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Jean L Patterson - One of the best experts on this subject based on the ideXlab platform.
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purification identification and biochemical characterization of a host encoded cysteine protease that cleaves a Leishmaniavirus gag pol polyprotein
Journal of Virology, 2003Co-Authors: Ricardo Carrion, Jean L PattersonAbstract: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.
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short report quantification of Leishmaniavirus rna in clinical samples and its possible role in pathogenesis
American Journal of Tropical Medicine and Hygiene, 2003Co-Authors: Monica M Ogg, J Ricardo R Carrion, Ana Cristina De Carvalho Botelho, Wilson Mayrink, Rodrigo Correaoliveira, Jean L PattersonAbstract:Leishmaniavirus (LRV) is a double-stranded RNA virus that infects the protozoa Leishmania and has been identified in numerous strains of Leishmania braziliensis and L. braziliensis guyanensis. In general, the species of Leishmania dictates disease manifestation except in the case of L. braziliensis, which is capable of causing either cutaneous or mucocutaneous leishmaniasis. We wanted to determine 1) the quantity of LRV RNA present in a clinical sample and 2) if infection with LRV was associated with a specific disease manifestation. A real-time reverse transcriptase-polymerase chain reaction assay was used to assay clinical samples for the presence of LRV. Of 47 samples tested, 12 positive samples were obtained from patients with cutaneous lesions, lesions in the process of scarring, and cutaneous scars. This is the first study to examine the prevalence of LRV RNA within a small cohort from Brazil.
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host cell proteins bind specifically to the capsid cleaved 5 end of Leishmaniavirus rna
Journal of Biochemistry, 1999Co-Authors: Margarita Saiz, Dyann F Wirth, Jean L PattersonAbstract:Leishmaniavirus (LRV) is a double-stranded RNA (dsRNA) virus that persistently infects some strains of the protozoan parasite, Leishmania. LRV generates a short transcript, corresponding to the 5' end of the positive-sense RNA (320 nt), via a cleavage event mediated by the viral capsid protein on the full-length positive sense RNA transcript. To address the possibility that the RNA cleavage represents a regulatory mechanism for maintaining persistent infection, the interactions between Leishmania cytoplasmic proteins and in vitro synthesized viral transcripts were studied. In gel mobility shift experiments, three specific RNA/protein complexes were formed between cellular proteins and the cleaved viral transcript, and three major proteins were labeled by UV cross-linking. No protein binding activity was observed for either the short (320 nt) or full-length RNA transcripts. However, the two cleavage reaction products were able to form stable RNA/RNA complexes. We present a model in which the virus is targeting its own transcript for cleavage to promote binding of host factors to cryptic domains inaccessible in the full-length transcript.
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generation of the short rna transcript in Leishmaniavirus correlates with the growth of its parasite host leishmania
Molecules and Cells, 1998Co-Authors: In Kwon Chung, Timothy Currie Armstrong, Scott M Scheffter, Joo Hun Lee, Young Min Kim, Jean L PattersonAbstract:Leishmaniavirus 1 is a double-stranded RNA virus that infects the New World kinetoplastid parasites, Leishmania braziliensis, and Leishmania guyanensis. The isolated virus particles contain an RNA-dependent RNA polymerase which exhibits both transcriptase activity for genome-length plus-strand synthesis and replicase activity for genome-length minus-strand synthesis. Recently, we identified a 320 nucleotide short RNA transcript of Leishmaniavirus 1-4, derived from the 5' end of the viral plus-strand, which is generated by the virus capsid via site-specific cleavage of the full-length positive single-stranded RNA. We have hypothesized that this short RNA transcript functions to regulate the virus life cycle during the growth of its parasite host, Leishmania guyanensis. To address this hypothesis, we measured the relative amount of short RNA transcripts and the absolute number of viral genomes per infected cell from log through stationary phase of the parasite growth cycle. In vitro assays of the viral polymerase showed an overall increase in viral polymerase activity from log growth into stationary phase which mirrored an in vivo increase in the quantity of double-stranded genome as measured by agarose gel electrophoresis. We have developed competitive reverse transcription-polymerase chain reaction (RT-PCR) assays to measure the relative amounts of viral transcripts in infected cells as well as the number of viral genomes per infected cell. The results of these assays show that the amount of full-length virus transcripts peaks in the parasite stationary phase (132 transcripts per cell), and that the short transcript is most abundant in the early stationary phase cells (24 transcripts per cell).
