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Biochemical characterization of the fidelity of poliovirus RNA-dependent RNA polymerase.

Freistadt MS, Vaccaro JA, Eberle KE - Virol. J. (2007)

Bottom Line: To derive kpol/Kd for correct base incorporation, we performed conventional pre-steady state single turnover measurements, yielding values that range from 0.62 to 9.4 microM-1 sec-1.Overall, we found that fidelity for poliovirus polymerase 3Dpol ranges from 1.2 x 10(4) to 1.0 x 10(6) for transition mutations and 3.2 x 10(5) to 4.3 x 10(7) for transversion mutations.Based on unusual enzyme behavior that we observed, we speculate that RNA mismatches either directly or indirectly cause enzyme RNA dissociation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology, Immunology and Parasitology; Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112, USA. mfreis@tulane.edu

ABSTRACT

Background: Putative high mutation rates of RNA viruses are believed to mediate undesirable phenomena, such as emergence of drug resistance. However, very little is known about biochemical fidelity rates for viral RNA-dependent RNA polymerases. Using a recently developed in vitro polymerase assay for poliovirus polymerase 3Dpol [Arnold and Cameron (2000) JBC 275:5329], we measured fidelity for each possible mismatch. Polymerase fidelity, in contrast to sequence error rate, is biochemically defined as kpol/Kd of {(correct plus incorrect) divided by incorrect} incorporations, such that a larger value connotes higher fidelity.

Results: To derive kpol/Kd for correct base incorporation, we performed conventional pre-steady state single turnover measurements, yielding values that range from 0.62 to 9.4 microM-1 sec-1. Pre-steady state measurements for incorrect base incorporation were less straightforward: several anomalous phenomena interfered with data collection. To obtain pre-steady state kinetic data for incorrect base incorporation, three strategies were employed. (1) For some incorrect bases, a conventional approach was feasible, although care was taken to ensure that only single turnovers were being assessed. (2) Heparin or unlabeled RNA traps were used to simulate single turnover conditions. (3) Finally, for some incorrect bases, incorporation was so poor that single datapoints were used to provide kinetic estimates. Overall, we found that fidelity for poliovirus polymerase 3Dpol ranges from 1.2 x 10(4) to 1.0 x 10(6) for transition mutations and 3.2 x 10(5) to 4.3 x 10(7) for transversion mutations.

Conclusion: These values are unexpectedly high showing that high RNA virus sequence variation is not due to intrinsically low polymerase fidelity. Based on unusual enzyme behavior that we observed, we speculate that RNA mismatches either directly or indirectly cause enzyme RNA dissociation. If so, high sequence variation of RNA viruses may be due to template-switch RNA recombination and/or unknown fitness/selection phenomena. These findings may lead to a mechanistic understanding of RNA virus error catastrophe and improved anti-viral strategies.

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Single turnover analysis (rapid quench) of UTP (correct) incorporation into SSA. (A) Product yield as a function of time for three concentrations of UTP. A series of single turnover, rapid quench reactions were performed and analyzed described in Methods. Product concentration of resulting 11 mer (in nM) as a function of time in seconds is shown for 75 (circles), 150 (squares) and 500 (triangles) μM UTP. Other reagents' concentrations were: 3Dpol: 1 μM, Sym/Sub A: 1 μM (= 0.5 μM duplex). Data from each UTP concentration were fit to a single exponential. Amplitudes and observed reaction rates are given in Table 1. (B) Observed reaction rates as a function of UTP concentration. Data were fit to a hyperbola. Kd and kpol are given in Table 1.
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Figure 2: Single turnover analysis (rapid quench) of UTP (correct) incorporation into SSA. (A) Product yield as a function of time for three concentrations of UTP. A series of single turnover, rapid quench reactions were performed and analyzed described in Methods. Product concentration of resulting 11 mer (in nM) as a function of time in seconds is shown for 75 (circles), 150 (squares) and 500 (triangles) μM UTP. Other reagents' concentrations were: 3Dpol: 1 μM, Sym/Sub A: 1 μM (= 0.5 μM duplex). Data from each UTP concentration were fit to a single exponential. Amplitudes and observed reaction rates are given in Table 1. (B) Observed reaction rates as a function of UTP concentration. Data were fit to a hyperbola. Kd and kpol are given in Table 1.

Mentions: For each correct incorporation reaction, a pre-steady state kinetic analysis was conducted under single turnover, rapid quench conditions, in which enzyme was in slight excess of RNA substrate. Confirmation of enzyme excess was determined as described in Methods. In correct reactions, only 11 mers were produced. Quantified product yield at each timepoint (7–10 points under 1 second) was used to derive amplitude and observed reaction rate (kobsd) by fitting data to a single exponential. (shown for Sym/Sub A with UTP in Fig 2A). A series of these pre-steady reactions were then performed at various NTP concentrations (Table 1). With increasing NTP concentration, observed reaction rates increased. Observed reaction rates were then plotted as a function of NTP concentration and data fit to a hyperbola to derive Kd and kpol (Fig. 2B and Table 1). The values obtained for incorporation of UMP into Sym/Sub A appeared similar to published values for 3Dpol. We performed correct incorporation measurements for the four correct reactions as controls for incorrect measurements. However, detailed experiments were not pursued since these values have already been reported. [15,19].


