Abstract
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.
Highlights
Leishmania is a widespread trypanosomatid protozoan parasite causing significant morbidity and mortality in humans
Many isolates of Leishmania within the subgenus Viannia, including Leishmania braziliensis (Lbr) and Leishmania guyanensis (Lgy), bear Leishmaniavirus, a single-segmented dsRNA Totivirus known as Leishmania RNA virus 1 (LRV1) (5, 7–9)
RNA-dependent RNA polymerase (RDRP) assays were carried out with Lgy strain M4147 LRV1 virions purified by CsCl equilibrium gradient centrifugation (7, 33)
Summary
RDRP assays were carried out with Lgy strain M4147 LRV1 virions purified by CsCl equilibrium gradient centrifugation (7, 33). IC50 values were calculated by fitting the inhibition data with a logistic dose-response curve (Table 1) These ranged from 150 M for full-length product synthesis by LD virions to 910 M for Figure 5. We compared the nucleotide profiles of LRV1ϩ Lgy grown in the presence or absence of 10 M extracellular 2CMA for 20 h, a time corresponding to more than two rounds of parasite replication Under these conditions, we observed a peak co-eluting with synthetic 2CMA-TP that was absent from untreated parasites (Fig. 6B). When propagated in the subinhibitory dose of 2CMA (1 M), the intracellular 2CMA-TP concentration was 78 Ϯ 9 M (Fig. 7B, n ϭ 4), well below the minimal IC50 for RDRP inhibition (150 M for full-length products from LD virions; Table 1). Cellular 2CMA-TP rose progressively to values exceeding the minimal RDRP IC50, which in turn corresponded reasonably well to the observed effects on LRV1 inhibition
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