Nucleosides for the treatment of respiratory RNA virus infections

Jordan, Paul C; Stevens, Sarah K; Deval, Jerome

The CoV are part of the larger nidovirus family and have exceptionally large genomes of up to 32 kilobases in length. 98 CoV are positive sense RNA viruses, with a notable example being SARS as one of the most pathogenic member of this viral family. 99 The CoV genome has a 5 0 -cap, is polyadenylated on the 3 0 end, and generates a total of 16 non-structural proteins (nsp1 to nsp16). The 5 0 two-thirds of its genome encodes for non-structural proteins that combine to form a replication and transcription complex that completes viral RNA synthesis. 100 The Nsp12 protein is the RdRp and is typically composed of a N-terminal domain composed of a nidovirusspecific RdRp-associated nucleotidyltransferase (NiRAN) and a C-terminal containing the RdRp domain, which contains a set of six conserved motifs (motifs A-F) responsible for recognizing substrates and template. 98, 101 The NiRAN domain has only been identified in nidoviruses and is approximately 300 residues long in CoV. 101 In SARS-CoV, a reverse genetic system identified this motif as a requirement for replication of its viral genome. While NiRAN activity has not been directly observed outside of a reverse genetic system for CoV, based on the nucleotidylation activity of EAV nsp9, the NiRAN domain is hypothesized to play a role in protein priming, capping, or as a potential universal ligation mechanism. 98 The active site of the polymerase is located within motif C and is composed of conserved (within the Nidovirus family) ser-asp-asp residues. Importantly, conserved aspartates found in motif A of SARS-CoV, which combined with those found in motif C, contribute to the polymerase and RNA synthesis activities. This is different from other positive strand RNA viruses which contain a GDD active site. Motif A along with motif C aid in coordinating the metal ions necessary for catalysis. The SARS-CoV harbors a signature sequence in motif G necessary for primer-dependent RNA synthesis. 102, 103 Due to difficulties in obtaining large enough amounts of highly pure protein, the structures of CoV nsp12 have not been solved either by X-ray crystallography or cryo-EM. Therefore, the structural information currently available is based solely on structural models obtained via sequence alignment and homology modeling techniques. These models indicate a right-handed fold composed of fingers, palm, and thumb subdomains with clearly defined entry and exit channels, consistent with RdRp domains for other structurally characterized positive sense RNA polymerases (for example, foot-and-mouth disease virus) but clearly distinct from the known molecular topology of negative-stranded RdRps. No structural models predict the presence of a priming loop. These data combined with biochemical data indicating no de novo initiation of RNA synthesis may account for the functionality of motif G.