Characterization of influenza virus variants induced by treatment with the endonuclease inhibitor baloxavir marboxil

Figures

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  Figure 1. Chemical structure of baloxavir acid (BXA, active form) and baloxavir marboxil (prodrug form). The full chemical name of BXA is (12aR)-12-[(11 S)-7,8-difluoro-6,11-dihydrodibenzo[b,e] thiepin-11-yl]-7-hydroxy-3,4,12,12a-tetrahydro-1H-[1,4] oxazino[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione.

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  Figure 2. Replicative capacity of variant viruses with indicated AA substitutions in PA protein. Canine MDCK cells (A–C) or human RPMI2650 cells (D,E) were infected with WT or I38x viruses based on rgA/WSN/33 (H1N1) (A,D), rgA/Victoria/3/75 (H3N2) (B,E), or B/Maryland/1/59 (C,F). The culture supernatants were collected at the indicated time points and viral titers (TCID50/mL) were determined in MDCK cells. Each symbol represents the mean and standard deviation of triplicate experiments. The lower limit of quantification of the virus titers was indicated by a dashed line.

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  Figure 3. In vitro endonuclease activity and inhibition of PA variants and thermal stabilization induced by the binding of BXA. (A) For endonuclease activity (lane marked - in all panels), FAM-RNA was incubated with either 0.5–5 μM PA-A-WT, 5–50 μM PA-A-I38T, 5–50 µM PA-B-WT or 50–500 µM PA-B-I38T. Reactions were carried out for 2 hours in the presence of 2.5 mM MnCl2 at 37 °C and were stopped by adding 1 mM EDTA. For inhibition assays, all four proteins were pre-incubated with increasing concentrations of BXA for 30′ at RT and then tested for activity. In all cases, four compound concentrations were tested, maintaining the molar ratio to protein equal to 0.1–0.2–0.5–1 (in all panels, WT: lanes 4–7, I38T: lanes 9-12). The reaction products were loaded on an 8 M 20% acrylamide gel and detected at 520 nm using standard emission filter in Gel Doc™ XR+ (BioRad). Images were processed using Image Lab software (v 6.0.0.25, BioRad) as detailed in Methods. Gels were cropped (removal of gel wells and band-less edges) for clarity. Black triangles (▼) compare the RNase activity of WT and I38T forms at equal protein concentration for FluA (5 µM, left) and FluB (50 µM right). Red asterisks (*) indicate…

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  Figure 4. BXA binding to influenza A/H1N1 PA endonuclease. BXA interacts with (A) PA-A WT and (B) PA-A I38T by chelating the two manganese ions in the active site. Top panels represent the 3D structure of BXA-bound PA-A endonuclease, whereas the bottom ones are the corresponding 2D interaction ligplots. In the structures, both BXA (in TV red for PA WT and yellow for PA I38T) and the interacting side chains (in chartreuse green for WT and TV blue for I38T) are represented as sticks, Mn ions (purple) and water molecules (red) as spheres. In blue mesh, the omit Fo-Fc density map for BXA at 3.0 σ. In the ligplots, dash lines represent hydrogen bonds, red crowns hydrophobic interactions and red circles highlight the mutation site at position 38. Structural figures were prepared using Pymol (www.pymol.org), ligand interaction plots with LigPlot+ (EMBL−EBI).

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  Figure 5. BXA binding to influenza B/Memphis PA endonuclease As Fig. 4, but for (A) PA-B WT and (B) PA-B I38T. Top panels: 3D structures of BXA-bound PA-B. Bottom panels: corresponding 2D interaction ligplots. Both BXA (in green for PA WT and light magenta for PA I38T) and the interacting side chains (in teal for WT and orange for I38T) are represented as sticks, Mn ions (purple) and water molecules (red) as spheres. The protein main chain is drawn as a cartoon. Represented by the blue mesh is the omit Fo-Fc density map for BXA at 3.0 σ. In the ligplots, dash lines represent hydrogen bonds and red crowns hydrophobic interactions. Red circles highlight the mutation site, blue circles the additional hydrophobic contacts that characterize binding of BXA to PA-B. Ligand interaction plots were prepared with LigPlot+ (EMBL-EBI).

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  Figure 6. Comparison of PA endonuclease from Flu A and Flu B bound to BXA in either WT or I38T form. Superposition of PA-BXA complexes: (A) PA-A WT and PA-A I38T, (B) PA-B WT and PA-B I38T, (C) PA-A WT and PA-B WT, (D) PA-A I38T and PA-B I38T. Complexes PA/BXA are represented in sticks and coloured in chartreuse/TV red for A WT, TV blue/yellow for A I38T, teal/green for B WT, orange/light magenta for B I38T. Manganese ions and water residues involved in protein-inhibitor interactions are depicted as spheres, in purple and red, respectively. Protein main chain is represented as a cartoon. Highlighted in red rectangles are the mutation sites. The binding of the inhibitor does not differ significantly among the four protein constructs, which share high conservation in the local network of interactions with BXA, except locally when residue 38 is mutated. Additional contacts in the case of Influenza B are shown in blue rectangles in B, C and D.

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  Figure 7. Local interactions of residue 38 in apo- and BXA-bound FluB PA (A) Superposition of ligand-free PA-B WT (PDB:5FML, in hotpink) and bound to BXA (green sticks for BXA, teal sticks/cartoon for PA). (B) Superposition of ligand-free (forest green) and BXA-bound PA-B I38T (light magenta sticks for BXA, orange sticks/cartoon for PA). For the WT, BXA binding does not alter the disposition of Ile38 and neighbouring Met34. For the I38T mutant, both side-chains change rotamer and the contacts with both the main chain and the compound hydrophobic pocket are altered.