Fig. 5: Functional role of the NβHL in PCT recognition.
A HMPV polymerase activity in the presence of mutations in N relative to strain NL/00/1, assessed by minigenome assay in BSRT7/5 cells. TM-A denotes a triple mutant with alanines in positions 100, 103, and 111. TM-E denotes a triple mutant with glutamates in positions 100, 103, and 111. Data points represent the values of quadruplicates from two independent experiments. Data are presented as mean values +/- SD. B GST-PCT and 6xHis-tagged N proteins (wt and mutants as in A) were co-expressed and co-purified using a GST-tag (one independent repeat of the purification was performed). A SDS-PAGE analysis of the purification products is shown. C SDS-PAGE analysis of N (wt and mutants as in A) purified via a 6xHis-tag, demonstrating that these are soluble (one independent repeat of the purification was performed). D Fluorescence microscopy of BSRT7/5 cells transfected with plasmids encoding a blue fluorescent protein (BFP)-tagged HMPV P and HMPV N (wt and mutants as in A). Cells were fixed 24 h post-transfection. N protein was stained by labeling with rabbit polyclonal anti-N antibody, and nuclei were stained with Hoechst 33342. Scale bars, 20 µm. The experiment was repeated independently twice with similar results. Source data are provided as a Source Data file.
