Table 1.
Crystallographic data collection and refinement statistics.
Fig 1.
Structural analysis of CTD binding to influenza B polymerase.
A. Overall view of the crystal structure of influenza B polymerase, with bound vRNA 5’ hook (violet) and CTD peptide mimic (slate blue spheres) in site 2B. Ribbon diagram of the polymerase with PA (green), PB1 (cyan), PB2-N (red), PB2-cap-binding (orange), PB2-midlink (magenta), PB2-627 (deep salmon). B. Overall view of the crystal structure of influenza B polymerase with bound promoter (violet and yellow), capped primer (blue) and CTD peptide mimic (slate blue spheres) bound in sites 1AB and 2B. The polymerase is coloured as in (A). The N and C-termini of the two CTD fragments are marked and the red dotted line shows the shortest connection between them with directionality indicated by the arrow. C. Overall view of the crystal structure of bat influenza A polymerase with bound promoter and CTD peptide mimic (slate blue spheres) bound in sites 1AB and 2A ([32], PDB: 5M3H). The colour code is as in (A). The N and C-termini of the two CTD fragments are marked and the red dotted line shows the shortest connection between them with directionality indicated by the arrow. D. Details of the interaction between key residues of the influenza B polymerase PA subunit (green), PB2-N (red) and PB2-627 (deep-salmon) with the CTD peptide (slate blue sticks) in site 2B. Three CTD repeats denoted a (orange), b (cyan) and c (dark green) are involved in this interaction. Hydrogen bonds are indicated as dotted green lines. E. Sequence alignment of the CTD binding sites in the A/WSN/33 (A0A2Z5U3X0) and B/Memphis/13/2003 (Q5V8X3) polymerase subunits PA and PB2. Protein sequences were obtained from UniProt (https://www.uniprot.org/) and aligned with SnapGene 6.0. Key residues for CTD binding are indicated in bold. Identical, similar and non-similar residues are highlighted in purple, light blue and orange, respectively. Grey boxes indicate residues that form a loop. Residues submitted to mutagenesis in this study are indicated with their numbers above (FluPolA) and below (FluPolB) the alignment, respectively.
Fig 2.
Gaussia princeps luciferase-based FluPol—CTD binding assay.
A-B. G2-CTD was expressed by transient transfection in HEK-293T cells together with PB2, PB1 and PA of FluPolB (B/Memphis/13/2003, blue bars) or FluPolA (A/WSN/1933, grey bars). Either PB2 (A, hatched bars) or PA (B, filled bars) were C-terminally tagged in frame with G1. As controls, the wild-type (wt) PB1 was replaced by the catalytic inactive PB1 D444A D445A mutant (i) or was omitted (-). Luciferase activities were measured in cell lysates at 24 hpt. Normalised luciferase ratios (NLRs) were calculated as described in the Materials and Methods section. The data shown are the mean ± SD of at least three independent experiments performed in technical triplicates. **p ≤ 0.002; ***p ≤ 0.001 (two-way ANOVA; Dunnett’s multiple comparisons test). C-D. The CTD binding of FluPolA mutants PA K289A and R638A was investigated. HEK-293T cells were transfected as described in (A) and (B), respectively. Relative Light Units (RLUs) are expressed as percentages relative to the FluPolA PA wt. The data shown are the mean ± SD of three independent experiments performed in technical triplicates. **p ≤ 0.002; ***p ≤ 0.001 (one-way ANOVA; Dunnett’s multiple comparisons test). The dotted line labelled “Ctrl” indicates the background signal i.e. the sum of the luminescence activities measured in control samples co-transfected with either the FluPol-G1 and G2 plasmids, or the G1 and G2-CTD plasmids. E. Schematic representation of the CTD constructs used in (F) and (G): the wild-type G2-CTD (wt, top) and the G2-CTD in which all serine 5 residues were replaced with an alanine (S5A, bottom). F-G. The interaction of the wt or the S5A mutated CTD to FluPolB (F) or FluPolA (G) was investigated by transient transfection in HEK-293T cells as described in (A-B). The data shown are the mean ± SD of four independent experiments performed in technical triplicates. *p ≤ 0.033; ***p ≤ 0.001 (two-way ANOVA; Sidak’s multiple comparisons test). In parallel, cell lysates were analysed by western blot using antibodies specific for the pS5 or pS2 CTD, G.princeps luciferase (Gluc) and tubulin. The slow- and fast-migrating bands detected with the pS2 CTD antibody, which likely correspond to the hyperphosphorylated and hypophosphorylated forms of the CTD, respectively, are indicated by a star and a triangle, respectively. The smeared signal in the G2-S5A-CTD samples likely corresponds to the detection of phosphorylation intermediates.
