Figure 1.
Solution structure of the core domain of RSV M2-1.
(A) Cartoon representation of the NMR structure of the α-helical domain of M2-158–177 (model of lowest energy). The color is ramped from blue to red from the N- to the C-terminus. (B) Electrostatic surface potential of M2-158–177 calculated with DELPHI [58] using PARSE parameters [59]. Two opposite faces of the protein are shown. The left-hand view is the same as for panel A. Colors for charges are red to blue for potential energies −6 to +6 kBT. Basic residues belonging to the main cluster are labeled in black bold letters. Basic residues belonging to the 3 minor clusters are indicated in blue, green and purple letters. (C) Schematic representation of the tetramer of full-length M2-1.
Table 1.
Apparent M2-158–177:oligonucleotide dissociation constants determined from amide (1H and 15N), methyl (1H and 13C) and R151-Hδ chemical shift variations (at 14.1 T and 293 K).
Figure 2.
Chemical shift perturbations of M2-158–177 by RNA reveal a continuous RNA binding surface.
Panels A, B and C show close-ups of the superposition of HSQC spectra of 15N13C-labeled M2-158–177 (50 µM, 14.1 T, 293 K) in the presence of increasing amounts of the synthetic short polyA RNA (5′-CCAAAAAAAU-3′). The spectra are shown in colors ranging from red to purple with 0/0.1/0.2/0.4/0.6/0.8/1.0/1.5/2.5/4.0/6.0 RNA equivalents. (A) 1H-15N HSQC spectrum, (B) methyl region of the 1H-13C HSQC spectrum and (C) arginine side chain region of 1H-13C HSQC. (D) Per residue difference plot of weighted averaged chemical shifts Δδ1H15N of 15N-M2-158–177 in the presence of short polyA determined with a 6∶1 RNA∶protein molar ratio. Bars are color coded from red to blue: lowest to highest value. The mean value and mean+1 standard deviation (sd) are indicated with solid and dashed lines. Panels (E) and (F) show the mapping of chemical shift variations (Δδ1H15N) on the M2-158–177 structure, in cartoon and surface representation respectively. Amide 15N atoms of residues with Δδ1H15N>mean+1sd for all tested RNA sequences (recapitulated in Table 1) are indicated with red spheres. Side chains of R151 and K92, for which RNA induces 13Cδ-1Hδ and 13Cε-1Hε chemical shift variations, are in stick representation. The surface formed by all these residues is colored in red. P153, for which no information is available from 1H-15N HSQC data, is shown in dark grey.
Figure 3.
Probing phosphoprotein P binding to M2-158–177 by NMR perturbation experiments.
(A, B, C and D) Results of transferred cross-saturation (TCS) experiments carried out with 150 µM 2H15N-M2-158–177 in the presence of P (15 µM, 91% D2O, 14.1 T, 293 K). (A) Close-up of the 1H-15N HSQC spectra with methyl proton saturation of P (blue) and without (orange). (B) Per residue plot of reduced 1H-15N HSQC cross-peak intensities, calculated as a ratio between intensities with (Isat) and without (I0) methyl proton saturation and corrected by the reduced cross-peak intensities measured in the absence of P. (C) and (D) Mapping of the TCS effect on the structure. 15N atoms of residues with Δ(Isat/I0)>mean+1sd are shown as purple spheres. The surface formed by these residues is colored in purple. (E, F, G and H) 1H-15N HSQC cross-peak intensity perturbation experiments of 15N13C-M2-158–177 (50 µM) in the presence of P100–166 (100 µM, 14.1 T, 298 K). (E) Close-up of the 1H-15N HSQC spectra with P100–166 (blue) and without (red). (F) Per residue plot of the resulting cross-peak intensity reduction relative to the reference intensity. (G) and (H) Mapping of the intensity variations on the structure. 15N atoms are shown as spheres for residues with ΔI/I0>75% (blue) and >70% (cyan). The same color code is used for the surface representation.
Figure 4.
Effect of M2-1 mutations on RSV transcription and association with the nucleocapsid.
(A) Analysis of RSV specific M2-1-controlled transcription with WT and M2-1 substitution mutants. BSRT7/5 cells were transfected with RSV pP, pN, pL, and pM2-1 plasmids and an RSV specific minigenome containing the firefly luciferase reporter gene, together with p-β-Gal constitutively expressing β-galactosidase. Luciferase activity, measured 24 h after transfection, was normalized by β-galactosidase activity, and the luciferase activity gained with WT M2-1 set to 100%. The mean value and confidence intervals (error bars) result from 3 separate experiments performed in duplicate. A control was run without M2-1. (B) Expression of M2-1 mutant proteins in BSRT7 cells. Cells were co-transfected with plasmids encoding M2-1 mutants and N. Cell extracts were analyzed by Western blotting with rabbit polyclonal antibodies against M2-1 and N. Expression levels of M2-1 were normalized against N expression and compared to tubulin. (C, D) Colocalization studies of M2-1 with N-P complexes. Plasmids encoding N, P, and M2-1 mutants were transfected into BSRT7/5 cells. Immunofluorescence analysis was performed on cells fixed 24 h after transfection, by using rabbit polyclonal anti-N (1∶100) or anti-P (1∶500) and Alexa Fluor 594 goat anti-rabbit (1∶1000) antibodies, and mouse monoclonal anti-M2-1 (1∶40 dilution) and Alexa Fluor 488 goat anti-mouse (1∶1000) antibodies. Horizontal bars correspond to 10 µm.
Figure 5.
Effect of mutations affecting M2-1-controlled transcription on M2-158–177:RNA and M2-1:P complex formation in vitro.
(A and B) Electrophoretic mobility shift assay (EMSA) of M2-1:RNA complex formation. Eluted GST-M2-158–177 (WT and mutants selected using the minigenome assay, 100 µM final concentration) were incubated with yeast tRNA (∼50 µM final concentration) for 1 h at room temperature. (A) Complexes were resolved by agarose gel electrophoresis stained with ethidium bromide. (B) Proteins were revealed by amido black staining. M2-1 mutations are indicated above each lane. (C and D) GST pull-down of purified P by GST-M2-1 (WT and the same mutants as in A and B). (C) GST-M2-1 or GST were incubated alone (−) or in the presence of P (+), washed, and analyzed by SDS-PAGE. P was also run alone (lane P). (D) Coomassie blue-stained gels were scanned and M2-1:P binding was quantified using ImageJ software and corrected for nonspecific binding to GST. Errors were estimated to ±5%. (E) The surfaces formed by the 8 residues, for which mutants were analyzed, are indicated on the M2-1 structure according to their binding partner: in red (RNA binding) and blue (P binding).
Figure 6.
Structural alignment of RSV M2-158–177 and EBOV VP30CTD.
Cartoon representations of aligned RSV M2-158–177 (residues L74 to T172) and EBOV VP30CTD (pdb 2I8B, [38]). Disordered N- and C-termini are not shown. Structural alignment with M2-158–177 was generated by the Dali server [39] (Z-score = 5.7; rmsd = 3.9 Å; 92 aligned residues; 9% sequence identity). VP30CTD helix α7, which has no counterpart in M2-158–177, is not represented. Identical hydrophobic residues in helices α5 and α6 are represented with sticks for M2-158–177 and VP30CTD.