Nucleosides for the treatment of respiratory RNA virus infections

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

A complete characterization of the in vitro nsp12 RdRp activity has demonstrated overall weak activity. Initial evidence suggested that a previously uncharacterized N-terminal domain may have been required for polymerase activity. 104 However, the addition of this domain using a C-terminally tagged protein still yielded protein with poor activity and processivity. Based on these results and to increase the in vitro activity of the nsp12, two other non-structural proteins, nsp7 and nsp8, were added to the nsp12 protein in a primer extension mode. 100 The addition of nsp7 and nsp8 to nsp12 appear to activate and increase the processivity of the polymerase allowing it to produce an RNA synthesis product of 340 nucleotides in the presence of Mg 2þ . Linking the nsp7 and nsp8 subunits together also increased the polymerase reaction efficiency suggesting that nsp7-nsp8 complex formation may influence the reaction rate. Importantly, nsp14 can interact with an nsp7-nsp8-nsp12 complex without influencing RNA synthesis activities. 100 Nsp14 contains an exoribonuclease domain that has been shown to decrease nucleotide mismatch, in many ways similar to the proofreading exoribonuclease activities correlated with a polymerase. 105