Phosphatase PP1 binds to the "RVxF" motif of RSV P.

<p>(A) Sequence alignment of <i>Pneumoviridae</i> P showing the conservation of the "RVxF" motif. Human RSV A strain (HRSV), bovine RSV (BRSV), ovine RSV (ORSV), pneumonia virus of mice (PVM), canine pneumovirus (CPV), swine orthopneumovirus (SOV), human metapneumovirus (HMPV) and avian metapneumovirus (AMPV) (accession codes AAX23990.1, NP_048051.1, Q83956.1, Q5MKM7.1, AHF88957.1, <a target="_blank">ANO40516.1</a>, <a target="_blank">YP_012606.1</a>, and <a target="_blank">AAF05910.1</a>, respectively) P sequences were aligned by Clustal Omega and prepared with ESPript 3. Numbers correspond to RSV P amino acid residues. (B) BSRT7/5 cells were co-transfected with pN and pHA-P or p-HA-P[F87A]. HA-P (WT and variant) was immunoprecipitated from cell lysates and the presence of PP1 in the precipitate was analyzed by Western blot. L, cell lysate; IP, immunoprecipitated products. (C) NMR analysis of GST-PP1α binding to RSV P. Intensity ratios (I/I₀), measured for each peak in ¹H-¹⁵N BEST-TROSY spectra of 25 μM ¹⁵N-labeled P[1–126] or ¹⁵N-labeled full-length P, alone and in the presence of 2 molar equivalents of GST-PP1α, are represented in the bar diagrams. Straight and broken lines indicate mean and mean ± rmsd values. Regions with transient α-helical or β-sheet secondary structure [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006920#ppat.1006920.ref044" target="_blank">44</a>] are highlighted by a colored background with the same color code as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006920#ppat.1006920.g001" target="_blank">Fig 1</a>. The hatched area corresponds to a P region encompassing the OD and C-terminal helices for which peaks cannot be observed in the free form. (D) Proteins GST-PP1, M2-1, P WT and F87A mutant were expressed in <i>E</i>. <i>coli</i> and purified separately. Beads coated with GST or GST-PP1 were saturated with 3% BSA, and incubated with, P WT or F87A mutant and washed. Complexes were pulled down, resolved by SDS-PAGE and stained with Coomassie blue.