This Article
Right arrow Full Text
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 Amberg, S. M.
Right arrow Articles by Rice, C. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Amberg, S. M.
Right arrow Articles by Rice, C. M.

 Previous Article  |  Next Article 

Journal of Virology, October 1999, p. 8083-8094, Vol. 73, No. 10
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Mutagenesis of the NS2B-NS3-Mediated Cleavage Site in the Flavivirus Capsid Protein Demonstrates a Requirement for Coordinated Processing

Sean M. Amberg and Charles M. Rice*

Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110-1093

Received 11 March 1999/Accepted 6 July 1999

Analysis of flavivirus polyprotein processing has revealed the presence of a substrate for the virus-encoded NS2B-NS3 protease at the carboxy-terminal end of the C (capsid or core) protein. Cleavage at this site has been implicated in the efficient generation of the amino terminus of prM via signal peptidase cleavage. Yellow fever virus has four basic residues (Arg-Lys-Arg-Arg) in the P1 through P4 positions of this cleavage site. Multiple alanine substitutions were made for these residues in order to investigate the substrate specificity and biological significance of this cleavage. Mutants were analyzed by several methods: (i) a cell-free trans processing assay for direct analysis of NS2B-NS3-mediated cleavage; (ii) a trans processing assay in BHK-21 cells, using a C-prM polyprotein, for analysis of prM production; (iii) an infectivity assay of full-length transcripts to determine plaque-forming ability; and (iv) analysis of proteins expressed from full-length transcripts to assess processing in the context of the complete genome. Mutants that exhibited severe defects in processing in vitro and in vivo were incapable of forming plaques. Mutants that contained two adjacent basic residues within the P1 through P4 region were processed more efficiently in vitro and in vivo, and transcripts bearing these mutations were fully infectious. Furthermore, two naturally occurring plaque-forming revertants were analyzed and shown to have restored protein processing phenotypes in vivo. Finally, the efficient production of prM was shown to be dependent on the proteolytic activity of NS3. These data support a model of two coordinated cleavages, one that generates the carboxy terminus of C and another that generates the amino terminus of prM. A block in the viral protease-mediated cleavage inhibits the production of prM by the signal peptidase, inhibits particle release, and eliminates plaque formation.


* Corresponding author. Mailing address: Department of Molecular Microbiology, Washington University School of Medicine, Box 8230, 660 S. Euclid Ave., St. Louis, MO 63110-1093. Phone: (314) 362-2842. Fax: (314) 362-1232. E-mail: rice{at}borcim.wustl.edu.


Journal of Virology, October 1999, p. 8083-8094, Vol. 73, No. 10
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.



This article has been cited by other articles:

