Residues within the foot-and-mouth disease virus 3dpol nuclear localization signal affect polymerase fidelity

Authors: KLOC, ANNA - Oak Ridge Institute For Science And Education (ORISE); RAI, DEVENDRA - University Of Minnesota; Kenney, Mary; Schafer, Elizabeth; Rieder, Aida - Elizabeth; Gladue, Douglas

Submitted to: Journal of Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/11/2020
Publication Date: 6/24/2020
Citation: Kloc, A., Rai, D.K., Kenney, M.A., Schafer, E.A., Rieder, A.E., Gladue, D. 2020. Residues within the foot-and-mouth disease virus 3dpol nuclear localization signal affect polymerase fidelity. Journal of Virology. https://doi.org/10.1128/JVI.00833-20.
DOI: https://doi.org/10.1128/JVI.00833-20

Interpretive Summary: Foot-and-mouth disease virus (FMDV) is the most devastating disease affecting livestock worldwide. Here we report the identification of mutations in the viral protein (enzyme) responsible for viral replication (3Dpol) that affect the accuracy of the enzyme during viral replication (i.e. fidelity) resulting in decreased virus growth in host cells. The alteration of 3Dpol fidelity described in this study uncovers a novel strategy to derive safer and more stable attenuated FMDV vaccine candidates.

Technical Abstract: The low fidelity of the viral polymerase allows for generation of genetically diverse populations that can adapt to changing environment and thwart anti-viral therapies. The RNA dependent RNA polymerase (RdRp), or 3Dpol, of the Foot-and-Mouth disease virus (FMDV) is responsible for replication of the viral genomes. The 3Dpol N-terminus encodes a nuclear localization signal (NLS) sequence – MRKTKLAPT - important for import of the protein to host nucleus. Residues of the NLS have been reported to be play a role in nucleotide recognition during synthesis of a new RNA strand. Specifically, substitutions at residues 18 and 20 have a defect in proper incorporation of nucleotides and RNA binding ability. Here, using an alanine scanning mutagenesis approach, we identify two residues of the 3Dpol NLS -T19 and L21- that are important for maintenance of the enzyme fidelity. Alanine substitution of 3DT19 and 3DL21 results in aberrant incorporation of nucleoside analogs, conferring a low fidelity phenotype of the enzyme. A structural dynamics analysis reveals that the 3DT19 residue forms a hydrogen bond with D165 of the 3Dpol template-binding region. Consequently, the mutated 3DT19A residue can no longer bind to D165, inducing a conformational change of the 3Dpol active site, which changes its ability to incorporate proper nucleotides during viral replication. These findings demonstrate that the NLS of 3Dpol plays a vital role in faithful RNA synthesis and maintenance of the enzyme fidelity.