Fig 7.
PI constitutes the site of interaction with the PCTD. (A) Minigenome assay. Normalized luciferase activity was determined for the I53 and R132 mutants. Error bars represent standard deviations calculated based on data from three independent experiments performed in triplicate. (B) Observation of cellular colocalization between P and N mutants in cytoplasmic inclusion bodies. N and P proteins were coexpressed in BSRT7/5 cells; cells were then fixed at 24 h posttransfection and labeled with anti-P (green) and anti-N (red) antibodies; and the distribution of viral proteins was observed by fluorescence microscopy. Nuclei were stained with Hoechst 33342. Scale bars, 10 μm. (C) Western blot analysis of products of immunoprecipitation assays preformed with an anti-HA antibody on cells coexpressing HA-P and N mutant proteins. (D) Analysis of GST pulldown assays performed with GST-PCTD and I53 and R132 mutant recombinant N proteins assembled as N-RNA rings. (E) Far-UV CD spectra showing the absence of major conformational changes between wild-type N (black) and the R132A (light gray) or I53W (gray) mutant.