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

Abstract

SummaryRNA 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.

The antiviral compound remdesivir potently inhibits RNA-dependent RNA polymerase from Middle East respiratory syndrome coronavirus

“…Delayed chain termination could be based on inhibitor-induced structural changes of the newly synthesized dsRNA that at some point prevent a productive alignment of primer and incoming nucleotide, e.g. through primer/template repositioning (23) or backtracking mechanisms (24). Enzyme-specific interactions with the extended primer may likewise affect the continued extension of the primer, which helps to explain variations in the site of RNA synthesis arrest.…”

Complexities of Viral Mutation Rates

“…Here, the nucleotide misincorporation (or discrimination) rate of purified polymerases can be directly quantified in a biochemical reaction. A variety of techniques have been developed and used to investigate RdRp fidelity for many RNA viruses (11,23). Because these assays define misincorporation dynamics independent of the mutation's effect on the virus, they are less biased against lethal and deleterious mutations.…”

Temperature controlled high-throughput magnetic tweezers show striking difference in activation energies of replicating viral RNA-dependent RNA polymerases

“…The micrometric change in tether extension upon RdRp activity ( Figure 3A) is converted into numbers of transcribed nucleotides using the forceextension relationships for dsRNA and ssRNA constructs (15). RdRp activity traces are low-pass filtered at 0.5 Hz using a Kaiser-Bessel window, from which the dwell time were extracted as previously described (14,15,24). Stochastic-pausing model.…”

“…kilobases (kb), and cannot interrogate rare asynchronous events, such as nucleotide misincorporation or antiviral nucleotide analogue incorporation in the presence of cognate canonical NTPs. Our recent single molecule studies on Φ6 and PV RdRps elongation kinetics have partly filled this gap, shedding light on the kinetic of elongation pauses of various biochemical origins (14)(15)(16). This work relied on the concomitant development of high throughput magnetic tweezers, a powerful single molecule force and torque spectroscopy technique enabling the characterization of protein-nucleic acids interactions at both high throughput (15,(17)(18)(19)(20) and high-resolution (21)(22)(23), and of a new analysis approach based on First-Passage statistics (24,25).…”