Nucleobase but not Sugar Fidelity is Maintained in the Sabin I RNA-Dependent RNA Polymerase
Abstract
:1. Introduction
2. Results
2.1. Sabin PV RdRp Discriminates against Incorrect Nucleobases, but not against Incorrect Sugars, to the Same Extent as WT RdRp
2.2. The K250E Substitution is Unstable in Cell Culture
Variant | NTP | kpol (s−1) | Kd,app (μM) | kpol/Kd,app (μM−1·s−1) | kpol,corr./ kpol,incorr. | (kpol/Kd,app)corr./ (kpol/Kd,app)incorr. |
---|---|---|---|---|---|---|
WT | ATP | 5.9 ± 0.1 × 101 | 36 ± 2 | 1.6 | – | – |
D53N | 6.2 ± 0.1 × 101 | 39 ± 2 | 1.6 | – | – | |
Y73H | 4.6 ± 0.1 × 101 | 40 ± 2 | 1.2 | – | – | |
K250E | 7.6± 0.1 × 101 | 58 ± 3 | 1.3 | – | – | |
T362I | 6.7 ± 0.1 × 101 | 33 ± 2 | 2.0 | – | – | |
D53N/T362I | 7.2 ± 0.1 × 101 | 44 ± 2 | 1.6 | – | – | |
D53N/Y73H/T362I | 8.4 ± 0.2 × 101 | 47 ± 4 | 1.8 | – | – | |
Sabin | 7.4 ± 0.1 × 101 | 39 ± 2 | 1.9 | – | – | |
WT | 2′-dATP | 8.9 ± 0.1 × 10-1 | 134 ± 4 | 6.7 × 10−3 | 70 | 240 |
D53N | 9.3 ± 0.2 × 10-1 | 101 ± 9 | 9.3 × 10−3 | 70 | 170 | |
Y73H | 7.3 ± 0.1 × 10-1 | 117 ± 5 | 6.2 × 10−3 | 60 | 190 | |
K250E | 1.4 ± 0.0 | 174 ± 6 | 8.0 × 10−3 | 50 | 160 | |
T362I | 1.5 ± 0.0 | 112 ± 4 | 1.3 × 10−2 | 40 | 150 | |
D53N/T362I | 1.5 ± 0.0 | 132 ± 6 | 1.1 × 10−2 | 50 | 150 | |
D53N/Y73H/T362I | 1.4 ± 0.0 | 101 ± 9 | 1.4 × 10−2 | 60 | 130 | |
Sabin | 2.0 ± 0.0 | 145 ± 4 | 1.4 × 10−2 | 40 | 140 | |
WT | GTP | 1.1 ± 0.1 × 10−2 | 142 ± 15 | 7.7 × 10−5 | 5400 | 21,000 |
D53N | 7.3 ± 0.8 × 10−3 | 91 ± 35 | 8.0 × 10−5 | 8400 | 20,000 | |
Y73H | 8.2 ± 0.5 × 10−3 | 154±31 | 5.3 × 10−5 | 5600 | 23,000 | |
K250E | 9.9 ± 0.8 × 10−3 | 160±41 | 6.1 × 10−5 | 7700 | 21,000 | |
T362I | 1.8 ± 0.1 × 10−2 | 149 ± 25 | 1.2 × 10−4 | 3700 | 17,000 | |
D53N/T362I | 1.2 ± 0.1 × 10−2 | 115 ± 21 | 1.0 × 10−4 | 6000 | 16,000 | |
D53N/Y73H/T362I | 1.1 ± 0.1 × 10−2 | 128±12 | 8.6 × 10−5 | 7600 | 21,000 | |
Sabin | 1.3 ± 0.1 × 10−2 | 127 ± 16 | 1.0 × 10−4 | 5700 | 19,000 | |
WT | 2′-C-methyl ATP | 1.2 ± 0.0 | 160 ± 9 | 7.5 × 10−3 | 50 | 210 |
Sabin | 1.9 ± 0.0 | 129 ± 5 | 1.5 × 10−2 | 40 | 130 |
2.3. D53N, Y73H and K250E PV RdRp Present Different Fidelities for Sugar and Nucleobase Selection
2.4. Allosteric Effects among the Sabin Amino Acid Substitutions
2.5. Structural Dynamic Differences between the Sabin I RdRp and the Triple Variant D53/Y73H/T362I
2.6. The Sabin I RdRp is More Susceptible to 2′-Modified Nucleotides
3. Discussion
4. Materials and Methods
4.1. Materials
4.2. Plasmid Construction
4.3. Overexpression and Protein Purification
4.4. Kinetic Assays
4.5. Determination of Kinetic Constants (Kd,app, and kpol) for Nucleotide Incorporation Catalyzed by RdRp
4.6. NMR Sample Preparation and Spectroscopy
4.7. Construction of Mutated Viral cDNA Clones and Replicons
4.8. RNA Transcription
4.9. Infectious Center Assays
4.10. Virus Isolation, RNA Isolation, cDNA Synthesis, and Sequencing to Confirm the Presence of the Quadruple Mutation
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Liu, X.; Musser, D.M.; Lee, C.A.; Yang, X.; Arnold, J.J.; Cameron, C.E.; Boehr, D.D. Nucleobase but not Sugar Fidelity is Maintained in the Sabin I RNA-Dependent RNA Polymerase. Viruses 2015, 7, 5571-5586. https://doi.org/10.3390/v7102894
Liu X, Musser DM, Lee CA, Yang X, Arnold JJ, Cameron CE, Boehr DD. Nucleobase but not Sugar Fidelity is Maintained in the Sabin I RNA-Dependent RNA Polymerase. Viruses. 2015; 7(10):5571-5586. https://doi.org/10.3390/v7102894
Chicago/Turabian StyleLiu, Xinran, Derek M. Musser, Cheri A. Lee, Xiaorong Yang, Jamie J. Arnold, Craig E. Cameron, and David D. Boehr. 2015. "Nucleobase but not Sugar Fidelity is Maintained in the Sabin I RNA-Dependent RNA Polymerase" Viruses 7, no. 10: 5571-5586. https://doi.org/10.3390/v7102894