Incorporation fidelity of the viral RNA-dependent RNA polymerase: a kinetic, thermodynamic and structural perspective - PubMed (original) (raw)

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Incorporation fidelity of the viral RNA-dependent RNA polymerase: a kinetic, thermodynamic and structural perspective

Christian Castro et al. Virus Res. 2005 Feb.

Abstract

Positive-strand RNA viruses exist as a quasi-species due to the incorporation of mutations into the viral genome during replication by the virus-encoded RNA-dependent RNA polymerase (RdRP). Therefore, the RdRP is often described as a low-fidelity enzyme. However, until recently, a complete description of the kinetic, thermodynamic and structural basis for the nucleotide incorporation fidelity of the RdRP has not been available. In this article, we review the following: (i) the steps employed by the RdRP to incorporate a correct nucleotide; (ii) the steps that are employed by the RdRP for nucleotide selection; (iii) the structure-based hypothesis for nucleotide selection; (iv) the impact of sites remote from the active site on polymerase fidelity. Given the recent observation that RNA viruses exist on the threshold of error catastrophe, the studies reviewed herein suggest novel strategies to perturb RdRP fidelity that may lead ultimately to the development of antiviral agents to treat RNA virus infection.

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Figures

Fig. 1

Fig. 1

Symmetrical primer template substrate (sym/sub) used to study poliovirus polymerase 3Dpol-catalyzed nucleotide incorporation.

Scheme 1

Scheme 1

Complete kinetic mechanism for 3Dpol-catalyzed nucleotide incorporation.

Fig. 2

Fig. 2

Structural model for 3Dpol-catalyzed nucleotide incorporation: (A) ground-state binding of metal-complexed nucleotide; (B) reorientation of the triphosphate into the catalytically competent configuration; (C) phosphoryl transfer and pyrophosphate release. While the kinetic mechanism suggests a conformational change prior to pyrophosphate release, kinetic data do not provide any information to permit a molecular description of this step. Images were generated from the model previously described (Gohara et al., 2000). Nucleotide and side chain motions were derived from (Johnson et al., 2003) by approximate rotation and translation movements. Atom colors correspond to the following: red, oxygen; blue, nitrogen; gray, carbon; magenta, Mg2+ or Mn2+. The images were rendered with WebLab Viewer Pro (Accelrys Inc., San Diego, CA). Reproduced with permission from Biochemistry (2004), submitted. © 1998 Am. Chem. Soc.

Scheme 2

Scheme 2

Comparison of the conformational-change step and the phosphoryl-transfer step for 3Dpol-catalyzed correct and incorrect nucleotide incorporation in the presence of Mg2+ and Mn2+.

Fig. 3

Fig. 3

Comparison of the free energy profile for correct and incorrect 3Dpol-catalyzed nucleotide incorporation in the presence of Mg2+. The free energy profile for correct and incorrect nucleotide incorporation are shown as follows: solid line for AMP incorporation, small dotted line for 2′-dAMP incorporation, and large dotted line for GMP incorporation. The concentrations of the substrates and products used were 2000 μM NTP and 20 μM PP_i_. The free energy for each reaction step was calculated from Δ_G_ = RT [ln (kT/h) − ln (_k_obs,for)], where R = 1.99 cal K−1 mol−1, T = 303 K, k = 3.30 × 10−24 cal K−1, h = 1.58 × 10−34 cal s and k_obs is the first-order rate constant (Arnold et al., 2004). The free energy for each species was calculated from Δ_G = RT [ln (kT/h) − ln (_k_obs,for)] − RT [ln (kT/h) − ln (_k_obs,rev)]. Reproduced with permission from Biochemistry, 2004, submitted. © 1998 Am. Chem. Soc.

Fig. 4

Fig. 4

Structural model for 3Dpol-catalyzed nucleotide incorporation fidelity. Yellow molecules indicate important structural changes: (A) possible conformation of 2′-dATP bound to the NTP-binding pocket; the change here could be caused by the different sugar pucker. (B) Possible conformation of GTP bound to the NTP-binding pocket; the change here could be caused by the non-planar G:U basepair. Reproduced with permission from Biochemistry, 2004, submitted. © 1998 Am. Chem. Soc.

Fig. 5

Fig. 5

Nucleotide-binding pocket of 3Dpol: (A) residues located in the NTP-binding pocket as observed in the unliganded structure of 3Dpol (Hansen et al., 1997); Asp-233 and Asp-238 are from structural motif A; Ser-288, Thr-293, and Asn-297 are from motif B; Asp-328 is from motif C. (B) Model for interaction of 3Dpol with bound nucleotide (Gohara et al., 2004); ATP and metal ions required for catalysis are labeled. In this model, the side chains for Asp-233 and Asp-238 have been rotated to permit interactions with ATP. Asp-238, Ser-288 and Thr-293 have been positioned to interact. The image was created by using the program WebLab Viewer (Molecular Simulations Inc., San Diego, CA). Reproduced with permission from Biochemistry, 2004, submitted. © 1998 Am. Chem. Soc.

Fig. 6

Fig. 6

A conserved mechanism for linking binding of a correct nucleotide to the efficiency of phosphoryl transfer. The nucleotide-binding pocket of all nucleic acid polymerases with a canonical “palm”-based active site is highly conserved. The site can be divided into two parts: a region that has “universal” interactions mediated by conserved structural motif A that organizes the metals and triphosphate for catalysis and a region that has “adapted” interactions mediated by conserved structural motif B that dictate whether ribo- or 2′-deoxribonucleotides will be utilized. In the classical polymerase, there is a motif A residue located in the sugar-binding pocket capable of interacting with motif B residue(s) involved in sugar selection. This motif A residue in other polymerases could represent the link between the nature of the bound nucleotide (correct vs. incorrect) to the efficiency of phosphoryl transfer as described herein for Asp-238 of 3Dpol (Gohara et al., 2004). Reproduced with permission from Biochemistry, 2004, submitted. © 1998 Am. Chem. Soc.

Fig. 7

Fig. 7

Location of Gly-64 in the structural model of 3Dpol. Model of 3Dpol (complete) based upon sequence and structural homology to rabbit hemorrhagic disease virus 3Dpol (Ng et al., 2002). The conserved structural motifs in the palm subdomain are colored as follows: motif A, red; motif B, green; motif C, yellow; motif D, blue; motif E, purple. van der Waal's projection of Gly-64 (orange). The image was rendered using the program WebLab Viewer Pro (Molecular Simulations Inc., San Diego, CA).

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References

    1. Airaksinen A., Pariente N., Menendez-Arias L., Domingo E. Curing of foot-and-mouth disease virus from persistently infected cells by ribavirin involves enhanced mutagenesis. Virology. 2003;311:339–349. - PubMed
    1. Arnold J.J., Cameron C.E. Poliovirus RNA-dependent RNA polymerase (3Dpol): assembly of stable, elongation-competent complexes by using a symmetrical primer-template substrate (sym/sub) J. Biol. Chem. 2000;275:5329–5336. - PubMed
    1. Arnold, J.J., Cameron, C.E., 2004a, unpublished observations.
    1. Arnold J.J., Cameron C.E. Poliovirus RNA-dependent RNA polymerase (3Dpol): pre-steady-state kinetic analysis of ribonucleotide incorporation in the presence of Mg2+ Biochemistry. 2004;43:5126–5137. - PMC - PubMed
    1. Arnold J.J., Ghosh S.K., Cameron C.E. Poliovirus RNA-dependent RNA polymerase (3Dpol): divalent cation modulation of primer, template, and nucleotide selection. J. Biol. Chem. 1999;274:37060–37069. - PubMed

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