Nucleosides for the treatment of respiratory RNA virus infections

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

a linker connecting the two subdomains of PA. The PB1 possesses a b-hairpin loop within motif E from residues 641 to 657 in the thumb subdomain of influenza A. 46 In de novo initiation, it has been shown with other related polymerases that this priming loop may serve as a platform for the initial NTP on the 3 0 end of the template and ensure against double-stranded RNA. Biochemical investigations have shown that the proline found within the loop tip motif of 648-Ala-His-Gly-Pro is necessary for in vitro and cell-based RNA synthesis. This loop may also be necessary during replication mode for terminal de novo initiation but unnecessary for internal initiation and transcription. 55 The PA endonuclease domain or the PA subunit, as it will be named here, has little homology to other proteins and its exact enzymatic function was discovered only recently. The subunit was expressed in insect cells to reveal an N-terminal third (PA-Nter) and a C-terminal two-thirds (PA-Cter) subdomains. They have molecular weights of 25 and 55 kDa, respectively. These two subdomains are connected by a flexible linker. The endonuclease activity was originally thought to occur through the PB1 or PB2 domains but the structure of the PA-Nter was solved by two groups to reveal that the catalytic residues for endonuclease activity reside in the PA domain, not in the PB1 subunit as originally thought. 50, 51 The PA endonuclease domain contains a signature (P)DXN(D/E)XK motif that is conserved among influenza viruses and coordinate divalent cations such as magnesium or manganese. 56 Although the exact quantity and identity of ions present in the native influenza enzyme are unclear, the endonuclease activity is strongly activated by metal ion binding through hydrolysis of ssDNA and ssRNA substrates.