Structure of the extracellular domain of matrix protein 2 of influenza A virus in complex with a protective monoclonal antibody

Abstract

The extracellular domain of influenza A virus matrix protein 2 (M2e) is conserved and is being evaluated as a quasiuniversal influenza A vaccine candidate. We describe the crystal structure at 1.6 Å resolution of M2e in complex with the Fab fragment of an M2e-specific monoclonal antibody that protects against influenza A virus challenge. This antibody binds M2 expressed on the surfaces of cells infected with influenza A virus. Five out of six complementary determining regions interact with M2e, and three highly conserved M2e residues are critical for this interaction. In this complex, M2e adopts a compact U-shaped conformation stabilized in the center by the highly conserved tryptophan residue in M2e. This is the first description of the three-dimensional structure of M2e.

Importance: M2e of influenza A is under investigation as a universal influenza A vaccine, but its three-dimensional structure is unknown. We describe the structure of M2e stabilized with an M2e-specific monoclonal antibody that recognizes natural M2. We found that the conserved tryptophan is positioned in the center of the U-shaped structure of M2e and stabilizes its conformation. The structure also explains why previously reported in vivo escape viruses, selected with a similar monoclonal antibody, carried proline residue substitutions at position 10 in M2.

FIG 1

Structure of M2e in complex with Fab65. (A) (Left) Overall structure of the M2e (magenta) and Fab65 (green for light chain and blue for heavy chain) complex. (Top right) LCDR 1 (dark green) flanks M2e (magenta), contacting the 3₁₀ helix. (Bottom right) Top view with cartoon presentation of M2e (magenta) in complex with the heavy chain (blue) and light chain (green) of Fab65. (B) Surface representation of M2e bound to Fab65 viewed from the paratope toward M2e. M2e is color coded according to electrostatic potential. The color scale was set from −10 kT/e (red) to 10 kT/e (blue) (kT is the product of the Boltzmann constant, k, and the temperature, T; e is the electrostatic potential), as calculated by the APBS tool plugged into Pymol. LCDR and HCDR residues that interact with M2e are shown in stick presentation in green and blue, respectively. (C) Comparison of shape complementarity scores of different antibody-epitope complexes. Black bars represent shape complementarity (SC) scores deduced from cocrystalized influenza hemagglutinin HA and Fab fragments deposited in the PDB database. The red bar on the right indicates the SC of M2e/Fab65 (PDB code 4N8C).

FIG 2

Details of the main interactions between M2e and Fab65. (A) Closeup view of hydrophilic interactions between M2e-Glu6 and Fab65. Dotted lines represent hydrogen bonds and salt bridges. (B and C) Water-mediated interactions (B) and other hydrophilic interactions (C) between M2e amino acid residues (purple) and Fab65 amino acid residues (light chain in green and heavy chain in blue). (D) Hydrophobic interactions between M2e and MAb 65 with surface presentation of Fab65 (left) and M2e (right) color coded according to electrostatic potential. The color scale was set from −10 kT/e (red) to 10 kT/e (blue), as calculated by the APBS tool plugged into Pymol. (E) Reactivity of MAb 65 and 14C2 with M2 was analyzed by Western blotting of lysates of HEK-293T cells transfected with M2-Flag expression constructs carrying M2e-Ala mutants. Anti-Flag detection was used to normalize the amount of M2 in the cell lysates that were loaded. NC, negative control.

FIG 3

Binding of MAb 65 to cells infected with influenza A virus. (A) Confocal images of immunofluorescence staining of MDCK cells infected with influenza virus. M2 staining is shown in red fluorescence and was revealed using MAb 65 or MAb 148 (a TCN-031-like antibody that binds to the N terminus of M2). Goat polyclonal antibody against influenza virus ribonucleoprotein (BEI Resources, no. NR-3133) was used to stain viral RNPs (green). Cell nuclei were identified by DAPI staining (blue). (B) Amino acid sequence alignment of M2e from different influenza A viruses. The conserved Trp15 is in bold. (C) MAb 65 does not affect influenza A virus plaque size. PR8, WSN, A/USSR/77, and X31 viruses were used in a plaque assay on MDCK cells in the presence of antibodies, amantadine, or immune sera with virus neutralizing activity. The plaque assay was performed in a 96-well format, and the plaques were visualized by immune staining with goat anti-vRNP immune serum. MAb 65, MAb 148, and MAb 3G8 (an isotype-matched negative-control MAb) were used at a starting concentration of 10 μg/ml. Amantadine was used at a starting concentration of 100 μM. “Anti-HA” indicates goat antiserum against PR8 virus (BEI Resources, no. NR-3148) with strong neutralizing activity against PR8 and WSN viruses and some neutralizing activity against A/USSR/77 and X31 viruses. “Virus dilution” indicates serial 10-fold dilution of the amount of virus that was used to inoculate the cells in the presence of MAb, amantadine, or anti-HA serum, which were used as 10-fold serial dilutions, as indicated at the top.

FIG 4

Properties of binding of MAb 65 to M2e peptide and M2-expressing cells. (A) Binding of MAb 65 and MAb 148 to different M2e peptides. ELISA plates were coated with wild-type (WT), M2eW15G, or M2eP10L M2e peptides at 20 ng/ml or 200 ng/ml. (B) Cellular ELISA using HEK-293T cells that had been transfected with wild-type, M2Trp15Ala, or M2Pro10Ala M2-Flag expression vectors.

FIG 5

Structure of M2e. (A) (Top) M2eTrp15 occupies a central position in M2e. The distance between the main-chain oxygen of M2eLeu4 and the nitrogen of M2eGly16 is 3.1 Å (double arrow). (Bottom) The β-turn involving Thr5 to Glu8 in M2e (left) and a 3₁₀ helix from Ile11 to Trp15 (right). Dotted lines represent hydrophilic interactions. (B) The position and interactions of M2eTrp15 in M2e (top) resemble those of Trp in a Trp cage miniprotein (bottom). (C) Fourier transform infrared spectroscopy spectra of M2e peptides. Each spectrum was normalized and subtracted from the spectrum of the PBS buffer. Shown are FTIR spectra of wild-type M2e (red), M2eW15G (blue) and M2eP10L (purple) peptides. The data are averages of 256 recorded spectra with a resolution of 2 nm.

FIG 6

Recombinant PR/8 virus with M2-Trp15 substitution is highly attenuated. Wild-type PR/8 and PR/8 with M2 mutations that alter M2-Trp15 were generated by introducing the corresponding mutations in a plasmid-based reverse-genetics system. Sets of eight plasmids were transfected into HEK293T-MDCK cocultures to determine the expression of viral proteins and to try to rescue the virus. (A) Schematic representation of the codon usage and amino acid sequence near M2-Trp15 in M1 and M2 wild-type and mutant PR/8 viruses. (B) Western blot analysis of cell lysates prepared 24 h after transfection to evaluate expression of NP, M2 (revealed by staining with MAb 148 and MAb 65) and M1, as indicated. WT, wild type; NC, negative control (i.e., transfected with only pCAXL-PA, pCAXL-PB1, pCAXL-PB2, and pCAXL-NP); ST, mutant with stop codons introduced after M2-Asp24; NT, nontransfected. (C) Plaque assay to quantify rescued PR/8 viruses with introduced M1/M2Trp15 mutations. (D) Hemagglutinating activity in the supernatants of transfected HEK293T-MDCK cocultures. The supernatant and serial 3-fold dilutions were assayed for hemagglutination of chicken red blood cells on day 2 or 5 after transfection with the eight-plasmid system used for generating PR/8 virus.