Purification, identification, and biochemical characterization of a host-encoded cysteine protease that cleaves a leishmaniavirus gag-pol polyprotein.
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.
Carrion
Ricardo
R
Department of Virology and Immunology, Southwest Foundation for Biomedical Research, San Antonio, Texas 78227-5301, USA.
Ro
Young-Tae
YT
Patterson
Jean L
JL
D000818:Animals; D003546:Cysteine Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D015685:Fusion Proteins, gag-pol / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007891:Leishmania / Q000201:enzymology*; D019199:Leishmaniavirus / Q000378:metabolism*; D013058:Mass Spectrometry; D011233:Precipitin Tests; D015800:Protozoan Proteins / Q000302:isolation & purification*; D013696:Temperature
D015685:Fusion Proteins, gag-pol; D015800:Protozoan Proteins; D003546:Cysteine Endopeptidases