Research ArticleINFECTIOUS DISEASE

Structural basis for potent antibody-mediated neutralization of human cytomegalovirus

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Science Immunology  30 Jun 2017:
Vol. 2, Issue 12, eaan1457
DOI: 10.1126/sciimmunol.aan1457
  • Fig. 1 Crystal structure of HCMV Pentamer.

    Two 180°-rotated views of a cartoon (A) and surface (B) representation of Pentamer. gH, gL, UL128, UL130, and UL131A are colored and labeled in gray, green, magenta, yellow, and blue, respectively. Disulfide bonds are shown in (A) as black sticks, and Asn-linked oligomannose residues modeled using the GlyProt server (41) are shown as spheres colored in black, red, blue, and white for carbon, oxygen, nitrogen, and hydrogen atoms, respectively.

  • Fig. 2 Analysis of Pentamer interfaces.

    Interfaces of Pentamer with gH, gL, UL128, UL130, and UL131A are colored in gray, green, magenta, yellow, and blue, respectively. The N-terminal extension in gL is colored bright green, whereas only D-I of gH is shown for clarity. Starting with the top right and going clockwise, boxed subpanels show details of the interactions at the subunit interfaces of UL130-UL131A, gL-UL130, gH-gL, gL-UL128, and UL128-UL130-UL131A, with key residues depicted as sticks. Disulfides are shown as black sticks, and polar contacts are shown as dashed black lines. For gH/gL, only the interface involving the double disulfide between the respective N termini is shown.

  • Fig. 3 Pentamer–8I21 Fab and Pentamer–9I6 Fab interactions and structural superpositions.

    (A) Structure of the Pentamer–8I21 Fab complex, with 8I21 HC (H) and LC (L) colored in red and teal, respectively. The boxed region on the complex is shown on the right as an “open-book” view, revealing details of the Pentamer–8I21 Fab interface. Oligomannose residues linked to Asn85 and Asn118 of UL130 are shown as sticks. (B) Details of Pentamer–8I21 Fab interactions. Residues involved in direct H-bond interactions are shown with sticks, and direct bonds are depicted with blue dashes. (C) Structure of the Pentamer–9I6 Fab complex, with Pentamer depicted as cartoon and colored as in Fig. 1 and (A), and 9I6 HC and LC depicted as brown and light blue surfaces, respectively. A top view of the 9I6 epitope on Pentamer (boxed region) is shown as surface on the right and colored according to subunits (UL128, magenta; UL131A, blue), with residues contacting the HC and LC of 9I6 colored in brown and light blue, respectively. (D) gH-based superposition of the 4.0 Å–resolution (violet) and 3.0 Å–resolution (cyan) Pentamer–8I21 Fab and of the 9I6 (gold) and the 3.0 Å–resolution 8I21 (cyan) complexes viewed from the side and from the top. A UL130/ UL131A-based superposition of the 9I6 (gold) and the 3.0 Å–resolution 8I21 (cyan) Fab complexes is also shown in the upper right panel. Blue semitransparent arrows in (D) show the movements of secondary structure elements.

  • Fig. 4 Neutralizing epitopes and cell binding of Pentamer.

    EM maps of Pentamer-Fab complexes from (21) are shown as gray semitransparent isosurfaces, and crystal structures of Pentamer-Fab complexes described in this work are shown as cartoons and colored as in previous figures. Red surfaces mapped on the crystal structures show Pentamer peptides identified by HDX-MS that are closest to the relative Fab for 15D8 (A), 10F7 (B), 10P3 (C), and 2C12 (D) in the EM-fitted models. Red labels show the specific Fab for which the epitopes are shown as red surfaces. Peptides that are part of the epitopes are labeled below each fitting.

  • Fig. 5 Binding of Pentamer to cells.

