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Journal of Virology, October 2003, p. 10448-10455, Vol. 77, No. 19
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.19.10448-10455.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Purification, Identification, and Biochemical Characterization of a Host-Encoded Cysteine Protease That Cleaves a Leishmaniavirus Gag-Pol Polyprotein

Ricardo Carrion Jr.,1,2 Young-Tae Ro,3 and Jean L. Patterson1*

Department of Virology and Immunology, Southwest Foundation for Biomedical Research, San Antonio, Texas 78227-5301,1 Department of Microbiology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229-3900,2 Laboratory of Biochemistry, College of Medicine, Konkuk University, Chungju, Korea3

Received 10 April 2003/ Accepted 9 July 2003

Leishmania RNA virus (LRV) is a double-stranded RNA virus that infects some strains of the protozoan parasite Leishmania. As with other totiviruses, LRV presumably expresses its polymerase by a ribosomal frameshift, resulting in a capsid-polymerase fusion protein. We have demonstrated previously that an LRV capsid-polymerase polyprotein is specifically cleaved by a Leishmania-encoded cysteine protease. This study reports the purification of this protease through a strategy involving anion-exchange chromatography and affinity chromatography. By using a Sepharose-immobilized lectin, concanavalin A, we isolated a fraction enriched with LRV polyprotein-specific protease activity. Analysis of the active fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoreses and silver staining revealed a 50-kDa protein that, upon characterization by high-pressure liquid chromatography electrospray tandem mass spectrometry (electrospray ionization/MS/MS), was identified as a cysteine protease of trypanosomes. A partial amino acid sequence derived from the MS/MS data was compared with a protein database using BLAST software, revealing homology with several cysteine proteases of Leishmania and other trypanosomes. The protease exhibited remarkable temperature stability, while inhibitor studies characterized the protease as a trypsin-like cysteine protease—a novel finding for Leishmania. To elucidate substrate preferences, a panel of deletion mutations and single-amino-acid mutations were engineered into a Gag-Pol fusion construct that was subsequently transcribed and translated in vitro and then used in cleavage assays. The data suggest that there are a number of cleavage sites located within a 153-amino-acid region spanning both the carboxy-terminal capsid region and the amino-terminal polymerase domain, with LRV capsid exhibiting the greatest susceptibility to proteolysis.

* Corresponding author. Mailing address: Southwest Foundation for Biomedical Research, Department of Virology and Immunology, 7620 NW Loop 410, San Antonio, TX 78227-5301. Phone: (210) 258-9431. Fax: (210) 670-3329. E-mail: jpatters{at}sfbr.org.

Journal of Virology, October 2003, p. 10448-10455, Vol. 77, No. 19
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.19.10448-10455.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Vitovski, S., Sayers, J. R. (2007). Relaxed Cleavage Specificity of an Immunoglobulin A1 Protease from Neisseria meningitidis. Infect. Immun. 75: 2875-2885 [Abstract] [Full Text]  


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