Triphosphate Re-orientation of the Incoming Nucleotide as a Fidelity Checkpoint in Viral RNA-dependent RNA polymerases [RNA]

The nucleotide incorporation fidelity of the viral RNA-dependent RNA polymerase (RdRp) is important for maintaining functional genetic information, but at the same time for generating sufficient genetic diversity to escape the bottlenecks of the host's anti-viral response. We have previously shown that the structural dynamics of the motif-D loop is closely related to nucleotide discrimination. Previous studies have also suggested that there is a re-orientation of the triphosphate of the incoming nucleotide, which is essential prior to nucleophilic attack from the primer RNA 3'-hydroxyl. Here, we have used 31P NMR with poliovirus RdRp to show that the binding environment of the triphosphate is different when correct versus incorrect nucleotide binds. We also show that amino acid substitutions at residues known to interact with the triphosphate can alter the binding orientation/environment of the nucleotide, sometimes lead to protein conformational changes, and lead to substantial changes in RdRp fidelity. The analysis of other fidelity variants also show that changes in the triphosphate binding environment are not always accompanied by changes in the structural dynamics of the motif-D loop or other regions known to be important for RdRp fidelity, including motif B. Altogether, our studies suggest that the conformational changes in motif B and motif D, and the nucleoside triphosphate reorientation represent separable, 'tuneable' fidelity checkpoints.

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