Biochemical characterization of the fidelity of poliovirus RNA-dependent RNA polymerase.

Freistadt MS, Vaccaro JA, Eberle KE - Virol. J. (2007)

Single turnover analysis (rapid quench) of UTP (correct) incorporation into SSA. (A) Product yield as a function of time for three concentrations of UTP. A series of single turnover, rapid quench reactions were performed and analyzed described in Methods. Product concentration of resulting 11 mer (in nM) as a function of time in seconds is shown for 75 (circles), 150 (squares) and 500 (triangles) μM UTP. Other reagents' concentrations were: 3Dpol: 1 μM, Sym/Sub A: 1 μM (= 0.5 μM duplex). Data from each UTP concentration were fit to a single exponential. Amplitudes and observed reaction rates are given in Table 1. (B) Observed reaction rates as a function of UTP concentration. Data were fit to a hyperbola. Kd and kpol are given in Table 1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1904441&req=5

Figure 2: Single turnover analysis (rapid quench) of UTP (correct) incorporation into SSA. (A) Product yield as a function of time for three concentrations of UTP. A series of single turnover, rapid quench reactions were performed and analyzed described in Methods. Product concentration of resulting 11 mer (in nM) as a function of time in seconds is shown for 75 (circles), 150 (squares) and 500 (triangles) μM UTP. Other reagents' concentrations were: 3Dpol: 1 μM, Sym/Sub A: 1 μM (= 0.5 μM duplex). Data from each UTP concentration were fit to a single exponential. Amplitudes and observed reaction rates are given in Table 1. (B) Observed reaction rates as a function of UTP concentration. Data were fit to a hyperbola. Kd and kpol are given in Table 1.
Mentions: For each correct incorporation reaction, a pre-steady state kinetic analysis was conducted under single turnover, rapid quench conditions, in which enzyme was in slight excess of RNA substrate. Confirmation of enzyme excess was determined as described in Methods. In correct reactions, only 11 mers were produced. Quantified product yield at each timepoint (7–10 points under 1 second) was used to derive amplitude and observed reaction rate (kobsd) by fitting data to a single exponential. (shown for Sym/Sub A with UTP in Fig 2A). A series of these pre-steady reactions were then performed at various NTP concentrations (Table 1). With increasing NTP concentration, observed reaction rates increased. Observed reaction rates were then plotted as a function of NTP concentration and data fit to a hyperbola to derive Kd and kpol (Fig. 2B and Table 1). The values obtained for incorporation of UMP into Sym/Sub A appeared similar to published values for 3Dpol. We performed correct incorporation measurements for the four correct reactions as controls for incorrect measurements. However, detailed experiments were not pursued since these values have already been reported. [15,19].

Bottom Line: To derive kpol/Kd for correct base incorporation, we performed conventional pre-steady state single turnover measurements, yielding values that range from 0.62 to 9.4 microM-1 sec-1.Overall, we found that fidelity for poliovirus polymerase 3Dpol ranges from 1.2 x 10(4) to 1.0 x 10(6) for transition mutations and 3.2 x 10(5) to 4.3 x 10(7) for transversion mutations.Based on unusual enzyme behavior that we observed, we speculate that RNA mismatches either directly or indirectly cause enzyme RNA dissociation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology, Immunology and Parasitology; Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112, USA. mfreis@tulane.edu

ABSTRACT

Background: Putative high mutation rates of RNA viruses are believed to mediate undesirable phenomena, such as emergence of drug resistance. However, very little is known about biochemical fidelity rates for viral RNA-dependent RNA polymerases. Using a recently developed in vitro polymerase assay for poliovirus polymerase 3Dpol [Arnold and Cameron (2000) JBC 275:5329], we measured fidelity for each possible mismatch. Polymerase fidelity, in contrast to sequence error rate, is biochemically defined as kpol/Kd of {(correct plus incorrect) divided by incorrect} incorporations, such that a larger value connotes higher fidelity.

Results: To derive kpol/Kd for correct base incorporation, we performed conventional pre-steady state single turnover measurements, yielding values that range from 0.62 to 9.4 microM-1 sec-1. Pre-steady state measurements for incorrect base incorporation were less straightforward: several anomalous phenomena interfered with data collection. To obtain pre-steady state kinetic data for incorrect base incorporation, three strategies were employed. (1) For some incorrect bases, a conventional approach was feasible, although care was taken to ensure that only single turnovers were being assessed. (2) Heparin or unlabeled RNA traps were used to simulate single turnover conditions. (3) Finally, for some incorrect bases, incorporation was so poor that single datapoints were used to provide kinetic estimates. Overall, we found that fidelity for poliovirus polymerase 3Dpol ranges from 1.2 x 10(4) to 1.0 x 10(6) for transition mutations and 3.2 x 10(5) to 4.3 x 10(7) for transversion mutations.

Conclusion: These values are unexpectedly high showing that high RNA virus sequence variation is not due to intrinsically low polymerase fidelity. Based on unusual enzyme behavior that we observed, we speculate that RNA mismatches either directly or indirectly cause enzyme RNA dissociation. If so, high sequence variation of RNA viruses may be due to template-switch RNA recombination and/or unknown fitness/selection phenomena. These findings may lead to a mechanistic understanding of RNA virus error catastrophe and improved anti-viral strategies.

Show MeSH
Related in: MedlinePlus