  • Schrauf, S., Mandl, C. W., Bell-Sakyi, L., Skern, T. (2009). Extension of Flavivirus Protein C Differentially Affects Early RNA Synthesis and Growth in Mammalian and Arthropod Host Cells. J. Virol. 83: 11201-11210 [Abstract] [Full Text]  
  • Suzuki, R., Winkelmann, E. R., Mason, P. W. (2009). Construction and Characterization of a Single-Cycle Chimeric Flavivirus Vaccine Candidate That Protects Mice against Lethal Challenge with Dengue Virus Type 2. J. Virol. 83: 1870-1880 [Abstract] [Full Text]  
  • Sangiambut, S., Keelapang, P., Aaskov, J., Puttikhunt, C., Kasinrerk, W., Malasit, P., Sittisombut, N. (2008). Multiple regions in dengue virus capsid protein contribute to nuclear localization during virus infection. J. Gen. Virol. 89: 1254-1264 [Abstract] [Full Text]  
  • Schrauf, S., Schlick, P., Skern, T., Mandl, C. W. (2008). Functional Analysis of Potential Carboxy-Terminal Cleavage Sites of Tick-Borne Encephalitis Virus Capsid Protein. J. Virol. 82: 2218-2229 [Abstract] [Full Text]  
  • Yoshii, K., Goto, A., Kawakami, K., Kariwa, H., Takashima, I. (2008). Construction and application of chimeric virus-like particles of tick-borne encephalitis virus and mosquito-borne Japanese encephalitis virus. J. Gen. Virol. 89: 200-211 [Abstract] [Full Text]  
  • Roosendaal, J., Westaway, E. G., Khromykh, A., Mackenzie, J. M. (2006). Regulated Cleavages at the West Nile Virus NS4A-2K-NS4B Junctions Play a Major Role in Rearranging Cytoplasmic Membranes and Golgi Trafficking of the NS4A Protein. J. Virol. 80: 4623-4632 [Abstract] [Full Text]  
  • Barba-Spaeth, G., Longman, R. S., Albert, M. L., Rice, C. M. (2005). Live attenuated yellow fever 17D infects human DCs and allows for presentation of endogenous and recombinant T cell epitopes. JEM 202: 1179-1184 [Abstract] [Full Text]  
  • Huang, C. Y.-H., Silengo, S. J., Whiteman, M. C., Kinney, R. M. (2005). Chimeric Dengue 2 PDK-53/West Nile NY99 Viruses Retain the Phenotypic Attenuation Markers of the Candidate PDK-53 Vaccine Virus and Protect Mice against Lethal Challenge with West Nile Virus. J. Virol. 79: 7300-7310 [Abstract] [Full Text]  
  • Chappell, K. J., Nall, T. A., Stoermer, M. J., Fang, N.-X., Tyndall, J. D. A., Fairlie, D. P., Young, P. R. (2005). Site-directed Mutagenesis and Kinetic Studies of the West Nile Virus NS3 Protease Identify Key Enzyme-Substrate Interactions. J. Biol. Chem. 280: 2896-2903 [Abstract] [Full Text]  
  • Tao, D., Barba-Spaeth, G., Rai, U., Nussenzweig, V., Rice, C. M., Nussenzweig, R. S. (2005). Yellow fever 17D as a vaccine vector for microbial CTL epitopes: protection in a rodent malaria model. JEM 201: 201-209 [Abstract] [Full Text]  
  • Pryor, M. J., Azzola, L., Wright, P. J., Davidson, A. D. (2004). Histidine 39 in the dengue virus type 2 M protein has an important role in virus assembly. J. Gen. Virol. 85: 3627-3636 [Abstract] [Full Text]  
  • Nall, T. A., Chappell, K. J., Stoermer, M. J., Fang, N.-X., Tyndall, J. D. A., Young, P. R., Fairlie, D. P. (2004). Enzymatic Characterization and Homology Model of a Catalytically Active Recombinant West Nile Virus NS3 Protease. J. Biol. Chem. 279: 48535-48542 [Abstract] [Full Text]  
  • Scholle, F., Girard, Y. A., Zhao, Q., Higgs, S., Mason, P. W. (2004). trans-Packaged West Nile Virus-Like Particles: Infectious Properties In Vitro and in Infected Mosquito Vectors. J. Virol. 78: 11605-11614 [Abstract] [Full Text]  
  • Briese, T., Rambaut, A., Lipkin, W. I. (2004). Analysis of the medium (M) segment sequence of Guaroa virus and its comparison to other orthobunyaviruses. J. Gen. Virol. 85: 3071-3077 [Abstract] [Full Text]  
  • Agapov, E. V., Murray, C. L., Frolov, I., Qu, L., Myers, T. M., Rice, C. M. (2004). Uncleaved NS2-3 Is Required for Production of Infectious Bovine Viral Diarrhea Virus. J. Virol. 78: 2414-2425 [Abstract] [Full Text]  
  • Wang, A., Han, S., Sanfacon, H. (2004). Topogenesis in membranes of the NTB-VPg protein of Tomato ringspot nepovirus: definition of the C-terminal transmembrane domain. J. Gen. Virol. 85: 535-545 [Abstract] [Full Text]  
  • Lobigs, M., Lee, E. (2004). Inefficient Signalase Cleavage Promotes Efficient Nucleocapsid Incorporation into Budding Flavivirus Membranes. J. Virol. 78: 178-186 [Abstract] [Full Text]  
  • Bredenbeek, P. J., Kooi, E. A., Lindenbach, B., Huijkman, N., Rice, C. M., Spaan, W. J. M. (2003). A stable full-length yellow fever virus cDNA clone and the role of conserved RNA elements in flavivirus replication. J. Gen. Virol. 84: 1261-1268 [Abstract] [Full Text]  
  • Lorenz, I. C., Allison, S. L., Heinz, F. X., Helenius, A. (2002). Folding and Dimerization of Tick-Borne Encephalitis Virus Envelope Proteins prM and E in the Endoplasmic Reticulum. J. Virol. 76: 5480-5491 [Abstract] [Full Text]  
  • Kummerer, B. M., Rice, C. M. (2002). Mutations in the Yellow Fever Virus Nonstructural Protein NS2A Selectively Block Production of Infectious Particles. J. Virol. 76: 4773-4784 [Abstract] [Full Text]  
  • Hope, R. G., Murphy, D. J., McLauchlan, J. (2002). The Domains Required to Direct Core Proteins of Hepatitis C Virus and GB Virus-B to Lipid Droplets Share Common Features with Plant Oleosin Proteins. J. Biol. Chem. 277: 4261-4270 [Abstract] [Full Text]  
  • Mackenzie, J. M., Westaway, E. G. (2001). Assembly and Maturation of the Flavivirus Kunjin Virus Appear To Occur in the Rough Endoplasmic Reticulum and along the Secretory Pathway, Respectively. J. Virol. 75: 10787-10799 [Abstract] [Full Text]  
  • Matusan, A. E., Kelley, P. G., Pryor, M. J., Whisstock, J. C., Davidson, A. D., Wright, P. J. (2001). Mutagenesis of the dengue virus type 2 NS3 proteinase and the production of growth-restricted virus. J. Gen. Virol. 82: 1647-1656 [Abstract] [Full Text]  
  • Khromykh, A. A., Sedlak, P. L., Westaway, E. G. (2000). cis- and trans-Acting Elements in Flavivirus RNA Replication. J. Virol. 74: 3253-3263 [Abstract] [Full Text]