FIG 7.

Accumulation of intracellular viral nucleocapsid inactivates PP5. (A) HEK293 cells were transfected with expression plasmids encoding MeV-N, -P, and -L genes (N, P, and L) or control empty plasmid, together with phRG-B as an internal reference. The following day, the cells were transfected with mock or negative-strand minigenomic RNA (minigenome). The cells were harvested at 24, 36, and 48 h after RNA transfection, and their lysates were subjected to luciferase activity analysis (upper panel), immunoblotting for MeV-N (middle panel), and an in vitro phosphatase assay with anti PP5 antibody (lower panel), as described above. (B) 293SLAM cells were transfected with N, P, and L expression plasmids and then transfected with mock or minigenomic RNA the following day. At 48 h after RNA transfection, cell lysates were immunoprecipitated with anti-PP5 or anti-PP2A antibody. Phosphatase activity in the immunocomplexes was determined as described above. (C) Nuclear extracts prepared from cells under the same conditions as panel B were subjected to EMSA. (D) The cell lysates from panel B and mock-treated cell lysates were immunoblotted to detect c-Myc, MeV-N, and GAPDH.