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single site cleavage in the 5 untranslated region of Leishmaniavirus rna is mediated by the viral capsid protein
Proceedings of the National Academy of Sciences of the United States of America, 1995Co-Authors: Kyle J Macbeth, Jean L PattersonAbstract:Abstract Leishmaniavirus (LRV) is a double-stranded RNA virus that persistently infects the protozoan parasite Leishmania. LRV produces a short RNA transcript, corresponding to the 5' end of positive-sense viral RNA, both in vivo and in in vitro polymerase assays. The short transcript is generated by a single site-specific cleavage event in the 5' untranslated region of the 5.3-kb genome. This cleavage event can be reproduced in vitro with purified viral particles and a substrate RNA transcript possessing the viral cleavage site. A region of nucleotides required for cleavage was identified by analyzing the cleavage sites yielding the short transcripts of various LRV isolates. A 6-nt deletion at this cleavage site completely abolished RNA processing. In an in vitro cleavage assay, baculovirus-expressed capsid protein possessed an endonuclease activity identical to that of native virions, showing that the viral capsid protein is the RNA endonuclease. Identification of the LRV capsid protein as an RNA endonuclease is unprecedented among known viral capsid proteins.
Sebastien Briolant - One of the best experts on this subject based on the ideXlab platform.
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Leishmaniavirus genetic diversity is not related to leishmaniasis treatment failure
Clinical Microbiology and Infection, 2021Co-Authors: Marine Ginouves, Eliane Bourreau, Pierre Couppie, S Simon, S Rogier, Paul Brousse, P Travers, Pommier V De Santi, Magalie Demar, Sebastien BriolantAbstract:Abstract Objectives The outcome of American tegumentary leishmaniasis (ATL) may depend on the presence of the Leishmania RNA virus (LRV). This virus may be involved in treatment failure. We aimed to determine whether genetic clusters of LRV1 are involved in this therapeutic outcome. Methods The presence of LRV1 was assessed in 129 Leishmania guyanensis isolates from patients treated with pentamidine in French Guiana. Among the 115 (89%) isolates found to carry LRV1, 96 were successfully genotyped. Patient clinical data were linked to the LRV data. Results The rate of treatment failure for LRV1-positive isolates was 37% (15/41) versus 40% (2/5) among LRV1-negative isolates (p 0.88). Concerning LRV1 genotypes, two predominant LRV1 groups emerged, groups A (23% (22/96)) and B (70% (67/96)). The treatment failure rate was 37% (3/8) for group A and 45% (9/20) for group B (p 0.31). Discussion Neither the presence nor genotype of LRV1 in patients with L. guyanensis seemed to correlate with pentamidine treatment failure.
Pierre Couppie - One of the best experts on this subject based on the ideXlab platform.
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Leishmaniavirus genetic diversity is not related to leishmaniasis treatment failure
Clinical Microbiology and Infection, 2021Co-Authors: Marine Ginouves, Eliane Bourreau, Pierre Couppie, S Simon, S Rogier, Paul Brousse, P Travers, Pommier V De Santi, Magalie Demar, Sebastien BriolantAbstract:Abstract Objectives The outcome of American tegumentary leishmaniasis (ATL) may depend on the presence of the Leishmania RNA virus (LRV). This virus may be involved in treatment failure. We aimed to determine whether genetic clusters of LRV1 are involved in this therapeutic outcome. Methods The presence of LRV1 was assessed in 129 Leishmania guyanensis isolates from patients treated with pentamidine in French Guiana. Among the 115 (89%) isolates found to carry LRV1, 96 were successfully genotyped. Patient clinical data were linked to the LRV data. Results The rate of treatment failure for LRV1-positive isolates was 37% (15/41) versus 40% (2/5) among LRV1-negative isolates (p 0.88). Concerning LRV1 genotypes, two predominant LRV1 groups emerged, groups A (23% (22/96)) and B (70% (67/96)). The treatment failure rate was 37% (3/8) for group A and 45% (9/20) for group B (p 0.31). Discussion Neither the presence nor genotype of LRV1 in patients with L. guyanensis seemed to correlate with pentamidine treatment failure.
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Leishmaniavirus dependent metastatic leishmaniasis is prevented by blocking il 17a
PLOS Pathogens, 2016Co-Authors: Maryanne Hartley, P Castiglioni, Eliane Bourreau, Matteo Rossi, Remzi Onur Eren, Florence Prevel, Pierre Couppie, Suzanne M Hickerson, Pascal Launois, Stephen M BeverleyAbstract:Cutaneous leishmaniasis has various outcomes, ranging from self-healing reddened papules to extensive open ulcerations that metastasise to secondary sites and are often resistant to standard therapies. In the case of L. guyanensis (L.g), about 5–10% of all infections result in metastatic complications. We recently showed that a cytoplasmic virus within L.g parasites (LRV1) is able to act as a potent innate immunogen, worsening disease outcome in a murine model. In this study, we investigated the immunophenotype of human patients infected by L.g and found a significant association between the inflammatory cytokine IL-17A, the presence of LRV1 and disease chronicity. Further, IL-17A was inversely correlated to the protective cytokine IFN-γ. These findings were experimentally corroborated in our murine model, where IL-17A produced in LRV1+ L.g infection contributed to parasite virulence and dissemination in the absence of IFN-γ. Additionally, IL-17A inhibition in mice using digoxin or SR1001, showed therapeutic promise in limiting parasite virulence. Thus, this murine model of LRV1-dependent infectious metastasis validated markers of disease chronicity in humans and elucidated the immunologic mechanism for the dissemination of Leishmania parasites to secondary sites. Moreover, it confirms the prognostic value of LRV1 and IL-17A detection to prevent metastatic leishmaniasis in human patients.