Fig 3.
FluPolB and FluPolA CTD-binding mode at site 1AB.
A. Superposition of the similar CTD binding in sites 1AB on the PA subunit for influenza B (B/Memphis/13/2003, green) and bat influenza A (A/little yellow-shouldered bat/Guatemala/060/2010(H17N10), light grey) polymerases with the CTD peptide as a thin tube (respectively slate blue and light grey). Key conserved residues are indicated in their respective colours, as well as the FluPolB-specific insertion (PA 608 loop) that is important for part of site 2B. See sequence alignment in Fig 1E. B. HEK-293T cells were transfected with the indicated FluPolA (A/WSN/1933) and FluPolB (B/Memphis/13/2003) site 1AB mutants, which were C-terminally tagged with the G1 fragment. Cells were lysed at 24 hpt and analysed by western blot using antibodies specific for G.princeps luciferase (PA-G1) and tubulin. The residue numbering corresponds to FluPolA (A/WSN/1933). C. In vivo CTD binding of the indicated mutants of FluPolA (A/WSN/1933, grey bars) and FluPolB (B/Memphis/13/2003, blue bars). The G2-tagged CTD was expressed by transient transfection in HEK-293T cells together with PB2, PB1 and PA-G1. RLUs are expressed as percentages relative to wt FluPolA/B. The data shown are mean ± SD of four independent experiments performed in technical triplicates. The dotted line labelled “Ctrl” indicates the background signal i.e. the sum of the luminescence activities measured in control samples co-transfected with either the FluPol-G1 and G2 plasmids or the G1 and G2-CTD plasmids. ***p ≤ 0.001 (two-way ANOVA; Dunnett’s multiple comparisons test). D. Characterisation of recombinant IAV (A/WSN/1933, grey dots) and IBV (B/Brisbane/60/2008, blue dots) viruses. Recombinant viruses with the indicated mutations were generated by reverse genetics as described in the Materials and Methods section. Reverse genetic supernatants were titrated on MDCK cells, stained at 72 hpi and plaque diameters were determined using the Fiji software. Each dot represents the diameter of a viral plaque relative to the mean plaque size of IAV wt or IBV wt recombinant virus. (#) not measurable pinhead-sized plaque diameter; (✞) no viral rescue. E. Polymerase activity of CTD-binding site 1AB mutants. FluPolA (A/WSN/1933, grey bars) or FluPolB (B/Memphis/13/2003, blue bars) was reconstituted in HEK-293T cells by transient transfection of PB2, PB1, PA, NP and a model RNA encoding the Firefly luciferase flanked by the 5’ and 3’ non-coding regions of the IAV or IBV NS segments, respectively. As an internal control, a RNA-Polymerase II promotor driven Renilla plasmid was used. Luminescence was measured at 24 hpt as described in the Materials and Methods section. Firefly activity was normalised to Renilla activity and is shown as percentages relative to wt FluPolA/B. The data shown are the mean ± SD of three independent experiments performed in technical duplicates. The dotted lines indicate the background signals in cells transfected with FluPolA or FluPolB plasmids minus the PB2A or PB2B plasmid, respectively. ***p ≤ 0.001 (two-way ANOVA; Dunnett’s multiple comparisons test).
Fig 4.
FluPolB and FluPolA CTD-binding mode at site 2A.
A. Superposition of CTD peptide (slate blue tube) bound at site 2A of the PA subunit of FluPolA (A/Zhejiang/DTID-ZJU01/2013(H7N9), green) with the equivalent region of FluPolB (B/Memphis/13/2003, wheat), showing similarities and differences in CTD interacting residues. See sequence alignment in Fig 1E. B-E. Protein expression (B), in vivo CTD binding (C), characterisation of recombinant IAV and IBV viruses (D) and polymerase activity (E) of CTD-binding site 2A mutants. Experiments were performed as described in Fig 3B–3E for FluPolB and FluPolA site 1AB mutations. C. ***p ≤ 0.001 (two-way ANOVA; Dunnett’s multiple comparisons test). D. (#) not measurable pinhead-sized plaque diameter; (✞) no viral rescue, (n.d.) not determined. E. ***p ≤ 0.001 (two-way ANOVA; Dunnett’s multiple comparisons test).
Fig 5.
FluPolA and FluPolB CTD-binding mode at site 2B.
