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.

Viral RNA-dependent RNA polymerases (RdRps) responsible for the replication of single-strand RNA virus genomes exert their function in the context of complex replication machineries. Within these replication complexes the polymerase activity is often highly regulated by RNA elements, proteins or other domains of multi-domain polymerases. Here, we present data of the influence of the methyltransferase domain (NS5-MTase) of dengue virus (DENV) protein NS5 on the RdRp activity of the polymerase domain (NS5-Pol). The steady-state polymerase activities of DENV-2 recombinant NS5 and NS5-Pol are compared using different biochemical assays allowing the dissection of the de novo initiation, transition and elongation steps of RNA synthesis. We show that NS5-MTase ensures efficient RdRp activity by stimulating the de novo initiation and the elongation phase. This stimulation is related to a higher affinity of NS5 toward the single-strand RNA template indicating NS5-MTase either completes a high-affinity RNA binding site and/or promotes the correct formation of the template tunnel. Furthermore, the NS5-MTase increases the affinity of the priming nucleotide ATP upon de novo initiation and causes a higher catalytic efficiency of the polymerase upon elongation. The complex stimulation pattern is discussed under the perspective that NS5 adopts several conformations during RNA synthesis.

The dengue virus (DV) is an important human pathogen from the Flavivirus genus, whose genome- and antigenome RNAs start with the strictly conserved sequence pppAG. The RNA-dependent RNA polymerase (RdRp), a product of the NS5 gene, initiates RNA synthesis de novo, i.e., without the use of a pre-existing primer. Very little is known about the mechanism of this de novo initiation and how conservation of the starting adenosine is achieved. The polymerase domain NS5Pol_DV of NS5, upon initiation on viral RNA templates, synthesizes mainly dinucleotide primers that are then elongated in a processive manner. We show here that NS5Pol_DV contains a specific priming site for adenosine 5′-triphosphate as the first transcribed nucleotide. Remarkably, in the absence of any RNA template the enzyme is able to selectively synthesize the dinucleotide pppAG when Mn²⁺ is present as catalytic ion. The T794 to A799 priming loop is essential for initiation and provides at least part of the ATP-specific priming site. The H798 loop residue is of central importance for the ATP-specific initiation step. In addition to ATP selection, NS5Pol_DV ensures the conservation of the 5′-adenosine by strongly discriminating against viral templates containing an erroneous 3′-end nucleotide in the presence of Mg²⁺. In the presence of Mn²⁺, NS5Pol_DV is remarkably able to generate and elongate the correct pppAG primer on these erroneous templates. This can be regarded as a genomic/antigenomic RNA end repair mechanism. These conservational mechanisms, mediated by the polymerase alone, may extend to other RNA virus families having RdRps initiating RNA synthesis de novo. The 5′- and 3′-ends of RNA virus genomes have evolved towards efficient replication, translation, and escape from defense mechanisms of the host cell. Little is known about how RNA viruses conserve or restore the correct ends of their genomes. The Flavivirus genus of positive-strand RNA viruses contains important human pathogens such as yellow fever virus, West Nile virus, Japanese encephalitis virus and dengue virus (DV). The Flavivirus genome ends are strictly conserved as 5′-AG…CU-3′. We demonstrate here the primary role of the DV polymerase in the conservation of the first and last genomic residue. We show that DV polymerase contains an ATP-specific priming site, which imposes a strong preference for the de novo synthesis of a dinucleotide primer starting with an ATP. Furthermore, the polymerase is able to indirectly correct erroneous sequences by producing the correct primer in the absence of template and on templates containing incorrect nucleotides at the 3′-end. The correct primer is productively elongated on either correct or incorrect templates. Our findings provide a direct demonstration of the implication of a viral RNA polymerase in the conservation and repair of genome ends. Other polymerases from other RNA virus families are likely to employ similar mechanisms.

The RNA-dependent RNA polymerase (RdRp) is a central piece in the replication machinery of RNA viruses. In picornaviruses this essential RdRp activity also uridylates the VPg peptide, which then serves as a primer for RNA synthesis. Previous genetic, binding, and biochemical data have identified a VPg binding site on poliovirus RdRp and have shown that is was implicated in VPg uridylation. More recent structural studies have identified a topologically distinct site on the closely related foot-and-mouth disease virus RdRp supposed to be the actual VPg-primer-binding site. Here, we report the crystal structure at 2.5-Å resolution of active coxsackievirus B3 RdRp (also named 3D ^pol ) in a complex with VPg and a pyrophosphate. The pyrophosphate is situated in the active-site cavity, occupying a putative binding site either for the coproduct of the reaction or an incoming NTP. VPg is bound at the base of the thumb subdomain, providing first structural evidence for the VPg binding site previously identified by genetic and biochemical methods. The binding mode of VPg to CVB3 3D ^pol at this site excludes its uridylation by the carrier 3D ^pol . We suggest that VPg at this position is either uridylated by another 3D ^pol molecule or that it plays a stabilizing role within the uridylation complex. The CVB3 3D ^pol /VPg complex structure is expected to contribute to the understanding of the multicomponent VPg-uridylation complex essential for the initiation of genome replication of picornaviruses.

Viral RNA-dependent RNA polymerases (RdRps) play a central role not only in viral replication, but also in the genetic evolution of viral RNAs. After binding to an RNA template and selecting 5′-triphosphate ribonucleosides, viral RdRps synthesize an RNA copy according to Watson-Crick base-pairing rules. The copy process sometimes deviates from both the base-pairing rules specified by the template and the natural ribose selectivity and, thus, the process is error-prone due to the intrinsic (in)fidelity of viral RdRps. These enzymes share a number of conserved amino-acid sequence strings, called motifs A–G, which can be defined from a structural and functional point-of-view. A co-relation is gradually emerging between mutations in these motifs and viral genome evolution or observed mutation rates. Here, we review our current knowledge on these motifs and their role on the structural and mechanistic basis of the fidelity of nucleotide selection and RNA synthesis by Flavivirus RdRps.

The flavivirus RNA genome contains a conserved cap-1 structure, 7MeGpppA2′OMeG, at the 5′ end. Two mRNA cap methyltransferase (MTase) activities involved in the formation of the cap, the (guanine-N7)- and the (nucleoside-2′O)-MTases (2′O-MTase), reside in a single domain of non-structural protein NS5 (NS5MTase). This study reports on the biochemical characterization of the 2′O-MTase activity of NS5MTase of dengue virus (NS5MTaseDV) using purified, short, capped RNA substrates (7MeGpppAC n or GpppAC n ). NS5MTaseDV methylated both types of substrate exclusively at the 2′O position. The efficiency of 2′O-methylation did not depend on the methylation of the N7 position. Using 7MeGpppAC n and GpppAC n substrates of increasing chain lengths, it was found that both NS5MTaseDV 2′O activity and substrate binding increased before reaching a plateau at n=5. Thus, the cap and 6 nt might define the interface providing efficient binding of enzyme and substrate. K m values for 7MeGpppAC5 and the co-substrate S-adenosyl-l-methionine (AdoMet) were determined (0.39 and 3.26 μM, respectively). As reported for other AdoMet-dependent RNA and DNA MTases, the 2′O-MTase activity of NS5MTaseDV showed a low turnover of 3.25×10−4 s−1. Finally, an inhibition assay was set up and tested on GTP and AdoMet analogues as putative inhibitors of NS5MTaseDV, which confirmed efficient inhibition by the reaction product S-adenosyl-homocysteine (IC50 0.34 μM) and sinefungin (IC50 0.63 μM), demonstrating that the assay is sufficiently sensitive to conduct inhibitor screening and characterization assays.