    (A) Binding of Pentamer, gH/gL, Shiga toxin B (positive control), or TEV protease (negative control) to ARPE-19, HUVEC, and MRC5 cell surface. Abs, absorbance at 450 nm. (B) Effect of neutralizing antibodies on binding of Pentamer to HUVEC cell surface. The graph represents relative binding compared with the control sample with no antibodies (100%). (C) Two 180°-rotated surface views of Pentamer colored in white (gH), and four shades of gray from dark to light for gL, UL128, UL130, and UL131A. The location of the epitopes for neutralizing antibodies, as determined from the combination of x-ray crystal structures, NS-EM, and HDX-MS (this study), is depicted with colored surface patches (blue for site 1, red for site 2, green for site 3/7, pink for site 4/6, and orange for site 5). Additional epitopes located on gH and previously described (21) are mapped in cyan (site A; mAb 3G16), yellow (site B; mAb 13H11), and dark red (site C; mAb MSL-109). Glycosylation sites on the surface of Pentamer, modeled as oligomannose using the GlyProt server, are depicted with spheres, colored in black, red, blue, and white for carbon, oxygen, nitrogen, and hydrogen atoms, respectively. Notably, several of the neutralizing sites on the ULs are in the vicinity of, and potentially partially protected by, N-linked glycans.

  • Table 1 X-ray data collection and refinement statistics.

    Values in parentheses are for the outer shell. Rwork = ∑||F(obs)| − |F(calc)||/∑|F(obs)|. Rfree = Rwork but calculated for 5% of the total reflections, chosen at random, and omitted from refinement. APS, Advanced Photon Source; ESRF, European Synchrotron Radiation Facility; Rmsd, root mean square deviation.

    Pentamer–8I21 FabPentamer–9I6 Fab
    SeMetNativeNative
    Data collection statistics
    BeamlineAPS LS-CATESRF ID30A1 MASSIF-1APS LS-CAT
    Wavelength (Å)0.9780.9660.978
    Resolution range (Å)34.7–4.0 (4.1–4.0)49.4–3.0 (3.1–3.0)82.4–5.9 (6.1–5.9)
    Space groupP212121P212121I212121
    Unit cells a, b, and c (Å)124.41, 146.53, 190.94124.16, 145.42, 173.65148.25, 208.82, 267.97
    Total reflections442,847 (65,700)797,563 (72,320)87,283 (24,949)
    Unique reflections30,418 (4,347)62,329 (5,985)11,302 (3,153)
    Multiplicity14.6 (15.1)12.8 (11.8)7.7 (7.9)
    Completeness (%)100 (100)100 (100)100 (100)
    Mean I/sigma (I)14.0 (2.6)8.87 (1.07)8.6 (2.4)
    Wilson B-factor (Å2)113.69103.93327.76
    Rmerge0.149 (1.066)0.277 (3.28)0.129 (0.805)
    Rmeas0.160 (1.141)0.289 (3.432)0.134 (0.857)
    CC1/20.99 (0.898)0.995 (0.362)0.968 (0.869)
    Phasing*
    Figure of merit0.443
    Overall score75.49 ± 3.41
    R-factor32.71
    Map skew0.94
    Refinement
    Reflections used30,299 (2,910)62,185 (4,425)11,268 (2,653)
    Rwork0.2590.1900.256
    Rfree0.2760.2370.300
    Number of atoms13,70114,05714,004
    Macromolecules13,59213,92013,668
    Ligands/heterogens109179267
    Protein residues1,7061,7531,727
    Rmsd (bonds)0.010.010.01
    Rmsd (angles)1.231.181.29
    Ramachandran favored (%)919187
    Ramachandran outliers (%)2.11.73.6
    Rotamer outliers (%)141315
    Clashscore9.87.5512.67
    Average B-factor (Å2)121.0294.1675.52

    *Values from Phenix.autosol.

