of the NS3 polypeptide. In vitro cotranslational cleavage at the 2-3 junction is stimulated by the presence of microsomal membranes and ultimately results in the membrane insertion of the NS2 polypeptide. To characterize the biochemical properties of this viral protease, we have established an in vitro assay whereby the NS2-3 protease of HCV BK can be activated posttranslationally by the addition of detergents. The cleavage proficiency of several deletion and single point mutants was the same as that observed with microsomal membranes, indicating that the overall sequence requirements for proper cleavage at this site are maintained even under these artificial conditions. The processing efficiency of the NS2-3 protease varied according to the type of detergent used and its concentration. Also, the incubation temperature affected the cleavage at the 2-3 junction. The autoproteolytic activity of the NS2-3 protease could be inhibited by alkylating agents such as iodoacetamide and N-ethylmaleimide. Metal chelators such as EDTA and phenanthroline also inhibited the viral enzyme. The EDTA inhibition of NS2-3 cleavage could be reversed, at least in part, by the addition of ZnCl2 and CdCl2. Among the common protease inhibitors tested, tosyl phenylalanyl chloromethyl ketone and soybean trypsin inhibitor inactivated the NS2-3 protease. By means of gel filtration analysis, it was observed that the redox state of the reaction mixture greatly influenced the processing efficiency at the 2- 3 site and that factors present in the rabbit reticulocyte lysate, wheat germ extract, and HeLa cell extract were required for efficient processing at this site. Thus, the in vitro assay should allow further characterization of the biochemical properties of the NS2-3 protease of HCV and the identification of host components that contribute to the efficient processing at the 2-3 junction.


This article has been cited by other articles:

  • Schregel, V., Jacobi, S., Penin, F., Tautz, N. (2009). Hepatitis C virus NS2 is a protease stimulated by cofactor domains in NS3. Proc. Natl. Acad. Sci. USA 106: 5342-5347 [Abstract] [Full Text]  
  • Yi, M., Ma, Y., Yates, J., Lemon, S. M. (2007). Compensatory Mutations in E1, p7, NS2, and NS3 Enhance Yields of Cell Culture-Infectious Intergenotypic Chimeric Hepatitis C Virus. J. Virol. 81: 629-638 [Abstract] [Full Text]  
  • Welbourn, S., Green, R., Gamache, I., Dandache, S., Lohmann, V., Bartenschlager, R., Meerovitch, K., Pause, A. (2005). Hepatitis C Virus NS2/3 Processing Is Required for NS3 Stability and Viral RNA Replication. J. Biol. Chem. 280: 29604-29611 [Abstract] [Full Text]  
  • Svitkin, Y. V., Pause, A., Lopez-Lastra, M., Perreault, S., Sonenberg, N. (2005). Complete Translation of the Hepatitis C Virus Genome In Vitro: Membranes Play a Critical Role in the Maturation of All Virus Proteins except for NS3. J. Virol. 79: 6868-6881 [Abstract] [Full Text]  
  • Erdtmann, L., Franck, N., Lerat, H., Le Seyec, J., Gilot, D., Cannie, I., Gripon, P., Hibner, U., Guguen-Guillouzo, C. (2003). The Hepatitis C Virus NS2 Protein Is an Inhibitor of CIDE-B-induced Apoptosis. J. Biol. Chem. 278: 18256-18264 [Abstract] [Full Text]  
  • Dimitrova, M., Imbert, I., Kieny, M. P., Schuster, C. (2003). Protein-Protein Interactions between Hepatitis C Virus Nonstructural Proteins. J. Virol. 77: 5401-5414 [Abstract] [Full Text]  
  • Yamaga, A. K., Ou, J.-h. (2002). Membrane Topology of the Hepatitis C Virus NS2 Protein. J. Biol. Chem. 277: 33228-33234 [Abstract] [Full Text]  
  • Whitney, M., Stack, J. H., Darke, P. L., Zheng, W., Terzo, J., Inglese, J., Strulovicil, B., Kuo, L. C., Pollok, B. A. (2002). A Collaborative Screening Program for the Discovery of Inhibitors of HCV NS2/3 cis-Cleaving Protease Activity. J Biomol Screen 7: 149-154 [Abstract]  
  • Thibeault, D., Maurice, R., Pilote, L., Lamarre, D., Pause, A. (2001). In Vitro Characterization of a Purified NS2/3 Protease Variant of Hepatitis C Virus. J. Biol. Chem. 276: 46678-46684 [Abstract] [Full Text]  
  • Pallaoro, M., Lahm, A., Biasiol, G., Brunetti, M., Nardella, C., Orsatti, L., Bonelli, F., Orru, S., Narjes, F., Steinkuhler, C. (2001). Characterization of the Hepatitis C Virus NS2/3 Processing Reaction by Using a Purified Precursor Protein. J. Virol. 75: 9939-9946 [Abstract] [Full Text]  
  • Rinck, G., Birghan, C., Harada, T., Meyers, G., Thiel, H.-J., Tautz, N. (2001). A Cellular J-Domain Protein Modulates Polyprotein Processing and Cytopathogenicity of a Pestivirus. J. Virol. 75: 9470-9482 [Abstract] [Full Text]  
  • Fowler, A., Price-Jones, M., Hughes, K., Anson, J., Lingham, R., Schulman, M. (2000). Development of a High Throughput Scintillation Proximity Assay for Hepatitis C Virus NS3 Protease That Reduces the Proportion of Competitive Inhibitors Identified. J Biomol Screen 5: 153-158 [Abstract]  
  • Darke, P. L., Jacobs, A. R., Waxman, L., Kuo, L. C. (1999). Inhibition of Hepatitis C Virus NS2/3 Processing by NS4A Peptides. IMPLICATIONS FOR CONTROL OF VIRAL PROCESSING. J. Biol. Chem. 274: 34511-34514 [Abstract] [Full Text]  
  • Neddermann, P., Clementi, A., De Francesco, R. (1999). Hyperphosphorylation of the Hepatitis C Virus NS5A Protein Requires an Active NS3 Protease, NS4A, NS4B, and NS5A Encoded on the Same Polyprotein. J. Virol. 73: 9984-9991 [Abstract] [Full Text]  
  • Filocamo, G., Pacini, L., Nardi, C., Bartholomew, L., Scaturro, M., Delmastro, P., Tramontano, A., De Francesco, R., Migliaccio, G. (1999). Selection of Functional Variants of the NS3-NS4A Protease of Hepatitis C Virus by Using Chimeric Sindbis Viruses. J. Virol. 73: 561-575 [Abstract] [Full Text]  
  • Urbani, A., Bazzo, R., Nardi, M. C., Cicero, D. O., De Francesco, R., Steinkuhler, C., Barbato, G. (1998). The Metal Binding Site of the Hepatitis C Virus NS3 Protease. A SPECTROSCOPIC INVESTIGATION. J. Biol. Chem. 273: 18760-18769 [Abstract] [Full Text]  
  • Waxman, L., Whitney, M., Pollok, B. A., Kuo, L. C., Darke, P. L. (2001). Host cell factor requirement for hepatitis C virus enzyme maturation. Proc. Natl. Acad. Sci. USA 98: 13931-13935 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»