Specific in vitro cleavage of a Leishmania virus capsid-RNA-dependent RNA polymerase polyprotein by a host cysteine-like protease

Abstract

Antibodies raised against baculovirus-expressed RNA-dependent RNA polymerase (RDRP) recognized a 95-kDa antigen and two smaller proteins in sucrose-purified Leishmania virus particles isolated from infected parasites. The 95-kDa antigen is similar in size to one predicted by translation of the RDRP open reading frame (ORF) alone. In an effort to reconcile in vitro observations of translational frameshifting on Leishmania RNA virus 1-4 transcripts, we have developed an in vitro cleavage assay system to explore the possibility that the fusion polyprotein is proteolytically processed. We show that coincubation a synthetic Cap-Pol fusion protein with lysates from Leishmania parasites yields major cleavage products similar in size to those encoded by the individual capsid and RDRP genes as well as the antigens detected in vivo. The major 82-and 95-kDa major cleavage products are specifically immunoprecipitated by capsid-or polymerase-specific antibodies, respectively, showing that cleavage occurs at or near the junction of the two functional domains. Protease inhibitor studies suggest that a cysteine-like protease is responsible for cleavage in the in vitro assay system developed here. From these results, we suggest that failure to detect a capsid-polymerase fusion protein produced by translational frameshifting in vivo may be due to specific proteolytic processing. MATERIALS AND METHODSParasite strain and cell culture. L. guyanensis M4147 (MHOM/BR/75/M4147) and L. braziliensis M6244 (MTAM/BR/80/M6244) served as virus (LRV1-4)infected and uninfected strains, respectively. Cells were grown at room temperature in M199 semidefined medium (GIBCO-BRL) supplemented with 5% fetal bovine serum (HyClone, Inc.) and 1% fresh, filter-sterilized human urine (12).Construction of a recombinant RDRP baculovirus expression vector. First, two overlapping subclones, pCRE1-T12 and pCRE3-IIIЈ, representing a com-

Hygromycin B resistance mediates elimination of Leishmania virus from persistently infected parasites

“…Plasmid pX63-HYG encodes a selectable hph gene, conferring resistance to hygromycin B (20). A 5.3-kb fragment from XbaI/XhoI-cut pBSK-FULL14 or pBSK-INFRAME (35) was end filled with Klenow fragment (Promega) and subsequently purified from a lowmelting-point agarose gel (NuSieve GTG agarose; FMC Bioproducts). The purified fragment was ligated into pX63-HYG previously cut with SmaI and dephosphorylated by alkaline phosphatase (calf intestine; New England Biolabs).…”

“…in 1988 (40), similar viruses have been found in at least 13 strains of Leishmania braziliensis, L. guyanensis, or L. major (6,33,37,39). Much of the initial work on Leishmania RNA virus (LRV) focused on characterization of the viral RNA genome (36,37,39) and identification of viral genes and proteins, specifically the capsid and the RNA-dependent RNA polymerase (RDRP) (5,6,35).…”

“…However, definitive evidence of an LRV fusion product in virus-infected parasites is lacking. Recently, we used an in vitro cleavage assay to suggest that a cellular cysteine-like protease may cleave the predicted capsid-polymerase fusion protein in vivo (35).…”

“…To investigate the possibility of in vivo proteolytic cleavage of an LRV polyprotein by a cellular protease (35), we transfected cultured parasites with derivatives of a recombinant expression vector, pX63-HYG (9), encoding the relevant LRV1-4 gene sequences. These constructs, which also encode a hygromycin phosphotransferase (hph) gene (20), were electroporated into the LRV1-4-infected Leishmania strain designated M4147.…”

Purification, characterization, and physiological response of a catalase-peroxidase inMycobacteriumsp. strain JC1 DSM 3803 grown on methanol

“…Double immunodi¡usion analysis was performed in 1.2% agarose gel by the method of Ouchterlony and Nilson [20]. Western blot analysis was performed as described previously [21], using alkaline phosphatase color development reaction mixtures (Gibco BRL) after transfer of the proteins in the denaturing gel to a polyvinylidene di£uoride membrane.…”