A. Superposition of CTD peptide (slate blue tube) bound in site 2B of FluPolB (B/Memphis/13/2003, PA green, PB2-N red, PB2-627 deep salmon) with the equivalent region of FluPolA (A/NT/60/1968 (H3N2), [58], PDB: 6RR7, PA light green, PB2 pink), showing similarities and differences in CTD interacting residues. See sequence alignment in Fig 1E. B. Protein expression (left), in vivo CTD binding (middle), and polymerase activity (right) of FluPolB PA R608A. Experiments were performed as described in Fig 3. The data shown are mean ± SD of three independent experiments performed in technical triplicates. ***p ≤ 0.001 (unpaired t test).C-D. Protein expression (C) and in vivo CTD binding (D) of CTD-binding site 2B mutants. Experiments were performed as described in Fig 3B and 3C for FluPolB and FluPolA site 1AB mutations. D. **p ≤ 0.002, ***p ≤ 0.001 (two-way ANOVA; Dunnett’s multiple comparisons test). E. In vivo CTD binding of PB2 Δ627 domain deletion mutants was investigated. G2-CTD was expressed by transient transfection in HEK-293T cells together with PB2-G1, PB1 and PA of FluPolA (A/WSN/1933, grey bars) or FluPolB (B/Memphis/13/2003, blue bars). Luciferase activities were measured in cell lysates at 24 hpt. Normalised luciferase ratios (NLRs) were calculated as described in the Materials and Methods section. ***p ≤ 0.001 (two-way ANOVA; Sidak’s multiple comparisons test). Cell lysates were analysed in parallel by western blot with antibodies specific for the pS5 CTD, G. princeps luciferase (PB2-G1) and tubulin. F-G. Characterisation of recombinant IAV and IBV viruses (F) and polymerase activity (G) of CTD-binding site 2B mutants. Experiments were performed as described in Fig 3D-E for FluPolB and FluPolA site 1AB mutations. F. (✞) no viral rescue. G. ***p ≤ 0.001 (two-way ANOVA; Dunnett’s multiple comparisons test).
Fig 6.
Effect of CTD pS5 peptides on in vitro endonuclease and transcription activity of FluPolB and FluPolA.
A-B. CTD pS5 peptides of different lengths (two, four and six repeats) were added to (A) FluPolB (B/Memphis/13/2003) or (B) FluPolA (bat influenza A (A/little yellow-shouldered bat/Guatemala/060/2010(H17N10)) in vitro activity reactions as described in Materials and Methods. The left four lanes show endonuclease and right four lanes transcription reactions. Lanes 1 and 5: no CTD pS5 peptides. Lanes 2–6, 3–7 and 4–8: addition of two-, four- and six-repeat CTD pS5 peptides, respectively. The different RNA species are labeled. In the FluPolB samples, a secondary transcription product larger in size than those expected from the size of the template is detected, most likely resulting from stable hybridization of template and primary product as previously described [18]. Quantification of the reaction products of four independent experiments is shown below (FluPolB in blue and FluPolA in grey, respectively). The products of the reactions are normalised to the total RNA amount for each reaction and are presented as fractions of the activity of the reaction without peptide. *p ≤ 0.033, ***p ≤ 0.001 (one-way ANOVA; Dunnett’s multiple comparisons test).
Fig 7.
FluPolB and FluPolA binding to RPB1, RPB1ΔCTD and RPB2.
A. Binding of FluPolA (A/WSN/1933, grey bars) and FluPolB (B/Memphis/13/2003, blue bars) to RPB1, RPB1ΔCTD and RPB2 was evaluated. RPB1, RPB1ΔCTD and RPB2 were tagged with G2 and expressed by transient transfection in HEK-293T cells together with PB2, PB1 and PA-G1. Normalised luciferase activities (NLRs) were calculated as described in the Materials and Methods section. The data shown are the mean ± SD of five independent experiments performed in technical triplicates. *p ≤ 0.033, **p ≤ 0.002 (two-way ANOVA; Dunnett’s multiple comparisons test). Cell lysates were analysed in parallel by western blot with antibodies specific for the pS5 CTD, G. princeps luciferase and tubulin. Lanes 1 to 3 and 4 to 6 correspond to the co-transfection of FluPolA and FluPolB, respectively, with the G2-RPB1, -RPB1ΔCTD and -RPB2 plasmids as indicated. The band detected in lanes 2–3 and 5–6 with the pS5 CTD antibody correspond to the endogenous RPB1 protein. B-C. Binding of (B) FluPolB (B/Memphis/13/2003) and (C) FluPolA (A/WSN/1933) mutants in site 2B (PB2 K556/R555A), site 1 (PA K631A R634A/ PA K635A R638A) and site 2A (PA K450A / PA R454A) to RPB1, RPB1ΔCTD and RPB2 was evaluated as described in (A).