FIG 4.
Effect of AZ-27 on L-P interaction. RSV L and P proteins were expressed in HEp-2 cells, and L-P complexes were purified by virtue of a V5 tag on the L protein. V5-L-P complexes were subjected to five washes with transcription buffer containing either DMSO (lanes 3 to 8) or 10 μM AZ-27 (lanes 9 to 14) such that the concentration of DMSO was equivalent in both conditions. L-P complexes remaining attached to the columns were eluted with heated SDS-PAGE lysis buffer (lanes 3 and 9); the wash fractions are shown in lanes 4 to 8 and 10 to 14. Aliquots of each sample were analyzed by Western blotting using an RSV-specific polyclonal antibody (Ab) to detect P (top; note that this antibody does not detect L) or a V5-specific antibody to detect V5-L (bottom). Lanes 1 and 2 show control samples in which only V5-L or untagged P were expressed in cells (lanes 1 and 2, respectively). The band at 55 kDa is the heavy chain of the V5 antibody. The bands smaller than that of full-length V5-L detected by the V5 antibody are truncated L proteins (note that the L gene used for this experiment was not codon optimized). This result is representative of three independent experiments.