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.

FIG. 1.

Cleavage activity and protein profile of the highly purified CysP. (A) In vitro cleavage products were resolved on an SDS-10% polyacrylamide gel and visualized by autoradiography. Note the cleavage products in the positive control (lane 2) migrating at 82 and 95 kDa (capsid and polymerase, respectively). (B) Protein profile of active fractions from each purification step. Promastigote lysate, anion-exchange fraction, and ConA-eluted fraction were resolved on an SDS-10% polyacrylamide gel (lanes 1, 2, and 3). Note the 49-kDa CYSP and 30-kDa ConA contaminant (arrows). Lane M, prestained molecular mass marker (Invitrogen). CP, cysteine protease. (C) Substrate gel electrophoresis was performed as described in Materials and Methods. Lanes 1 to 3 represent the size marker, S15 fraction, and highly purified ConA-eluted CysP, respectively. The arrow indicates the location of the 50-kDa CYSP. CP, cysteine protease.

FIG. 2.

Characterization of the Gag-Pol cleavage products by immunoprecipitation with capsid- and polymerase-specific antisera. In vitro cleavage reactions were immunoprecipitated with preimmune antiserum, capsid-specific antiserum, and polymerase-specific antiserum (lanes 3 to 5, respectively). The immune complexes were resolved on an SDS-10% polyacrylamide gel and visualized by autoradiography. Note the cleavage products in the positive control (lane 2) migrating at 82 and 95 kDa (capsid and polymerase, respectively). Lane 1 is a buffer-only control.

FIG. 3.

Mass spectroscopy and amino acid determination. The highly purified CysP fraction was resolved by SDS-PAGE, and then the bands were excised and digested with trypsin. The resulting digests were analyzed by HPLC-ESI/MS/MS on a Thermo Finnigan LCQ ion trap mass spectrometer in conjunction with a Michrom BioResources MAGIC 2002 micro-HPLC and a microspray interface MALDI-TOF (MS). (A) A graph of the electro-spray mass spectroscopy data eluting at 17.3 min; (B) the tandem mass spectroscopy data of m/z 443.4. Also illustrated on the graph is the deduced amino acid sequence for the CysP.

FIG. 4.

Temperature and pH optimization of purified CysP. (A) The purified CysP is heat stable. Highly purified enzyme was incubated at 4, 25, 37, 45, 55, and 65°C (lanes 1 to 6). Substrate gel electrophoresis was performed as described in Materials and Methods. Gelatinase activity was retained even after incubation at 65°C. The buffer-only control shows no gelatinase activity (lane 7). (B) In vitro cleavage assays were incubated at 4, 25, 37, 45, 55, and 65°C (lanes 2 to 7, respectively). Above 55°C, the substrate is hydrolyzed, though specificity is lost as evidenced by the absence of cleavage products. (C) pH optimization experiments. In vitro cleavage assays were incubated in buffers at pH 4.5, 5.5, 6.2, 7.2, 8.0, and 8.8 (lanes 2 to 7). The protease showed no preference for pH. Note that lane 1 contains the negative control at pH 5.5.

FIG. 5.

Protease inhibitor study. The following protease inhibitors (Roche) were examined for their ability to block polyprotein proteolysis (lanes 3 to 10, respectively): antipain dihydrochloride, aprotinin, E-64, EDTA, leupeptin, Pefabloc SC, phosphoramidon, and trypsin inhibitor. Cleavage was blocked by both CysP inhibitors (lanes 3, 5, and 7) and trypsin inhibitors (lanes 8 and 10). Lanes 1 and 2 represent the buffer-only control and the protease control, respectively.

FIG. 6.

Schematic representation of deletion mutant constructs and single-amino-acid-mutant constructs. Mutants were assembled either by PCR-directed mutagenesis or splicing of overlapping ends as described in Materials and Methods. The results of the cleavage assay are noted to the right.

FIG. 7.

Protease cleavage assay using polymerase protein and capsid protein as substrate. (A) A truncated polymerase protein is observed in a protease cleavage assay with polymerase protein as substrate (lane 2). Arrows represent the location of polymerase protein and cleavage product, respectively. Lane 1 is the buffer-only negative control. (B) A truncated capsid protein is evident in a cleavage assay with capsid protein as substrate (lane 2). Arrows represent the location of polymerase protein and cleavage product, respectively. The buffer-only control is in lane 1.

FIG. 8.

(A) Cleavage of single-amino-acid mutants and deletion mutants. Cleavage assays were performed with del 15 (726 to 741) without (lane 1) or with (lane 2) CysP, del 30 (726 to 756) without (lane 3) or with (lane 4) CysP, K731 mutant without or with CysP (lanes 5 and 6, respectively), F766 mutant without or with CysP (lanes 7 and 8), and E7 (746) mutant without or with CysP (lanes 9 and 10). The arrows represent the 95-kDa polymerase cleavage product and the 66-kDa species of the capsid cleavage product. (B) Cleavage is abolished by a deletion spanning both the capsid and polymerase domains. Wild-type Gag-Pol and Gag-Pol del (644 to 797) were expressed in an in vitro transcription and translation assay, and the products were incubated in cleavage assay. Lanes 1 and 4 are wild-type and deletion Gag-Pol buffer-only control. Lanes 2 and 3 are wild-type Gag-Pol with CysP added at (40 and 120 ng, correspondingly), while lanes 5 and 6 are Gag-Pol del (644 to 797) with CysP added (at the concentrations indicated above).