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Date of Award

Summer 8-15-2017

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Biochemistry)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Some Leishmania parasites in the Viannia sub-genus are persistently infected with Leishmania RNA virus 1 (LRV1), a single-segmented double-stranded RNA virus belonging to the family Totiviridae. Infected parasites cause greater pathology and reach higher populations in mouse models of Leishmania infection. In human disease, LRV1+ parasites are correlated with increased frequency of treatment failure and relapse. Efficient methods to detect LRV1 and eliminate it from parasites are required to better understand the role of LRV1 in Leishmania infection. We optimized multiple techniques to measure LRV1 levels in parasites, most notably using flow cytometry to measure the amount of viral capsid protein in individual parasites stained with an anti-capsid antibody. A slot-blot based assay allowed us to rapidly quantify the levels of viral capsid protein or viral dsRNA. Armed with these tools, we screened various known anti-viral compounds and nucleoside analogs for compounds that could inhibit LRV1. The two compounds we identified, 2’-C-methyl-adenosine (2CMA) and 7-deaza-2CMA (7d2CMA), can be utilized to efficiently cure parasites of LRV1 and generate matched LRV1+/LRV1- strains for further study. These pairs allowed us to confirm the role of LRV1 in exacerbating disease.

We then set out to determine the mechanism of LRV1 inhibition by 2CMA. I showed that the active form of 2CMA is its triphosphate (2CMA-TP), and that it directly inhibits the viral RNA polymerase activity with IC50 values ranging from 130 to >600 µM depending on the type of virus particle (empty, ssRNA, or dsRNA-containing) and the RNA product formed. However, this inhibition required much greater 2CMA-TP concentrations than expected based on the EC50 of 2CMA against LRV1 replication in parasites (3 µM). Leishmania are known to accumulate significant amounts of other purine analogs, so I measured the concentration of 2CMA-TP in parasites treated with 10 µM 2CMA and found that they accumulated 410 µM 2CMA-TP. I then created a simulation of LRV1 inhibition and used it to show that this level of 2CMA-TP accumulation was sufficient to replicate the LRV1 inhibition we previously observed experimentally. Monitoring 2CMA-TP levels in parasites after 2CMA was removed from their medium showed that they retained significant amounts of 2CMA-TP even after 8 hours. Furthermore, culturing LRV1+ parasites in a low concentration of 2CMA slowly generated a significant population of LRV1-low parasites. These results suggest that our existing LRV1 inhibitors may be sufficiently potent to inhibit LRV1 in mice infected with LRV1+ parasites and determine if this reduces or eliminates the increased pathogenicity associated with LRV1. The data summarized herein enables quantification of LRV1 and controlled study of its effects on both parasites themselves and the host immune system. The essential role of purine salvage for the potency of 2CMA highlights the importance of that pathway in drug design for Leishmania and suggests further methods for identifying LRV1 or Leishmania inhibitors.

Language

English (en)

Chair and Committee

Stephen M. Beverley

Committee Members

Adrianus Boon, Eric Galburt, Daniel Goldberg, Niraj Tolia,

Comments

Permanent URL: https://doi.org/10.7936/K7930SK6

Available for download on Thursday, September 05, 2019

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