Concentration of 2'C-methyladenosine triphosphate by Leishmania guyanensis enables specific inhibition of Leishmania RNA virus 1 via its RNA polymerase.

Leishmania is a widespread trypanosomatid protozoan parasite causing significant morbidity and mortality in humans. The endobiont dsRNA virus Leishmania RNA virus 1 (LRV1) chronically infects some strains, where it increases parasite numbers and virulence in murine leishmaniasis models, and correlates with increased treatment failure in human disease. Previously, we reported that 2'-C-methyladenosine (2CMA) potently inhibited LRV1 in Leishmania guyanensis (Lgy) and Leishmania braziliensis, leading to viral eradication at concentrations above 10 µm Here we probed the cellular mechanisms of 2CMA inhibition, involving metabolism, accumulation, and inhibition of the viral RNA-dependent RNA polymerase (RDRP). Activation to 2CMA triphosphate (2CMA-TP) was required, as 2CMA showed no inhibition of RDRP activity from virions purified on cesium chloride gradients. In contrast, 2CMA-TP showed IC50 values ranging from 150 to 910 µm, depending on the CsCl density of the virion (empty, ssRNA-, and dsRNA-containing). Lgy parasites incubated in vitro with 10 µm 2CMA accumulated 2CMA-TP to 410 µm, greater than the most sensitive RDRP IC50 measured. Quantitative modeling showed good agreement between the degree of LRV1 RDRP inhibition and LRV1 levels. These results establish that 2CMA activity is due to its conversion to 2CMA-TP, which accumulates to levels that inhibit RDRP and cause LRV1 loss. This attests to the impact of the Leishmania purine uptake and metabolism pathways, which allow even a weak RDRP inhibitor to effectively eradicate LRV1 at micromolar concentrations. Future RDRP inhibitors with increased potency may have potential therapeutic applications for ameliorating the increased Leishmania pathogenicity conferred by LRV1.

Generation of the short RNA transcript in Leishmaniavirus correlates with the growth of its parasite host, Leishmania.

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).