Purification, identification, and biochemical characterization of a host-encoded cysteine protease that cleaves a leishmaniavirus gag-pol polyprotein.

Abstract:

: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.

journal_name

J Virol

journal_title

Journal of virology

authors

Carrion R Jr,Ro YT,Patterson JL

doi

10.1128/jvi.77.19.10448-10455.2003

subject

Has Abstract

pub_date

2003-10-01 00:00:00

pages

10448-55

issue

19

eissn

0022-538X

issn

1098-5514

journal_volume

77

pub_type

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