RNA viruses pose a threat to public health that is exacerbated by the dearth of antiviral therapeutics. The RNA-dependent RNA polymerase (RdRp) holds promise as a broad-spectrum, therapeutic target because of the conserved nature of the nucleotide-substrate-binding and catalytic sites. Conventional, quantitative, kinetic analysis of antiviral ribonucleotides monitors one or a few incorporation events. Here, we use a high-throughput magnetic tweezers platform to monitor the elongation dynamics of a prototypical RdRp over thousands of nucleotide-addition cycles in the absence and presence of a suite of nucleotide analog inhibitors. We observe multiple RdRp-RNA elongation complexes; only a subset of which are competent for analog utilization. Incorporation of a pyrazine-carboxamide nucleotide analog, T-1106, leads to RdRp backtracking. This analysis reveals a mechanism of action for this antiviral ribonucleotide that is corroborated by cellular studies. We propose that induced backtracking represents a distinct mechanistic class of antiviral ribonucleotides. Dulin et al. find that a prototypical RNA-dependent RNA polymerase (RdRp) visits several states during nucleotide synthesis, of which only one incorporates nucleotide analogs with therapeutic potential. Different analogs exhibit distinct kinetic signatures, with an analog thought to induce chain termination actually promoting RdRp backtracking.
Bibliographical noteFunding Information:
We acknowledge Eric Snijder, Clara Posthuma, Bojk Berghuis, and Behrouz Eslami Mossalam for fruitful discussions. We thank Bojk Berghuis for experimental assistance, Richard Janissen for figure revisions, and Leo Beigelman and Jerome Deval from Alios BioPharma for providing T-705-TP. D.D. was supported by the Interdisciplinary Center for Clinical Research (IZKF) at the University Hospital of the University of Erlangen-Nuremberg . J.J.A. and C.E.C. were supported in part by the NIH/NIAID (grant AI045818 ). M.D. acknowledges support from a TU Delft startup grant. C.E.C. was the recipient of a Burroughs Wellcome Fund 2015 Collaborative Research Travel Grant. Funding to N.H.D. was provided by the Netherlands Organisation for Scientific Research (NWO) via its TOP-GO program and by the European Union via an ERC consolidator grant (DynGenome, 312221 ).
- RNA polymerase
- RNA virus
- magnetic tweezers
- nucleoside analog
- pyrazine carboxamide