The absence of ‘shovel-ready’ anti-coronavirus drugs during vaccine development has exceedingly worsened the SARS-CoV-2 pandemic. Furthermore, new vaccine-resistant variants and coronavirus outbreaks may occur in the near future, and we must be ready to face this possibility. However, efficient antiviral drugs are still lacking to this day, due to our poor understanding of the mode of incorporation and mechanism of action of nucleotides analogs that target the coronavirus polymerase to impair its essential activity. Here, we characterize the impact of remdesivir (RDV, the only FDA-approved anti-coronavirus drug) and other nucleotide analogs (NAs) on RNA synthesis by the coronavirus polymerase using a high-throughput, single-molecule, magnetic-tweezers platform. We reveal that the location of the modification in the ribose or in the base dictates the catalytic pathway(s) used for its incorporation. We show that RDV incorporation does not terminate viral RNA synthesis, but leads the polymerase into backtrack as far as 30 nt, which may appear as termination in traditional ensemble assays. SARS-CoV-2 is able to evade the endogenously synthesized product of the viperin antiviral protein, ddhCTP, though the polymerase incorporates this NA well. This experimental paradigm is essential to the discovery and development of therapeutics targeting viral polymerases.
|Number of pages||26|
|Early online date||7 Oct 2021|
|Publication status||Published - Oct 2021|
Bibliographical noteFunding Information:
National Institutes of HealthAI123498.
The authors thank Joy Feng from Gilead Sciences for providing RDV-TP, and Veronique Fattorini and Barbara Selisko for excellent technical assistance and help in SARS-CoV-1 proteins purification. DD was supported by the Interdisciplinary Center for Clinical Research (IZKF) at the University Hospital of the University of Erlangen-Nuremberg, the German Research Foundation grant DFG-DU-1872/ 3-1 and BaSyC – Building a Synthetic Cell’ Gravitation grant (024.003.019) of the Netherlands Ministry of Education, Culture and Science (OCW) and the Netherlands Organisation for Scientific Research (NWO). DD thanks OICE for providing office and lab space, and access to their molecular biology lab. RNK was supported by grant AI123498 from NIAID, NIH. JMW and LDH thank the Ministry of Business Innovation and Employment Contract UOOX1904 (NZ). YX was supported by the National Institutes of Health (NIH) grant AI151638. SARS-Related Coronavirus 2, Isolate USA-WA1/ 2020 (NR-52281) was deposited by the Centers for Disease Control and Prevention and obtained through BEI Resources, NIAID, NIH. PYS was supported by NIH grants AI134907 and UL1TR001439, and awards from the Sealy and Smith Foundation, Kleberg Foundation, the John S Dunn Foundation, the Amon G. Carter Foundation, the Gilson Longenbaugh Foundation, and the Summerfield Robert Foundation. JJA and CEC were supported by grant AI045818 from NIAID, NIH. AS, TTNL, and BC acknowledge grants by the Fondation pour la Recherche Médicale (Aide aux équipes), the SCORE project H2020 SC1-PHE-Coronavirus-2020 (grant#101003627), and the REACTing initiative (REsearch and ACTion targeting emerging infectious diseases).
© Seifert et al.