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

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.

Figure 1

RNA substrates used. (A) Sequence of "Symmetrical/Substrate U". Commercially synthesized RNA 10 mers were prepared and phosphorylated with isotopic [γ³²P] label (*) as described in Methods. After melting and annealing, the RNA molecules self-anneal to form a six base-pair duplex with identical 5' overhangs. For Sym/Sub U, the correct base at N + 1 position, A, and one incorrect base, U, is shown (incorporated bases shown in lighter type). (B) List of the four Sym/Subs used in this study. Four similar synthetic RNAs were used. The first template base is shown in bold, and the correct base indicated in the right-hand column.

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: 3D^pol: 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. K_d and k_pol are given in Table 1.

Figure 3

Incorrect incorporation into Sym/Sub U. (A) Apparent inhibition by high NTP concentrations. Products formed in the presence of 3000 (circles), 5000 (squares) and 6000 (triangles) μM GTP were quantified and plotted as a function of time in seconds. All products were included in the calculations for this graph. These data did not fit a single exponential: the line is provided only for improved viewing. (B) Apparent multiple turnovers with incorrect base. Quantified products (all) formed in the presence of 375 (circles), 700 (squares), 1500 (triangles) and 3000 (diamonds) μM UTP and plotted as a function of time in seconds. These data did not fit a single exponential: the line is provided only for improved viewing. (C) Formation of higher molecular weight products in the presence of incorrect, but not, correct NTP. Left five lanes: 1 μM Sym/Sub U incubated with 500 μM ATP (correct base) for the indicated times (in minutes). Right five lanes: 1 μM SSU incubated with 1500 μM UTP (incorrect base) for indicated time (minutes). Samples were denatured and electrophoresed in a 20% denaturing PAGE. Size of RNA species is indicated at the right of the gel. 10 mer is the starting material.

Figure 4

Traps to reduce multiple turnovers. (A) Titration of heparin inhibition of 3D^pol. 1 μM Sym/Sub U was incubated with 500 μM ATP, 1 μM 3D^pol and increasing amounts of heparin. Polymerase activity was assessed in 20% PAGE, as described in Methods. The data were fit to a single exponential curve for inhibition. (B) Apparent single turnover kinetics for 3D^pol with Sym/Sub U and incorrect base U in the presence heparin trap. 1 μM 3D^pol and 1 μM Sym/Sub U were incubated with 750 (squares) or 1500 μM UTP (circles) for the indicated time (in minutes) as described in Methods, except that 100 nM heparin was added immediately after reaction initiation. Data (from all products) were plotted as a function of time in seconds and fit to a single exponential (Table 2). (C) Apparent single turnover kinetics for 3D^pol with Sym/Sub U with incorrect base U in the presence of cold trap. 1 μM 3D^pol and 1 μM Sym/Sub U were incubated with 750 (squares) or 1500 μM UTP (circles) for the indicated time (in minutes) as described in Methods, except that ten-fold molar excess unlabeled Sym/Sub U was added immediately after reaction initiation. Data (from all products) were plotted as a function of time in seconds and fit to a single exponential (Table 2).

Figure 5

Simulation of Sym/Sub U with UTP. (A) Hypothesized mechanism used for simulation. E=Enzyme, R_n=Template/Primer RNA, ER_n=active complex. The nonvarying parameters were: ER_n: 500 nM, NTPs: 750 or 1500 μM, k₊₁ : 10 μM^-1 sec^-1 and k_off : 5 × 10⁴ sec^-1. In the simulation, which is based on the mechanism in [19]. k_pol and k_-1 (both in gray) were varied. (B) Data and simulated fit. Experimentally derived products formed with 750 (dark gray circles/blue) or 1500 μM (light gray circles/orange) UTP were plotted as a function of time (in seconds). Simulated fits (curves) were superimposed with same color scheme.