Signatures of Nucleotide Analog Incorporation by an RNA-Dependent RNA Polymerase Revealed Using High-Throughput Magnetic Tweezers

Signatures of Nucleotide Analog Incorporation by an RNA-Dependent RNA Polymerase Revealed Using High-Throughput Magnetic Tweezers

Dulin, D. (TU Delft BN/Nynke Dekker Lab; Friedrich-Alexander-Universität Erlangen-Nürnberg; Kavli institute of nanoscience Delft)
Arnold, Jamie J. (The Pennsylvania State University)
van Laar, T. (TU Delft BN/Nynke Dekker Lab; Kavli institute of nanoscience Delft)
Oh, Hyung Suk (The Pennsylvania State University)
Lee, Cheri (The Pennsylvania State University)
Perkins, Angela L. (University of Minnesota Twin Cities)
Harki, Daniel A. (University of Minnesota Twin Cities)
Depken, S.M. (TU Delft BN/Martin Depken Lab; Kavli institute of nanoscience Delft)
Cameron, Craig E. (The Pennsylvania State University)
Dekker, N.H. (TU Delft BN/Nynke Dekker Lab; Kavli institute of nanoscience Delft)

2017

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.

backtracking
inhibitor
magnetic tweezers
nucleoside analog
pyrazine carboxamide
RNA polymerase
RNA virus
T-1106
T-705

http://resolver.tudelft.nl/uuid:a57098b3-8e44-400f-b53f-172b282f6da3

https://doi.org/10.1016/j.celrep.2017.10.005

2211-1247

Cell Reports, 21 (4), 1063-1076

Institutional Repository

journal article

© 2017 D. Dulin, Jamie J. Arnold, T. van Laar, Hyung Suk Oh, Cheri Lee, Angela L. Perkins, Daniel A. Harki, S.M. Depken, Craig E. Cameron, N.H. Dekker