Fig. 2.
IFN-I binding properties of the variant E194 protein unable to interact with GAGs. a SPR sensorgrams and fitting obtained for the determination of the kinetic constants of the interaction of E194 and E194GAGmut recombinant proteins with mouse IFNαA. E194 or E194GAGmut were immobilised in a SPR Biacore SA sensor chip and binding and dissociation of several concentrations of mouse IFNαA at 30 μl/min were recorded and adjusted to a 1:1 Langmuir fitting (solid lines). The nanomolar concentration corresponding to each sensorgram is indicated. Kinetic parameters and calculated affinity constants of E194 and E194GAGmut are shown in the inset and compared to those previously described for the interaction of human IFNα-2b with VACV B18 and B18GAGmut (originally named IM15). b To determine the IFN-I blocking activities, in the preincubation assay (left), increasing amounts of recombinant proteins were incubated with 50 U of mouse IFNαA and the mixture added to L929 cells. After 16 h incubation, cells were infected with VSV and cell viability measured at 72 hpi. In the washing assay (right), cells were incubated with recombinant proteins, extensively washed, and then incubated with 50 U of mouse IFNαA as described for the previous assay. In both assays, untreated (IFN -) or IFN-I treated (IFN + ) cells, and also the addition of B18 recombinant protein were incorporated as controls. Data are means showing standard deviations of two independent experiments performed in triplicate