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Research Article

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

F. Matthew Kuhlmann, John I. Robinson, Gregory R. Bluemling, Catherine Ronet, Nicolas Fasel, and View ORCID ProfileStephen M. Beverley
  1. aDepartment of Molecular Microbiology, Washington University School of Medicine in St. Louis, St. Louis, MO 63110;
  2. bDepartment of Medicine, Division of Infectious Diseases, Washington University School of Medicine in St. Louis, St. Louis, MO 63110;
  3. cEmory Institute for Drug Development, Emory University, Atlanta, GA 30329;
  4. dDepartment of Biochemistry, University of Lausanne, 1066 Lausanne, Switzerland

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PNAS January 31, 2017 114 (5) E811-E819; first published January 17, 2017; https://doi.org/10.1073/pnas.1619114114
F. Matthew Kuhlmann
aDepartment of Molecular Microbiology, Washington University School of Medicine in St. Louis, St. Louis, MO 63110;
bDepartment of Medicine, Division of Infectious Diseases, Washington University School of Medicine in St. Louis, St. Louis, MO 63110;
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John I. Robinson
aDepartment of Molecular Microbiology, Washington University School of Medicine in St. Louis, St. Louis, MO 63110;
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Gregory R. Bluemling
cEmory Institute for Drug Development, Emory University, Atlanta, GA 30329;
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Catherine Ronet
dDepartment of Biochemistry, University of Lausanne, 1066 Lausanne, Switzerland
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Nicolas Fasel
dDepartment of Biochemistry, University of Lausanne, 1066 Lausanne, Switzerland
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Stephen M. Beverley
aDepartment of Molecular Microbiology, Washington University School of Medicine in St. Louis, St. Louis, MO 63110;
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  • ORCID record for Stephen M. Beverley
  • For correspondence: stephen.beverley@wustl.edu
  1. Contributed by Stephen M. Beverley, December 19, 2016 (sent for review November 21, 2016; reviewed by Buddy Ullman and C. C. Wang)

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Significance

The endogenous double-stranded RNA virus Leishmaniavirus (LRV1) has been implicated as a pathogenicity factor for leishmaniasis in rodent models and human disease, and associated with drug-treatment failures. As a first step toward the identification of therapeutic LRV1 inhibitors, we identified two adenosine analogs that selectively inhibited LRV1 replication. These analogs were used as tools to confirm that viral inheritance is by random segregation, as well as to generate LRV1-cured lines of Leishmania guyanensis, which correspondingly lost the increased pathogenicity conferred by LRV1. These compounds hold promise as leads to ameliorate the severity of LRV1-bearing Leishmania infections, and raise the possibility of targeting other protozoal infections whose pathogenicity may be exacerbated by similar endogenous viruses.

Abstract

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.

  • trypanosomatid protozoan parasite
  • endobiont virus
  • viral segregation
  • chemotherapy
  • virulence

Footnotes

  • ↵1To whom correspondence should be addressed. Email: stephen.beverley{at}wustl.edu.
  • Author contributions: F.M.K., N.F., and S.M.B. designed research; F.M.K., J.I.R., and C.R. performed research; G.R.B. contributed new reagents/analytic tools; F.M.K., J.I.R., C.R., and S.M.B. analyzed data; and F.M.K. and S.M.B. wrote the paper.

  • Reviewers: B.U., Oregon Health Sciences University; and C.C.W., University of California, San Francisco.

  • The authors declare no conflict of interest.

  • This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1619114114/-/DCSupplemental.

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Discovery of Leishmaniavirus LRV1 inhibitors
F. Matthew Kuhlmann, John I. Robinson, Gregory R. Bluemling, Catherine Ronet, Nicolas Fasel, Stephen M. Beverley
Proceedings of the National Academy of Sciences Jan 2017, 114 (5) E811-E819; DOI: 10.1073/pnas.1619114114

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Discovery of Leishmaniavirus LRV1 inhibitors
F. Matthew Kuhlmann, John I. Robinson, Gregory R. Bluemling, Catherine Ronet, Nicolas Fasel, Stephen M. Beverley
Proceedings of the National Academy of Sciences Jan 2017, 114 (5) E811-E819; DOI: 10.1073/pnas.1619114114
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