Structural and biochemical studies of HCMV gH/gL/gO and Pentamer reveal mutually exclusive cell entry complexes

Abstract

Human cytomegalovirus (HCMV) is a major cause of morbidity and mortality in transplant patients and the leading viral cause of birth defects after congenital infection. The glycoprotein complexes gH/gL/gO and gH/gL/UL128/UL130/UL131A (Pentamer) are key targets of the human humoral response against HCMV and are required for HCMV entry into fibroblasts and endothelial/epithelial cells, respectively. We expressed and characterized soluble forms of gH/gL, gH/gL/gO, and Pentamer. Mass spectrometry and mutagenesis analysis revealed that gL-Cys144 forms disulfide bonds with gO-Cys351 in gH/gL/gO and with UL128-Cys162 in the Pentamer. Notably, Pentamer harboring the UL128-Cys162Ser/gL-Cys144Ser mutations had impaired syncytia formation and reduced interference of HCMV entry into epithelial cells. Electron microscopy analysis showed that HCMV gH/gL resembles HSV gH/gL and that gO and UL128/UL130/UL131A bind to the same site at the gH/gL N terminus. These data are consistent with gH/gL/gO and Pentamer forming mutually exclusive cell entry complexes and reveal the overall location of gH/gL-, gH/gL/gO-, and Pentamer-specific neutralizing antibody binding sites. Our results provide, to our knowledge, the first structural view of gH/gL/gO and Pentamer supporting the development of vaccines and antibody therapeutics against HCMV.

Keywords: HCMV; Pentamer complex; gH/gL/gO; human cytomegalovirus; virus entry.

Fig. 1.

Analyses of gH/gL, gH/gL/gO, and Pentamer complexes. (A) SEC of affinity-purified gH/gL shows two peaks indicative of gH/gL homodimer and gH/gL monomer. SDS/PAGE analysis shows that DTT is required to completely disrupt the gH/gL homodimer, suggesting that disulfide bonds may cross-link two gH/gL heterodimers. (B) SEC of affinity-purified gH/gL/gO shows one major peak. Both heat and DTT are needed to dissociate the gH/gL/gO complex into gH, gL, and gO. (C) SEC of Pentamer shows one peak. SDS/PAGE has three distinct bands: one composed of gH/gL/UL128 held together by internal disulfide bonds and two others composed of either UL130 or UL131A. Heat and reducing agents disrupt gH/gL/UL128 and free UL128 has a similar size to UL131A. Individual protein identity was confirmed by N-terminal sequencing.

Fig. 2.

Anti-gL and -UL128 western blot analyses of purified mutant complexes under nonboiling and nonreducing conditions. (A) In the presence of SDS, purified WT gH/gL runs as a homodimer and monomer, whereas gH/gL–Cys-144–Ser mutant does not form homodimers. (B) The gH/gL/gO complex was affinity-purified by means of the Strep tag at the gO C terminus. The gL–Cys-144–Ser mutation completely abolishes the covalent interaction between gO and gH/gL. (C) Pentamer complexes purified by Strep affinity (Strep-tagged gH) showed that mutation of gL–Cys-144–Ser or UL128–Cys-162–Ser interferes with the covalent interaction between gL and UL128.

Fig. 3.

Effect of gL–Cys-144–Ser and UL128–Cys-162–Ser mutations on HCMV interference and cell fusion. (A) HCMV interference assay. HCMV IE expression in ARPE-19 epithelial cells transduced with replication-incompetent AdVs encoding individual HCMV VR1814 Pentamer subunits. HCMV WT Pentamer (black bar) interferes with subsequent HCMV VR1814 infection, whereas introduction of gL–Cys-144–Ser (green) or UL128–Cys-162–Ser (purple) single mutations attenuate interference. Transduction of gL–Cys-144–Ser/UL128–Cys-162–Ser double-mutant Pentamer (blue) completely abolishes HCMV interference. Bar graphs are normalized to LacZ and represent four independent experiments. Error bars represent SEM. *P < 0.05; **P < 0.01; ns, not significant. (B) Syncytia assay. Representative immunofluorescent images of ARPE-19 cells transduced with HCMV VR1814 glycoproteins are shown. Cytoplasm, whole-cell staining; DAPI, nuclear DNA staining with 4',6-diamidino-2-phenylindole; merge, combined cytoplasmic and nuclear staining together. Images represent four independent transductions. (C) Average Pentamer surface expression measured by FACS using MSL109, 10P3, or Cytotect antibodies is plotted as mean fluorescent intensity (MFI) for cells from A. MFI values represent four independent transductions. Error bars represent SEM. Color scheme and abbreviations are the same as in A.

Fig. 4.

Architecture of gH/gL, gH/gL/gO, and Pentamer alone and bound to MSL-109 Fab. (A) Stained 2D class averages of gH/gL homodimer alone (Left), bound to MSL-109 (Center), and model of the complex (Right). (B) Class averages of gH/gL/gO alone (Left), bound to MSL-109 (Center), and model of the complex (Right). (C) Class averages of Pentamer alone (Left), bound to MSL-109 (Center), and model of the entire complex (Right). The 2D analysis shows that both gO and the ULs bind the N-terminal region of gH/gL, consistent with gL–Cys-144 forming an intermolecular disulfide bond in each of the three complexes. The boot-like architecture of gH/gL is conserved in these complexes.

Fig. 5.

RCT reconstruction of gH/gL/gO/MSL-109 and Pentamer/MSL-109 complexes and epitope mapping of gH/gL/MSL-109 by HDX-MS. (A and B) RCT reconstruction was used to determine gH/gL/gO/MSL-109 (A) and Pentamer/MSL-109 (B) complex structures. (A and B, Upper) For both samples, the 3D model (Right) is shown alongside a 2D reference free class average (Left) and 3D model projection (Center). (A and B, Lower) Both RCT structures show the interaction between gO and ULs and the gH/gL N-terminal region. (C) HDX-MS analysis reveals MSL-109 gH/gL binding site at the amino acid level. Specifically two peptides are identified in the gH C-terminal region.

Fig. 6.

gH/gL/gO and Pentamer form mutually exclusive complexes exposing neutralizing sites. gH/gL complex interacts either with gO or with the ULs to form gH/gL/gO and Pentamer, respectively. Two major regions for neutralizing antibody binding can be identified: (i) the gH component (in light blue), which is targeted by antibodies neutralizing infection of both fibroblasts and endothelial /epithelial cells; and (ii) the UL region contains the binding sites for potently neutralizing antibodies of epithelial and endothelial cells infection. The latter region likely includes the receptor binding site for entry in epithelial/endothelial cells.