2'-C-methylated nucleotides terminate virus RNA synthesis by preventing active site closure of the viral RNA-dependent RNA polymerase.

The 2'-C-methyl ribonucleosides are nucleoside analogs representing an important class of antiviral agents, especially against positive-strand RNA viruses. Their value is highlighted by the highly successful anti-hepatitis C drug sofosbuvir. When appropriately phosphorylated, these nucleotides are successfully incorporated into RNA by the virally encoded RNA-dependent RNA polymerase (RdRp). This activity prevents further RNA extension, but the mechanism is poorly characterized. Previously, we had identified NMR signatures characteristic of formation of RdRp-RNA binary and RdRp-RNA-NTP ternary complexes for the poliovirus RdRp, including an open-to-closed conformational change necessary to prepare the active site for catalysis of phosphoryl transfer. Here we used these observations as a framework for interpreting the effects of 2'-C-methyl adenosine analogs on RNA chain extension in solution-state NMR spectroscopy experiments, enabling us to gain additional mechanistic insights into 2'-C-methyl ribonucleoside-mediated RNA chain termination. Contrary to what has been proposed previously, poliovirus RdRp that was bound to RNA with an incorporated 2'-C-methyl nucleotide could still bind to the next incoming NTP. Our results also indicated that incorporation of the 2'-C-methyl nucleotide does not disrupt RdRp-RNA interactions and does not prevent translocation. Instead, incorporation of the 2'-C-methyl nucleotide blocked closure of the RdRp active site upon binding of the next correct incoming NTP, which prevented further nucleotide addition. We propose that other nucleotide analogs that act as nonobligate chain terminators may operate through a similar mechanism.

Rational Control of Poliovirus RNA-Dependent RNA Polymerase Fidelity by Modulating Motif-D Loop Conformational Dynamics.

The conserved structural motif D is an important determinant of the speed and fidelity of viral RNA-dependent RNA polymerases (RdRps). Structural and computational studies have suggested that conformational changes in the motif-D loop that help to reposition the catalytic lysine represent critical steps in nucleotide selection and incorporation. Conformations of the motif-D loop in the poliovirus RdRp are likely controlled in part by noncovalent interactions involving the motif-D residue Glu364. This residue swivels between making interactions with Lys228 and Asn370 to stabilize the open and closed loop conformations, respectively. We show here that we can rationally control the motif-D loop conformation by breaking these interactions. The K228A variant favors a more active closed conformation, leading to increased nucleotide incorporation rates and decreased nucleotide selectivity, and the N370A variant favors a less active open conformation, leading to decreased nucleotide incorporation rates and increased nucleotide selectivity. Similar competing interactions likely control nucleotide incorporation rates and fidelity in other viral RdRps. Rational engineering of these interactions may be important in the generation of live, attenuated vaccine strains, considering the established relationships between RdRp function and viral pathogenesis.