Nucleosides for the treatment of respiratory RNA virus infections

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

Human rhinovirus (HRV), enterovirus 71 (EV71), and poliovirus (PV) are nonenveloped, positive strand RNA viruses and are all members of the Picornaviridae family. Picornaviruses replicate their genome using an RdRp, called 3D pol . 78 Replication takes place in one of two forms: primer-dependent format or de novo RNA synthesis. 79 De novo RNA synthesis, which uses a single initiation nucleotide, gives the 3 0 -hydroxyl group for adding the sequential nucleotide whereas a primer-dependent format uses a protein-based primer or an oligonucleotide for the hydroxyl group donor. 80 The polymerases from the picornavirus family only use a protein-primed mechanism of initiation. 80, 81 The 3D pol uses a small viral peptide (VPg) to initiate both plus and minus RNA synthesis in vivo, making Picornaviridae unique in their initiation mechanism. 78 RNA synthesis is initiated using a highly conserved tyrosine residue within VPg using cis-acting replication element as a template whose position varies depending on the genus. 78, 82 The 3D pol aids in the binding of two UMP molecules to the tyrosine hydroxyl group of VPg. [83] [84] [85] The product of this reaction, VPg-pU is then extended by an additional uridine to form VPg-pUpU. 86 The 3D pol is located at the C-terminal end of a longer viral polyprotein of approximately 250 kDa and the structures of 3D pol have been solved for EV71, HRV16, and PV, among many others. [86] [87] [88] The structures of these polymerases are largely similar and have sequence domains A-G indicating this conservation. 89 The 460-residue polymerase domain adopts a right-handed configuration with fingers, palm, and thumb subdomains providing an optimal arrangement for substrate and metal cation access during the catalytic cycle. The palm, fingers, and thumb subdomains contain these sequence motifs (motifs A-E in the palm subdomain shown in Figure 4) . 90, 91 These motifs have specialized roles in catalysis including nucleotide binding and overall structural integrity of the active site. 80 The palm contains the active site of all the RdRps and its structure consists of an antiparallel beta-sheet surrounded by three alpha helices. 90 Additional substructures within the fingers domain are referred to as the index, middle, ring, and pinky fingers. 82, 92 All but the pinky fingers build an extended beta-sheet that seems to be conserved. The index finger within the fingers subdomain makes an important contact with the thumb subdomain and pushes the ring finger down to trap it. This conformation results in the ring finger being the roof for NTP entry and making important interactions with the triphosphate. An additional structural feature of motif F is a positively charged tunnel that modulates the interactions of NTP. 93 This tunnel aides in the diffusion of nucleotides and is conserved among this family of viruses.