FIG. 4.

Minor truncations to the C-terminal end of PIV5 NP protein do not impair the formation of templates for minigenome reporter expression. (A) 293T cells were transfected to produce the indicated viral proteins. After metabolic labeling with ³⁵S-amino acids, cells were collected and Dounce-homogenized cell extracts were prepared. Cell extracts were placed at the tops of CsCl density gradients and centrifuged, and seven equal gradient fractions were collected. PIV5 proteins were collected from the fractions by immunoprecipitation, fractionated on SDS gels, and detected using a phosphorimager. (B) BSR-T7 cells, stably expressing T7 RNA polymerase, were transfected with a PIV5 minigenome plasmid that contains a Renilla luciferase reporter. Transcription by T7 polymerase results in the production of a negative-sense PIV5 minigenome. In addition to the minigenome plasmid, cells were transfected with additional plasmids to produce PIV5 P and L proteins, together with wt or truncated NP proteins, as indicated. An additional plasmid, encoding firefly luciferase, was included as a transfection efficiency control. At 24 h posttransfection, cell lysates were prepared and luciferase activities were measured using a luminometer. Relative luciferase activity was calculated as Renilla luciferase activity divided by firefly luciferase activity, normalized to the value obtained with wt NP protein. Values represent averages from three independent experiments, with error bars indicating standard deviations. (Inset) NP protein expression levels were verified from replicate transfections of BSR-T7 cells. After metabolic labeling of cells with ³⁵S-amino acids, lysates were prepared and subjected to immunoprecipitation using antibody specific to PIV5 NP protein, followed by SDS-PAGE and detection using a phosphorimager.