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Publication Abstract from PubMed

The N-terminal region of the FMDV 3D polymerase contains the sequence MRKTKLAPT (residues 16 to 24) that acts as a nuclear localization signal. A previous study showed that substitutions K18E and K20E diminished the transport to the nucleus of 3D and 3CD, and severely impaired virus infectivity. These residues have also been implicated in template binding as seen in the crystal structures of different 3D-RNA elongation complexes. Here we report the biochemical and structural characterization of different mutant polymerases harboring substitutions at residues 18 and 20, in particular K18E, K18A, K20E, K20A and the double mutant KAKA. All mutant enzymes exhibit low RNA binding activity, low processivity and alterations in nucleotide recognition, including increased incorporation of ribavirin monophosphate (RMP) relative to the incorporation of cognate nucleotides, as compared with the wild type enzyme. The structural analysis shows an unprecedented flexibility of the 3D mutant polymerases, including both, global rearrangements of the closed hand architecture and local conformational changes at loop beta9-alpha11 (within the polymerase motif B) and at the template-binding channel. Specifically, in 3D bound to RNA both K18E and K20E induced the opening of new pockets in the template channel where the downstream templating nucleotide at position +2 binds. The comparisons of free and RNA bound enzymes suggest that the structural rearrangements may occur in a concerted mode to regulate both RNA replication, processivity and fidelity. Thus, the N-terminal region of FMDV 3D that acts as a nuclear localization signal and in template binding, is also involved in nucleotide recognition, and can affect the incorporation of nucleotide analogues. IMPORTANCE: The study documents multifunctionality of a nuclear localization signal (NLS) located at the N-terminal region of the foot-and-mouth disease viral polymerase (3D). Amino acid substitutions at this polymerase region can impair the transport of 3D to the nucleus, reduce 3D binding to RNA, and alter the relative incorporation of standard nucleoside-monophosphate versus ribavirin-monophosphate. Structural data reveal that the conformational changes in this region, forming part of the template channel entry, would be involved in nucleotide discrimination. The results have implications for the understanding of viral polymerase function, and for lethal mutagenesis mechanisms.

Multifunctionality of a picornavirus polymerase domain: nuclear localization signal and nucleotide recognition.,Ferrer-Orta C, de la Higuera I, Caridi F, Sanchez-Aparicio MT, Moreno E, Perales C, Singh K, Sarafianos SG, Sobrino F, Domingo E, Verdaguer N J Virol. 2015 Apr 22. pii: JVI.03283-14. PMID:25903341^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.