Successful transient introduction of Leishmania RNA virus into a virally infected and an uninfected strain of Leishmania.

Armstrong, Timothy Currie; Keenan, Marie C.; Widmer, Giovanni

doi:10.1073/pnas.90.5.1736

Cited by 20 publications

(12 citation statements)

References 18 publications

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“…Previous siRNA studies in Leishmania analyzed RNAs using a tagged Argonaute inserted into an δago1 − knockout of Lbr M2903, which lacks LRV1 (9,29,38,39). Because the lines bearing LRV1 studied here had not been similarly modified, we sequenced total small RNAs (sRNAs) as an alternative.…”

Section: Resultsmentioning

confidence: 99%

Tilting the balance between RNA interference and replication eradicatesLeishmaniaRNA virus 1 and mitigates the inflammatory response

Brettmann

Shaik

Zangger

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Many Leishmania (Viannia) parasites harbor the double-stranded RNA virus Leishmania RNA virus 1 (LRV1), which has been associated with increased disease severity in animal models and humans and with drug treatment failures in humans. Remarkably, LRV1 survives in the presence of an active RNAi pathway, which in many organisms controls RNA viruses. We found significant levels (0.4 to 2.5%) of small RNAs derived from LRV1 in both Leishmania braziliensis and Leishmania guyanensis, mapping across both strands and with properties consistent with Dicer-mediated cleavage of the dsRNA genome. LRV1 lacks cis-or trans-acting RNAi inhibitory activities, suggesting that virus retention must be maintained by a balance between RNAi activity and LRV1 replication. To tilt this balance toward elimination, we targeted LRV1 using long-hairpin/stem-loop constructs similar to those effective against chromosomal genes. LRV1 was completely eliminated, at high efficiency, accompanied by a massive overproduction of LRV1-specific siRNAs, representing as much as 87% of the total. For both L. braziliensis and L. guyanensis, RNAi-derived LRV1-negative lines were no longer able to induce a Tolllike receptor 3-dependent hyperinflammatory cytokine response in infected macrophages. We demonstrate in vitro a role for LRV1 in virulence of L. braziliensis, the Leishmania species responsible for the vast majority of mucocutaneous leishmaniasis cases. These findings establish a targeted method for elimination of LRV1, and potentially of other Leishmania viruses, which will facilitate mechanistic dissection of the role of LRV1-mediated virulence. Moreover, our data establish a third paradigm for RNAi-viral relationships in evolution: one of balance rather than elimination.dsRNA virus | protozoan parasite | endosymbiont | trypanosomatid protozoa | microbial pathogenesis

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Section: Resultsmentioning

confidence: 99%

Tilting the balance between RNA interference and replication eradicatesLeishmaniaRNA virus 1 and mitigates the inflammatory response

Brettmann

Shaik

Zangger

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…11 Leishmania cultures that are free of LRV infection are capable of being transiently infected with RNA from LRV. 12 A reverse transcriptase−polymerase chain reaction (RT-PCR) assay was previously developed to detect LRV RNA in cutaneous leishmaniasis lesions from human biopsy tissues collected in Peru. 13 The fact that viral RNA could be detected in human samples led us to develop a quantitative assay system that could detect the presence of viral RNA in clinical samples.…”

Section: Introductionmentioning

confidence: 99%

Short Report: Quantification of Leishmaniavirus Rna in Clinical Samples and Its Possible Role in Pathogenesis

Ogg

Carrión²,

Botelho

et al. 2003

The American Journal of Tropical Medicine and Hygiene

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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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“…In the absence of infectious virions, direct infection or superinfection of promastigotes by electroporation of purified virions was attempted but was unsuccessful in establishing a persistent infection (1). Therefore, genetic techniques are being explored with the aim of either infecting an LRV Ϫ strain by introducing a viral cDNA copy or excluding an existing LRV infection.…”

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confidence: 99%

Suppression of Leishmania RNA virus replication by capsid protein overexpression

Widmer

1995

J Virol

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Some strains of the protozoan parasite genus Leishmania are persistently infected with single-segmented double-stranded RNA viruses, which are termed LRV. The function of these cytoplasmic viruses is unknown. In order to address the question of whether LRV affects the parasite's phenotype, pairs of isogenic LRV ؉ -LRV ؊ lines are required. Since the persistent nature of these viruses precludes de novo infection of virus-negative strains, LRV ؉ -LRV ؊ strains were transformed with a Leishmania expression vector expressing the LRV capsid protein with the aim of determining if LRV ؊ promastigotes support capsid assembly and if LRV replication is affected by excess capsid protein. I found that in LRV ؊ promastigotes, capsid protein was capable of selfassembly into virus-like capsids and that capsid overexpression in a naturally infected LRV ؉ line resulted in a progressive reduction in LRV copy number. Clonal lines derived from an LRV ؉ capsid overexpressor had no detectable levels of LRV. These results demonstrate that LRV replication can be inhibited and that a significant reduction of viral copy number has no effect on the parasite's viability in liquid medium.Viral infections have been found in several parasitic protozoa including Entamoeba histolytica (6), Eimeria species (7, 13), Babesia bovis (8), Giardia lamblia (19), Trichomonas vaginalis (20), and Leishmania species (16,22). In Leishmania species, double-stranded (ds) RNA viruses were discovered in New World strains originating from the Amazon basin and neighboring regions of South America (termed LRV1-1, LRV1-2, etc.) as well as in one L. major strain (5-ASKH) originating from the former Soviet Union. The virus isolated from this strain was shown to be related to LRV1 and was termed LRV2-1 (23). The LRV genome consists of two open reading frames (ORFs), ORF2 and ORF3. By use of a baculovirus system, ORF2 was shown to encode the capsid protein, which was capable of self-assembly in the cytoplasm of insect cells (5). ORF3, which contains sequence motifs characteristic of RNA-dependent RNA polymerases (RDRP), appears to be expressed as a fusion with the capsid protein by a ϩ1 translational frameshift (14,15). ORF1, which was initially identified in LRV1-1, was not found in a related LRV1 type and appears to be nonfunctional.Persistent fungal and protozoan dsRNA viruses were shown by phylogenetic analysis of the RDRP gene to form a closely related subgroup of dsRNA viruses (4). Two members of this subgroup, L-A/ScV in yeasts and TvV in T. vaginalis, are associated with distinct phenotypic properties of the host: the killer phenotype and the expression of a specific surface antigen, respectively. These observations suggest that LRV as well could be associated with a particular phenotype of Leishmania species. From the genomic organization of LRV, it is not obvious how such an effect could be mediated, since the two LRV1 genomes sequenced to date encode only the overlapping capsid and RDRP gene. However, in L-A/ScV, covalent attachment of viral capsid protein to...

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Successful transient introduction of Leishmania RNA virus into a virally infected and an uninfected strain of Leishmania.

Cited by 20 publications

References 18 publications

Tilting the balance between RNA interference and replication eradicatesLeishmaniaRNA virus 1 and mitigates the inflammatory response

Tilting the balance between RNA interference and replication eradicatesLeishmaniaRNA virus 1 and mitigates the inflammatory response

Short Report: Quantification of Leishmaniavirus Rna in Clinical Samples and Its Possible Role in Pathogenesis

Suppression of Leishmania RNA virus replication by capsid protein overexpression

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