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.

The ongoing Corona Virus Disease 2019 pandemic, caused by severe 20 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. Here we show that Favipiravir exerts an antiviral effect as a nucleotide analogue through a combination of chain termination, slowed RNA synthesis and lethal mutagenesis. The SARS-CoV RdRp 25 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 30 COVID-19.2 Coronaviruses (CoV) are large genome, positive-strand RNA viruses of the order 35 Nidovirales that have recently attracted global attention due to the ongoing COVID-19 pandemic. Despite significant efforts to control its spread, SARS-CoV-2 has caused substantial health and economic burden, emphasizing the immediate need for antiviral treatments. As with all positive strand RNA viruses, an RdRp lies at the core of the viral replication machinery and for CoVs this is the nsp12 protein. The pivotal role of nsp12 in the viral life-cycle, lack of host 40 homologues and high level of sequence and structural conservation makes it an optimal target for therapeutics. However, there has been remarkably little biochemical characterization of nsp12 and a lack of fundamental data to guide the design of antiviral therapeutics and study their mechanism of action (MoA). A promising class of RdRp inhibitors are nucleoside analogues (NAs), small molecule drugs that are metabolized intracellularly into their active ribonucleoside 45 5'-triphosphate (RTP) forms and incorporated into the nascent viral RNA by error-prone viral RdRps. This can disrupt RNA synthesis directly via chain termination, or can lead to the accumulation of deleterious mutations in the viral genome. For CoVs, the situation is complicated by the post-replicative repair capacity provided by the nsp14 exonuclease (ExoN) that is essential for maintaining the integrity of their large ~30 kb genomes 1-3 . Nsp14 has been 50 shown to remove certain NAs after insertion by the RdRp into the nascent RNA, thus reducing their antiviral effects 4-6 . Despite this, several NAs currently being used for the treatment of other viral infections have been identified as potential anti-CoV candidates 7-9 . Among these is the purine base analogue T-705 (Favipiravir, Avigan, Extended data Fig. 1a,b) that has broadspectrum activity against a number of RNA viruses and is currently licensed in Japan for use in 55

We here disclose chemical synthesis of ribonucleoside 5'-monophosphate (RMP), -diphosphate (RDP), and -triphosphate (RTP) and cycloSal-, Di PPro-, and Tri PPPro nucleotide prodrugs of the antiviral pseudobase T-1105. Moreover, we include one nucleoside diphosphate prodrug of the chemically less stable T-705. We demonstrate efficient T-1105-RDP and -RTP release from the Di PPro and Tri PPPro compounds by esterase activation. Using crude enzyme extracts, we saw rapid phosphorylation of T-1105-RDP into T-1105-RTP. In sharp contrast, phosphorylation of T-1105-RMP was not seen, indicating a yet unrecognized bottleneck in T-1105's metabolic activation. Accordingly, Di PPro and Tri PPPro compounds displayed improved cell culture activity against influenza A and B virus, which they retained in a mutant cell line incapable of activating the nucleobase parent. T-1105-RTP had a strong inhibitory effect against isolated influenza polymerase, and Di PPro-T-1105-RDP showed 4-fold higher potency in suppressing one-cycle viral RNA synthesis versus T-1105. Hence, our T-1105-RDP and -RTP prodrugs improve antiviral potency and achieve efficient metabolic bypass.