FIG 6.

Stem-loop B (SLB) of WNV is destabilized by AUF1 p45. (Top left) Scheme of fluorescence-based assay to detect AUF1 p45-mediated conformational rearrangement of WNV SLB by dequenching of Cy5. (Top right) The assay was performed with Cy5- and BHQ-labeled SLB incubated with the indicated concentrations of recombinant AUF1 p45 or AUF1 p45^aDMA. Following the addition of 3′UAR RNA, the fluorescence signals were measured, plotted as a function of time, and fitted according to a first-order reaction (no protein; equation 1) or second-order reaction (in the presence of protein; equation 2). When we performed the assay in the presence of 50 nM AUF1 p45 but without the complementary 3′UAR RNA, an increase of fluorescence was not detected. (Bottom left) Observed rate constants (k_obs) for the WNV 5′SLB-3′UAR interaction in the absence and presence of different concentrations of AUF1 p45 and AUF1 p45^aDMA. (Bottom right) The observed rate constants (k_obs) that were measured for the RNA-RNA interaction in the presence of the indicated concentrations of recombinant AUF1 p45, AUF1 p45^aDMA, or a control protein, hnRNPH1, were plotted as a function of protein concentration.