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Remdesivir (RDV, Veklury®) is the first FDA-approved antiviral treatment for COVID-19. It is a nucleotide analogue (NA) carrying a 1'-cyano (1'-CN) group on the ribose and a pseudo-adenine nucleobase whose contributions to the mode of action (MoA) are not clear. Here, we dissect these independent contributions by employing RDV-TP analogues. We show that while the 1'-CN group is directly responsible for transient stalling of the SARS-CoV-2 replication/transcription complex (RTC), the nucleobase plays a role in the strength of this stalling. Conversely, RNA extension assays show that the 1'-CN group plays a role in fidelity and that RDV-TP can be incorporated as a GTP analogue, albeit with lower efficiency. However, a mutagenic effect by the viral polymerase is not ascertained by deep sequencing of viral RNA from cells treated with RDV. We observe that once added to the 3' end of RNA, RDV-MP is sensitive to excision and its 1'-CN group does not impact its nsp14-mediated removal. A >14-fold RDV-resistant SARS-CoV-2 isolate can be selected carrying two mutations in the nsp12 sequence, S759A and A777S. They confer both RDV-TP discrimination over ATP by nsp12 and stalling during RNA synthesis, leaving more time for excision-repair and potentially dampening RDV efficiency. We conclude that RDV presents a multi-faced MoA. It slows down or stalls overall RNA synthesis but is efficiently repaired from the primer strand, whereas once in the template, read-through inhibition adds to this effect. Its efficient incorporation may corrupt proviral RNA, likely disturbing downstream functions in the virus life cycle., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Nucleotide analogues (NA) are currently employed for treatment of several viral diseases, including COVID-19. NA prodrugs are intracellularly activated to the 5'-triphosphate form. They are incorporated into the viral RNA by the viral polymerase (SARS-CoV-2 nsp12), terminating or corrupting RNA synthesis. For Coronaviruses, natural resistance to NAs is provided by a viral 3'-to-5' exonuclease heterodimer nsp14/nsp10, which can remove terminal analogues. Here, we show that the replacement of the α-phosphate of Bemnifosbuvir 5'-triphosphate form (AT-9010) by an α-thiophosphate renders it resistant to excision. The resulting α-thiotriphosphate, AT-9052, exists as two epimers (RP/SP). Through co-crystallization and activity assays, we show that the Sp isomer is preferentially used as a substrate by nucleotide diphosphate kinase (NDPK), and by SARS-CoV-2 nsp12, where its incorporation causes immediate chain-termination. The same -Sp isomer, once incorporated by nsp12, is also totally resistant to the excision by nsp10/nsp14 complex. However, unlike AT-9010, AT-9052-RP/SP no longer inhibits the N-terminal nucleotidylation domain of nsp12. We conclude that AT-9052-Sp exhibits a unique mechanism of action against SARS-CoV-2. Moreover, the thio modification provides a general approach to rescue existing NAs whose activity is hampered by coronavirus proofreading capacity., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The guanosine analog AT-527 represents a promising candidate against Severe Acute Respiratory Syndrome coronavirus type 2 (SARS-CoV-2). AT-527 recently entered phase III clinical trials for the treatment of COVID-19. Once in cells, AT-527 is converted into its triphosphate form, AT-9010, that presumably targets the viral RNA-dependent RNA polymerase (RdRp, nsp12), for incorporation into viral RNA. Here we report a 2.98 Å cryo-EM structure of the SARS-CoV-2 nsp12-nsp7-nsp8 2 -RNA complex, showing AT-9010 bound at three sites of nsp12. In the RdRp active-site, one AT-9010 is incorporated at the 3' end of the RNA product strand. Its modified ribose group (2'-fluoro, 2'-methyl) prevents correct alignment of the incoming NTP, in this case a second AT-9010, causing immediate termination of RNA synthesis. The third AT-9010 is bound to the N-terminal domain of nsp12 - known as the NiRAN. In contrast to native NTPs, AT-9010 is in a flipped orientation in the active-site, with its guanine base unexpectedly occupying a previously unnoticed cavity. AT-9010 outcompetes all native nucleotides for NiRAN binding, inhibiting its nucleotidyltransferase activity. The dual mechanism of action of AT-527 at both RdRp and NiRAN active sites represents a promising research avenue against COVID-19., (© 2022. The Author(s).)

The ongoing Corona Virus Disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has emphasized the urgent need for antiviral therapeutics. The viral RNA-dependent-RNA-polymerase (RdRp) is a promising target with polymerase inhibitors successfully used for the treatment of several viral diseases. We demonstrate here that Favipiravir predominantly exerts an antiviral effect through lethal mutagenesis. The SARS-CoV RdRp complex is at least 10-fold more active than any other viral RdRp known. It possesses both unusually high nucleotide incorporation rates and high-error rates allowing facile insertion of Favipiravir into viral RNA, provoking C-to-U and G-to-A transitions in the already low cytosine content SARS-CoV-2 genome. The coronavirus RdRp complex represents an Achilles heel for SARS-CoV, supporting nucleoside analogues as promising candidates for the treatment of COVID-19.