Journal Article

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

Ciferri, Claudio, Gerrein, Rachel, Barnett, Susan W., Donnarumma, Danilo, Norais, Nathalie, Carfi, Andrea, Rappuoli, Rino, Lilja, Anders E., Nikitin, Pavel A., Feire, Adam L., Chandramouli, Sumana, Settembre, Ethan C., Cianfrocco, Michael A.

2015

Overview

Significance Human cytomegalovirus (HCMV) is a major cause of morbidity and mortality in transplant patients and in fetuses after congenital infection. gH/gL/gO and Pentamer are targets for neutralizing antibodies. We show that gO and UL128/UL130/UL131A bind to the same site on gH/gL through formation of a disulfide bond with gL-Cys144. The alternative use of this binding site by either gO or the ULs may provide a mechanism for cell tropism modulation. Our analysis reveals that gH/gL antigenic sites are conserved among gH/gL, gH/gL/gO, and Pentamer, whereas gH/gL/gO- and Pentamer-specific neutralizing antibody-binding sites are located in the gH/gL N terminus protrusion that contains the gO and the UL subunits. These data support the development of vaccines and antibody therapeutics against HCMV. 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.

• Publisher: National Academy of Sciences
• Subject: Antibodies, Neutralizing - immunology / binding sites / Binding Sites - genetics / Biochemistry / Biological Sciences / Birth defects / Blotting, Western / Cells / Chromatography, Affinity / Conserved Sequence - genetics / Cytomegalovirus / Cytomegalovirus - immunology / Cytomegalovirus - metabolism / Cytomegalovirus - physiology / disulfide bonds / Disulfides - metabolism / fetus / Flow Cytometry / Human betaherpesvirus 5 / Humans / Image Processing, Computer-Assisted / Mass Spectrometry / Membrane Glycoproteins - chemistry / Membrane Glycoproteins - metabolism / Microscopy, Electron / morbidity / mortality / Multiprotein Complexes - chemistry / Multiprotein Complexes - metabolism / Mutagenesis / Mutagenesis, Site-Directed / Mutation - genetics / neutralization / neutralizing antibodies / patients / Protein Binding / Proteins / therapeutics / vaccine development / Viral Envelope Proteins - chemistry / Viral Envelope Proteins - metabolism / Virus Internalization