Fig. 5.

Mechanism of inhibition by Pus4. (A) The competitive binding of Pus4 and the BMV CP to SLD4. The purified BMV CP was printed on the glass slides as 12 arrays with quadruplicate per array. For each binding assay, duplicate arrays were incubated with Cy3-SLD4 and competitor proteins. The Cy3-SLD4 was premixed with BSA (0.2 mg/ml), Qri1, App1, or Pus4 at 1:1 or 2:1 (protein to SLD4) molar ratios. The median of fluorescence density (F532) was used as binding signal. Each data point was averaged from eight binding signals (±SEM). (B) The binding of Pus4 to SLD4 versus mSLD4. The purified Pus4 were printed on the glass slides as 12 arrays with quadruplicate per array. Each binding assay was performed in duplicate with Cy3-SLD4 or Cy3-mSLD4 at concentrations of 0.2, 0.5, 2.0, and 10 μM. The median of fluorescence density (F532) was taken as the binding signal. Each data point was averaged from binding signals of eight spots (±SEM). (C) The active site of Pus4 is not required for inhibition of the plus-strand BMV RNA3 and RNA4 accumulation. The Agrobacterium cultures expressing Pus4, mPus4, and App1 were infiltrated at an OD₅₉₅ of 0.5. (D) Electron micrographs of the uranyl acetate-stained images from BMV virion reassembly reactions performed in the presence of BSA or Pus4 at equal concentration to the CP. (Scale bar, 50 nm.) (Insets) Averaged images of 50 individual particles from the reassembly reactions. Numbers at the bottom of the micrographs are the means and one standard error from four independent micrographs of each reassembly reaction.