Discovering novel poxvirus-specific drug targets
Cadet, Valerie Elisabeth
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The goal of this work was to identify viral factors essential to orthopoxvirus (OPV) replication via a genome-wide library screen of individual short-interfering RNAs (siRNAs) targeting conserved OPV genes and to identify indispensable genes for OPV replication in mammalian cells. For the viral siRNA screen, 177 open reading frames (ORFs) associated with vaccinia virus (VACV)-WR strain, variola virus-China 48, and/or one of two strains of monkeypox virus were bioinformatically compared and the most conserved regions were analyzed. Four distinct siRNAs were designed for each of 138 ORFs, while for the remaining 39 ORFs, 1-3 siRNAs were designed with 94% targeted gene coverage. We have identified 26 siRNAs targeting 17 ORFs which, when depleted, inhibit cowpox (CWPV) virus replication in vitro. Upon further analysis, five siRNAs, each targeting a separate viral gene, showed superior inhibiting activity when administered prior to infection and all proved to be essential for both CWPV and VACV replication in vitro. Finally, experiments assessed in vivo efficacy of several targets in a murine model of poxvirus disease. Studies also address the deficiency in FDA-approved poxvirus drugs via utilization of a small molecule array comprised of 486 compounds. Nearly all compounds in the library have completed phases I-III trials and were selected based upon purity, solubility, commercial availability, and safety. Following in vitro screening in Vero E6 cells, we were able to identify four compounds currently in use as chemotherapeutics and/or immunosuppressives which directly protect cells from both VACV and CWPV infections: methotrexate, idarubicin, homoharringtonine and raltitrexed. These four compounds were further examined in a dose-response manner and methotrexate was chosen for in vivo drug efficacy analysis. These results identify a candidate from which derivatives can be studied for potential administration to protect against human OPV infections and highlight several other potential drugs to be further explored. We have also extended the work of previously published studies using methotrexate to protect against VACV by providing means to counter the drug toxicity observed. Taken together, these results show the utility of genome-wide siRNA screening for studying viral replication factors, along with identification of potential therapeutic targets for anti-orthopoxvirus therapies.