Structure and Functional Analysis of the RNA- and Viral Phosphoprotein-Binding Domain of Respiratory Syncytial Virus M2-1 Protein
Figure 5
Effect of mutations affecting M2-1-controlled transcription on M2-158–177:RNA and M2-1:P complex formation in vitro.
(A and B) Electrophoretic mobility shift assay (EMSA) of M2-1:RNA complex formation. Eluted GST-M2-158–177 (WT and mutants selected using the minigenome assay, 100 µM final concentration) were incubated with yeast tRNA (∼50 µM final concentration) for 1 h at room temperature. (A) Complexes were resolved by agarose gel electrophoresis stained with ethidium bromide. (B) Proteins were revealed by amido black staining. M2-1 mutations are indicated above each lane. (C and D) GST pull-down of purified P by GST-M2-1 (WT and the same mutants as in A and B). (C) GST-M2-1 or GST were incubated alone (−) or in the presence of P (+), washed, and analyzed by SDS-PAGE. P was also run alone (lane P). (D) Coomassie blue-stained gels were scanned and M2-1:P binding was quantified using ImageJ software and corrected for nonspecific binding to GST. Errors were estimated to ±5%. (E) The surfaces formed by the 8 residues, for which mutants were analyzed, are indicated on the M2-1 structure according to their binding partner: in red (RNA binding) and blue (P binding).