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. 2007 May 29;104(22):9463-8.
doi: 10.1073/pnas.0700518104. Epub 2007 May 21.

Sequential structures provide insights into the fidelity of RNA replication

Cristina Ferrer-Orta  1 , Armando Arias, Rosa Pérez-Luque, Cristina Escarmís, Esteban Domingo, Nuria Verdaguer
Affiliations

Sequential structures provide insights into the fidelity of RNA replication

Cristina Ferrer-Orta et al. Proc Natl Acad Sci U S A. .

Abstract

RNA virus replication is an error-prone event caused by the low fidelity of viral RNA-dependent RNA polymerases. Replication fidelity can be decreased further by the use of mutagenic ribonucleoside analogs to a point where viral genetic information can no longer be maintained. For foot-and-mouth disease virus, the antiviral analogs ribavirin and 5-fluorouracil have been shown to be mutagenic, contributing to virus extinction through lethal mutagenesis. Here, we report the x-ray structure of four elongation complexes of foot-and-mouth disease virus polymerase 3D obtained in presence of natural substrates, ATP and UTP, or mutagenic nucleotides, ribavirin triphosphate and 5-fluorouridine triphosphate with different RNAs as template-primer molecules. The ability of these complexes to synthesize RNA in crystals allowed us to capture different successive replication events and to define the critical amino acids involved in (i) the recognition and positioning of the incoming nucleotide or analog; (ii) the positioning of the acceptor base of the template strand; and (iii) the positioning of the 3'-OH group of the primer nucleotide during RNA replication. The structures identify key interactions involved in viral RNA replication and provide insights into the molecular basis of the low fidelity of viral RNA polymerases.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Structure of FMDV 3D catalytic complexes. Molecular surface of the polymerase (gray) is shown, with the position of the rNTP substrates and the trajectory of RNA template–primer and duplex product in two different complexes: the 3D·GCAUGGGCCC·ATP/UTP (A) and the 3D·GCAUGGGCCC-RTP (B). The N-terminal residues (residues 34–48) and residues at the top of the NTP tunnel (163–180) of 3D are omitted to show the substrate cavities. RNA molecules are shown in yellow (template strands) and green (primer strands). (A) The UTP substrate is shown in cyan. (B) The position of the antiviral mutagen RTP is shown in orange. Metal ions are shown as red spheres.
Fig. 2.
Fig. 2.
Conserved interactions between the FMDV 3D and the different RNA template–primers. The polymerase regions involved in contacts with the RNA molecule are explicitly labeled. The template and primer strands of the RNA molecule are shown in yellow and green, respectively; atoms are displayed in atom-type code, and hydrogen bonds are dashed lines in black. The template strand contacts mainly with residues in the fingers subdomain (blue). The 5′ overhang region of the template binds the template channel, where the different residues of the N-terminal region and the loop α4–β3 of the polymerase drive the ssRNA to the active-site cavity (Lower Left). The template strand of the dsRNA product contacts different residues of helix α7 and the loop β9–α11 in its exit through the central cavity of the enzyme (Upper Left). The primer strand interacts with motifs C and E of the palm subdomain, shown in magenta (Upper Right) and with helix α14 of the thumb, shown in red (Lower Right).
Fig. 3.
Fig. 3.
Nonconserved interactions established between the FMDV 3D, their template–primer RNAs, and the rNTP substrates in the polymerase active site of the four different complexes 3D·GCAUGGGCCC·ATP (A), 3D·GCAUGGGCCC·ATP/UTP (B), 3D·CGUAGGGCCC·FUTP (C), and 3D·GCAUGGGCCC·RTP (D). The 3D amino acids involved in direct interactions with the template, primer, and incoming nucleotides are shown as sticks in atom-type color code and explicitly labeled. Hydrogen bonds are shown as dashed lines in black. Template and primer RNA strands are shown in yellow and green, respectively. (A–C) Newly incorporated nucleotides are shown in light green. (A and C) Pyrophosphate product (PPi) is also shown in light green. (B) UTP substrate is shown in cyan. (D) Antiviral mutagen RTP is shown in orange. Metal ions are shown as red spheres.
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