Eliane Bourreau - One of the best experts on this subject based on the ideXlab platform.
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Leishmaniavirus genetic diversity is not related to leishmaniasis treatment failure
Clinical Microbiology and Infection, 2021Co-Authors: Marine Ginouves, Eliane Bourreau, Pierre Couppie, S Simon, S Rogier, Paul Brousse, P Travers, Pommier V De Santi, Magalie Demar, Sebastien BriolantAbstract:Abstract Objectives The outcome of American tegumentary leishmaniasis (ATL) may depend on the presence of the Leishmania RNA virus (LRV). This virus may be involved in treatment failure. We aimed to determine whether genetic clusters of LRV1 are involved in this therapeutic outcome. Methods The presence of LRV1 was assessed in 129 Leishmania guyanensis isolates from patients treated with pentamidine in French Guiana. Among the 115 (89%) isolates found to carry LRV1, 96 were successfully genotyped. Patient clinical data were linked to the LRV data. Results The rate of treatment failure for LRV1-positive isolates was 37% (15/41) versus 40% (2/5) among LRV1-negative isolates (p 0.88). Concerning LRV1 genotypes, two predominant LRV1 groups emerged, groups A (23% (22/96)) and B (70% (67/96)). The treatment failure rate was 37% (3/8) for group A and 45% (9/20) for group B (p 0.31). Discussion Neither the presence nor genotype of LRV1 in patients with L. guyanensis seemed to correlate with pentamidine treatment failure.
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Leishmaniavirus dependent metastatic leishmaniasis is prevented by blocking il 17a
PLOS Pathogens, 2016Co-Authors: Maryanne Hartley, P Castiglioni, Eliane Bourreau, Matteo Rossi, Remzi Onur Eren, Florence Prevel, Pierre Couppie, Suzanne M Hickerson, Pascal Launois, Stephen M BeverleyAbstract:Cutaneous leishmaniasis has various outcomes, ranging from self-healing reddened papules to extensive open ulcerations that metastasise to secondary sites and are often resistant to standard therapies. In the case of L. guyanensis (L.g), about 5–10% of all infections result in metastatic complications. We recently showed that a cytoplasmic virus within L.g parasites (LRV1) is able to act as a potent innate immunogen, worsening disease outcome in a murine model. In this study, we investigated the immunophenotype of human patients infected by L.g and found a significant association between the inflammatory cytokine IL-17A, the presence of LRV1 and disease chronicity. Further, IL-17A was inversely correlated to the protective cytokine IFN-γ. These findings were experimentally corroborated in our murine model, where IL-17A produced in LRV1+ L.g infection contributed to parasite virulence and dissemination in the absence of IFN-γ. Additionally, IL-17A inhibition in mice using digoxin or SR1001, showed therapeutic promise in limiting parasite virulence. Thus, this murine model of LRV1-dependent infectious metastasis validated markers of disease chronicity in humans and elucidated the immunologic mechanism for the dissemination of Leishmania parasites to secondary sites. Moreover, it confirms the prognostic value of LRV1 and IL-17A detection to prevent metastatic leishmaniasis in human patients.
Marine Ginouves - One of the best experts on this subject based on the ideXlab platform.
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Leishmaniavirus genetic diversity is not related to leishmaniasis treatment failure
Clinical Microbiology and Infection, 2021Co-Authors: Marine Ginouves, Eliane Bourreau, Pierre Couppie, S Simon, S Rogier, Paul Brousse, P Travers, Pommier V De Santi, Magalie Demar, Sebastien BriolantAbstract:Abstract Objectives The outcome of American tegumentary leishmaniasis (ATL) may depend on the presence of the Leishmania RNA virus (LRV). This virus may be involved in treatment failure. We aimed to determine whether genetic clusters of LRV1 are involved in this therapeutic outcome. Methods The presence of LRV1 was assessed in 129 Leishmania guyanensis isolates from patients treated with pentamidine in French Guiana. Among the 115 (89%) isolates found to carry LRV1, 96 were successfully genotyped. Patient clinical data were linked to the LRV data. Results The rate of treatment failure for LRV1-positive isolates was 37% (15/41) versus 40% (2/5) among LRV1-negative isolates (p 0.88). Concerning LRV1 genotypes, two predominant LRV1 groups emerged, groups A (23% (22/96)) and B (70% (67/96)). The treatment failure rate was 37% (3/8) for group A and 45% (9/20) for group B (p 0.31). Discussion Neither the presence nor genotype of LRV1 in patients with L. guyanensis seemed to correlate with pentamidine treatment failure.