Detection of Leishmania RNA Virus in Leishmania Parasites

Self Cite

The endogenous double-stranded RNA (dsRNA) virus Leishmaniavirus (LRV1) has been implicated as a pathogenicity factor for leishmaniasis in rodent models and human disease, and associated with drug-treatment failures in Leishmania braziliensis and Leishmania guyanensis infections. Thus, methods targeting LRV1 could have therapeutic benefit. Here we screened a panel of antivirals for parasite and LRV1 inhibition, focusing on nucleoside analogs to capitalize on the highly active salvage pathways of Leishmania, which are purine auxotrophs. Applying a capsid flow cytometry assay, we identified two 2′-C-methyladenosine analogs showing selective inhibition of LRV1. Treatment resulted in loss of LRV1 with first-order kinetics, as expected for random virus segregation, and elimination within six cell doublings, consistent with a measured LRV1 copy number of about 15. Viral loss was specific to antiviral nucleoside treatment and not induced by growth inhibitors, in contrast to fungal dsRNA viruses. Comparisons of drug-treated LRV1 + and LRV1 − lines recapitulated LRV1-dependent pathology and parasite replication in mouse infections, and cytokine secretion in macrophage infections. Agents targeting Totiviridae have not been described previously, nor are there many examples of inhibitors acting against dsRNA viruses more generally. The compounds identified here provide a key proof-ofprinciple in support of further studies identifying efficacious antivirals for use in in vivo studies of LRV1-mediated virulence.trypanosomatid protozoan parasite | endobiont virus | viral segregation | chemotherapy | virulence

Section: Resultssupporting

confidence: 86%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Antiviral screening identifies adenosine analogs targeting the endogenous dsRNALeishmaniaRNA virus 1 (LRV1) pathogenicity factor

Kuhlmann

Robinson

Bluemling

et al. 2017

Self Cite

“…The monoclonal anti-dsRNA J2 antibody binds dsRNAs at least 40 bp long in a sequence-independent fashion (12)(13)(14). The J2 dsRNA monoclonal antibody was previously used to detect viral dsRNA by dsRNA immunoblotting, immunohistochemistry, ELISA, and immunoprecipitation in eukaryotic cells (13)(14)(15)(16)(17)(18)(19)(20). In this study, we used the J2 dsRNA antibody in an immunoprecipitation assay to enrich dsRNA from total RNA extracted from E. coli.…”

Section: Resultsmentioning

confidence: 99%

The double-stranded transcriptome of Escherichia coli

Lybecker

Zimmermann

Bilusic

et al. 2014

104

146

Advances in high-throughput transcriptome analyses have revealed hundreds of antisense RNAs (asRNAs) for many bacteria, although few have been characterized, and the number of functional asRNAs remains unknown. We have developed a genomewide high-throughput method to identify functional asRNAs in vivo. Most mechanisms of gene regulation via asRNAs require an RNA-RNA interaction with its target RNA, and we hypothesized that a functional asRNA would be found in a double strand (dsRNA), duplexed with its cognate RNA in a single cell. We developed a method of isolating dsRNAs from total RNA by immunoprecipitation with a ds-RNA specific antibody. Total RNA and immunoprecipitated dsRNA from Escherichia coli RNase III WT and mutant strains were deep-sequenced. A statistical model was applied to filter for biologically relevant dsRNA regions, which were subsequently categorized by location relative to annotated genes. A total of 316 potentially functional asRNAs were identified in the RNase III mutant strain and are encoded primarily opposite to the 5′ ends of transcripts, but are also found opposite ncRNAs, gene junctions, and the 3′ ends. A total of 21 sense/antisense RNA pairs identified in dsRNAs were confirmed by Northern blot analyses. Most of the RNA steady-state levels were higher or detectable only in the RNase III mutant strain. Taken together, our data indicate that a significant amount of dsRNA is formed in the cell, that RNase III degrades or processes these dsRNAs, and that dsRNA plays a major role in gene regulation in E. coli.T he advent and development of high-throughput sequencing technologies has uncovered the presence of widespread antisense transcription in many bacteria, with the number of annotated genes associated with antisense RNA (asRNA) differing greatly among bacterial species (1, 2). asRNAs are encoded on the DNA strand opposite an annotated gene and overlap a portion of a gene or the entire gene, or span multiple genes with perfect complementarity. asRNAs range in size from tens to thousands of nucleotides. Although numerous chromosomally encoded asRNAs have been identified, few have been confirmed by traditional methods or functionally characterized. Raghavan et al. (3) reported that few asRNAs are conserved between Escherichia coli and Salmonella enterica, and that the predicted promoter sequences of the asRNAs are not conserved between these species, suggesting that most asRNA transcripts are products of spurious transcription and are not biologically functional RNAs.The majority of functionally characterized asRNAs are found on plasmids, phages, and transposons (4, 5). The mode of regulation by asRNAs can be classified according to molecular mechanism as transcription interference, transcription attenuation, alteration of transcript stability, and translation inhibition (1, 2, 6). With the exception of transcription interference, a physical RNA-RNA interaction between the sense RNAs and asRNAs is necessary for all of these mechanisms, requiring that both RNAs be expressed in the same...

“…Lbr siRNAs bear a 5′-P and 3′-OH, reflecting their origin through the action of cellular Dicer nucleases (39), and we used these properties to make siRNAfocused sRNA (<42 nt) libraries for next-generation sequencing (Table S1). For Lgy, we chose the established LRV1 + Lgy M4147 strain (7), and three different Lbr shown to bear LRV1 by PCR and/ or anti-dsRNA antibody tests (40).…”

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

Self Cite

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