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Multifunctionality of a picornavirus polymerase domain: nuclear localization signal and nucleotide recognition.

ABSTRACT: The N-terminal region of the foot-and-mouth disease virus (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 K18A K20A (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 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 ?9-?11 (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 RNA replication, processivity, and fidelity. Thus, the N-terminal region of FMDV 3D that acts as a nuclear localization signal (NLS) and in template binding is also involved in nucleotide recognition and can affect the incorporation of nucleotide analogues.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.

SUBMITTER: Ferrer-Orta C

PROVIDER: S-EPMC4468482 | biostudies-literature | 2015 Jul

REPOSITORIES: biostudies-literature

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Multifunctionality of a picornavirus polymerase domain: nuclear localization signal and nucleotide recognition.

Ferrer-Orta Cristina C de la Higuera Ignacio I Caridi Flavia F Sánchez-Aparicio María Teresa MT Moreno Elena E Perales Celia C Singh Kamalendra K Sarafianos Stefan G SG Sobrino Francisco F Domingo Esteban E Verdaguer Nuria N

Journal of virology 20150422 13

<h4>Unlabelled</h4>The N-terminal region of the foot-and-mouth disease virus (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 ...[more]

PMID: 25903341

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Project description:Many RNA viruses encode a proof-reading deficient, low-fidelity RNA-dependent polymerase (RdRp), which generates genetically diverse populations that can adapt to changing environments and thwart antiviral therapies. 3Dpol, the RdRp of the foot-and-mouth disease virus (FMDV), is responsible for replication of viral genomes. The 3Dpol N terminus encodes a nuclear localization signal (NLS) sequence,MRKTKLAPT, important for import of the protein to host nucleus. Previous studies showed that substitutions at residues 18 and 20 of the NLS are defective in proper incorporation of nucleotides and RNA binding. Here, we use a systematic alanine scanning mutagenesis approach to understand the role of individual residues of the NLS in nuclear localization and nucleotide incorporation activities of 3Dpol We identify two residues of 3Dpol NLS, T19 and L21, that are important for the maintenance of enzyme fidelity. The 3Dpol NLS alanine substitutions of T19 and L21 results in aberrant incorporation of nucleoside analogs, conferring a low fidelity phenotype of the enzyme. A molecular dynamics simulation of RNA- and mutagen (RTP)-bound 3Dpol revealed that the T19 residue participates in a hydrogen bond network, including D165 in motif F and R416 at the C terminus of the FMDV 3Dpol and RNA template-primer. Based on these findings and previous studies, we conclude that at least the first six residues of theMRKTKLAPT sequence motif play a vital role in the maintenance of faithful RNA synthesis activity (fidelity) of FMDV 3Dpol, suggesting that the role of the NLS motif in similar viral polymerases needs to be revisited.IMPORTANCE In this study, we employed genetic and molecular dynamics approaches to analyze the role of individual amino acids of the FMDV 3Dpol nuclear localization signal (NLS). The NLS residues were mutated to alanine using a type A full-genome cDNA clone, and the virus progeny was analyzed for defects in growth and in competition with the parental virus. We identified two mutants in 3Dpol, T19A and L21A, that exhibited high rate of mutation, were sensitive to nucleotide analogs, and displayed reduced replicative fitness compared to the parental virus. Using molecular dynamics simulation, we demonstrated that residues T19 and L21 played a role in the structural configuration of the interaction network at the 3Dpol palm subdomain. Cumulatively, our data suggest that the T19 and L21 3Dpol amino acids are important for maintaining the fidelity of the FMDV polymerase and ensuring faithful replication of the FMDV genome.

Dataset Information

Multifunctionality of a picornavirus polymerase domain: nuclear localization signal and nucleotide recognition.

Publications

Multifunctionality of a picornavirus polymerase domain: nuclear localization signal and nucleotide recognition.

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