    †The following residues had poor or no electron density and were not included in the final 3.0 Å–resolution model of Pentamer–8I21 Fab complex: gH 539–545 and 609–612, UL130 25–48, and UL131A 100–104. For the 4.0 Å–resolution Pentamer–8I21 Fab complex, the following residues were excluded from the final model: gH 175–179, 539–545, and 608–612; UL130 25–51; UL131A 100–104; 8I21 Fab HC 135–148 and 192–201; and LC 124–132, 149–155, and 181–189. The following residues were excluded from the final model of the Pentamer–9I6 Fab complex: gH 539–545 and 609–612, UL130 26–51, UL131A 100–104, and 9I6 Fab HC 153–162 and 210–221.

    Supplementary Materials

    • immunology.sciencemag.org/cgi/content/full/2/12/eaan1457/DC1

      Fig. S1. Comparison of HCMV gH/gL with related herpesvirus gH/gL.

      Fig. S2. Surface charge distribution on Pentamer.

      Fig. S3. Sequence conservation in gH and gL of HCMV and EBV.

      Fig. S4. Sequence conservation in gH and gL among β-herpesviruses.

      Fig. S5. Superposition of UL128 and ULl30 N-terminal domains with the most structurally similar chemokines, as identified by DALI.

      Fig. S6. Cavities in the Pentamer structure.

      Fig. S7. Pepsin digestion coverage of gH, gL, UL128, UL130, and UL131A.

      Fig. S8. Mapping of neutralizing epitopes by HDX-MS.

      Fig. S9. Regions of Pentamer stabilized by Fab binding.

      Fig. S10. Electron density maps of Pentamer complexes with Fabs 8I21 and 9I6.

      Fig. S11. Binding of soluble Pentamer to cell surfaces.

      Table S1. Analysis of Pentamer subunit interfaces.

      Table S2. Analysis of the Pentamer–8I21 Fab interface.

      Table S3. Molecular determinants of Pentamer–8I21 Fab binding.

      Table S4. Pentamer epitope mapping by HDX-MS.

      Movie S1. Pentamer structure.

      Movie S2. Pentamer is flexible and adopts different conformations.

    • Supplementary Materials

      Supplementary Material for:

      Structural basis for potent antibody-mediated neutralization of human cytomegalovirus

      Sumana Chandramouli, Enrico Malito, TuongVi Nguyen, Kate Luisi, Danilo Donnarumma, Yi Xing, Nathalie Norais, Dong Yu, Andrea Carfi*

      *Corresponding authors. Email: andrea.carfi{at}valeratx.com

      Published 30 June 2017, Sci. Immunol. 2, eaan1457 (2017)
      DOI: 10.1126/sciimmunol.aan1457

      This PDF file includes:

      • Fig. S1. Comparison of HCMV gH/gL with related herpesvirus gH/gL.
      • Fig. S2. Surface charge distribution on Pentamer.
      • Fig. S3. Sequence conservation in gH and gL of HCMV and EBV.
      • Fig. S4. Sequence conservation in gH and gL among β-herpesviruses.
      • Fig. S5. Superposition of UL128 and ULl30 N-terminal domains with the most structurally similar chemokines, as identified by DALI.
      • Fig. S6. Cavities in the Pentamer structure.
      • Fig. S7. Pepsin digestion coverage of gH, gL, UL128, UL130, and UL131A.
      • Fig. S8. Mapping of neutralizing epitopes by HDX-MS.
      • Fig. S9. Regions of Pentamer stabilized by Fab binding.
      • Fig. S10. Electron density maps of Pentamer complexes with Fabs 8I21 and 9I6.
      • Fig. S11. Binding of soluble Pentamer to cell surfaces.
      • Table S1. Analysis of Pentamer subunit interfaces.
      • Table S2. Analysis of the Pentamer–8I21 Fab interface.
      • Table S3. Molecular determinants of Pentamer–8I21 Fab binding.
      • Table S4. Pentamer epitope mapping by HDX-MS.
      • Legends for movies S1 and S2

      Download PDF

      Other Supplementary Material for this manuscript includes the following:

      • Movie S1 (.avi format). Pentamer structure.
      • Movie S2 (.avi format). Pentamer is flexible and adopts different conformations.

      Files in this Data Supplement:

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