Kinetic analysis of the effect of poliovirus receptor on viral uncoating: the receptor as a catalyst

Abstract

We examined the role of soluble poliovirus receptor on the transition of native poliovirus (160S or N particle) to an infectious intermediate (135S or A particle). The viral receptor behaves as a classic transition state theory catalyst, facilitating the N-to-A conversion by lowering the activation energy for the process by 50 kcal/mol. In contrast to earlier studies which demonstrated that capsid-binding drugs inhibit thermally mediated N-to-A conversion through entropic stabilization alone, capsid-binding drugs are shown to inhibit receptor-mediated N-to-A conversion through a combination of enthalpic and entropic effects.

FIG. 1

Differential scanning calorimetry profile of sPvR. For details, see Materials and Methods. The bold line corresponds to sPvR in the sample chamber; the lighter line corresponds to conversion buffer only in the sample chamber.

FIG. 2

Rate constant determination for receptor-mediated transitions. The published experimental procedure (22) was modified to include sPvR at 0.25 μM. The plots show the natural logarithm of the concentration of unconverted 160S particle versus time. Each point is the average of three separate experiments; bars indicate the standard deviation of the average of the measurements. The data for the low-temperature reactions for all graphs were truncated for the sake of clarity of the higher-temperature data. Plots were generated using Microsoft Excel.

FIG. 3

(A) Sucrose gradient analysis of virus products from conversions. ³H-labeled P1/M (1.8 μg; ∼20,000 cpm) and 0.25 μM sPvr were incubated under the most extreme conditions for each compound tested: 0.1% DMSO, 200 s and 37°C (squares); R77975, 110 s and 43°C (open circles); and R78206, 110 s and 45°C (triangles). (B) Positions of 80S, 135S, and 160S markers on equivalent gradients. The gradients were fractionated from the top.

FIG. 4

Arrhenius plots for conversion in the presence and absence of sPvR. The averaged values of the natural logarithm of k from Table 1 were plotted as a function of −1/RT, so that the slope of each line is equivalent to E_a. Solid lines correspond to data collected in the presence of sPvR; dashed lines represent data collected in the absence of sPvR. No drug (0.1% DMSO), R77975, and R78206 are denoted by ◊, ○, and ▵, respectively. Temperature increases from left to right on the chart.

FIG. 5

Model of the reaction pathway for the N-to-A transition. (A) Reaction pathway for the uncatalyzed (thermally mediated) conversion. The pathway proceeds through a single transition state, N†, whose E_a is independent of drug binding (25). (B) Reaction pathway for the receptor-mediated conversion. Binding of the receptor to N produces an initial virus receptor complex, NR. The receptor-mediated reaction goes through an intermediate, the activated virus receptor complex N∗R. R77975 and R78206 raise the E_a of the transition state for this step, N†R, such that it becomes rate limiting. The horizontal dashed line represents the energy barrier for the uncatalyzed reaction, which is 145 kcal/mol.