SARS-CoV-2 nsp14 Exoribonuclease Removes the Natural Antiviral 3'-Deoxy-3',4'-didehydro-cytidine Nucleotide from RNA.

The on-going global pandemic of COVID-19 is caused by SARS-CoV-2, which features a proofreading mechanism to facilitate the replication of its large RNA genome. The 3'-to-5' exoribonuclease (ExoN) activity of SARS-CoV-2 non-structural protein 14 (nsp14) removes nucleotides misincorporated during RNA synthesis by the low-fidelity viral RNA-dependent RNA polymerase (RdRp) and thereby compromises the efficacy of antiviral nucleoside/nucleotide analogues. Here we show biochemically that SARS-CoV-2 nsp14 can excise the natural antiviral chain-terminating nucleotide, 3'-deoxy-3',4'-didehydro-cytidine 5'-monophosphate (ddhCMP), incorporated by RdRp at the 3' end of an RNA strand. Nsp14 ExoN processes an RNA strand terminated with ddhCMP more efficiently than that with a non-physiological chain terminator 3'-deoxy-cytidine monophosphate (3'-dCMP), whereas RdRp is more susceptible to chain termination by 3'-dCTP than ddhCTP. These results suggest that nsp14 ExoN could play a role in protecting SARS-CoV-2 from ddhCTP, which is produced as part of the innate immune response against viral infections, and that the SARS-CoV-2 enzymes may have adapted to minimize the antiviral effect of ddhCTP.

Remdesivir: Quo vadis?

Remdesivir (GS-5734, Veklury®) has remained the only antiviral drug formally approved by the US FDA for the treatment of Covid-19 (SARS-CoV-2 infection). Its key structural features are the fact that it is a C-nucleoside (adenosine) analogue, contains a 1'-cyano function, and could be considered as a ProTide based on the presence of a phosphoramidate group. Its antiviral spectrum and activity in animal models have been well established and so has been its molecular mode of action as a delayed chain terminator of the viral RdRp (RNA-dependent RNA polymerase). Its clinical efficacy has been evaluated, but needs to be optimized with regard to timing, dosage and duration of treatment, and route of administration. Safety, toxicity and pharmacokinetics need to be further addressed, and so are its potential combinations with other drugs such as corticosteroids (i.e. dexamethasone) and ribavirin.

The mechanism of action of T-705 as a unique delayed chain terminator on influenza viral polymerase transcription.

Favipiravir (T-705) has been developed as a potent anti-influenza drug and exhibited a strong inhibition effect against a broad spectrum of RNA viruses. Its active form, ribofuranosyl-triphosphate (T-705-RTP), functions as a competitive substrate for the RNA-dependent RNA polymerase (RdRp) of the influenza A virus (IAV). However, the exact inhibitory mechanisms of T-705 remain elusive and subject to a long-standing debate. Although T-705 has been proposed to inhibit transcription by acting as a chain terminator, it is also paradoxically suggested to be a mutagen towards IAV RdRp by inducing mutations due to its ambiguous base pairing of C and U. Here, we combined biochemical assay with molecular dynamics (MD) simulations to elucidate the molecular mechanism underlying the inhibitory functions exerted by T-705 in IAV RdRp. Our in vitro transcription assay illustrated that IAV RdRp could recognize T-705 as a purine analogue and incorporate it into the nascent RNA strand. Incorporating a single T-705 is incapable of inhibiting transcription as extra natural nucleotides can be progressively added. However, when two consecutive T-705 are incorporated, viral transcription is completely terminated. MD simulations reveal that the sequential appearance of two T-705 in the nascent strand destabilizes the active site and disrupts the base stacking of the nascent RNA. Altogether, our results provide a plausible explanation for the inhibitory roles of T-705 targeting IAV RdRp by integrating the computational and experimental methods. Our study also offers a comprehensive platform to investigate the inhibition effect of antivirals and a novel explanation for the designing of anti-flu drugs.

Broad-Spectrum Antiviral Strategies and Nucleoside Analogues.

The emergence or re-emergence of viruses with epidemic and/or pandemic potential, such as Ebola, Zika, Middle East Respiratory Syndrome (MERS-CoV), Severe Acute Respiratory Syndrome Coronavirus 1 and 2 (SARS and SARS-CoV-2) viruses, or new strains of influenza represents significant human health threats due to the absence of available treatments. Vaccines represent a key answer to control these viruses. However, in the case of a public health emergency, vaccine development, safety, and partial efficacy concerns may hinder their prompt deployment. Thus, developing broad-spectrum antiviral molecules for a fast response is essential to face an outbreak crisis as well as for bioweapon countermeasures. So far, broad-spectrum antivirals include two main categories: the family of drugs targeting the host-cell machinery essential for virus infection and replication, and the family of drugs directly targeting viruses. Among the molecules directly targeting viruses, nucleoside analogues form an essential class of broad-spectrum antiviral drugs. In this review, we will discuss the interest for broad-spectrum antiviral strategies and their limitations, with an emphasis on virus-targeted, broad-spectrum, antiviral nucleoside analogues and their mechanisms of action.