Antiviral screening identifies adenosine analogs targeting the endogenous dsRNA Leishmania RNA virus 1 (LRV1) pathogenicity factor.

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-of-principle in support of further studies identifying efficacious antivirals for use in in vivo studies of LRV1-mediated virulence.

Tilting the balance between RNA interference and replication eradicates Leishmania RNA virus 1 and mitigates the inflammatory response.

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 Toll-like 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.