title: 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.
authors.affiliation: Novartis Vaccines, Cambridge, MA 02139;
authors.forename: Claudio
authors.initials: C
authors.lastname: Ciferri
authors.affiliation: Novartis Vaccines, Cambridge, MA 02139;
authors.forename: Sumana
authors.initials: S
authors.lastname: Chandramouli
authors.affiliation: Novartis Vaccines, 53100 Siena, Italy;
authors.forename: Danilo
authors.initials: D
authors.lastname: Donnarumma
authors.affiliation: Novartis Institutes for Biomedical Research, Emeryville, CA 94608;
authors.forename: Pavel A
authors.initials: PA
authors.lastname: Nikitin
authors.affiliation: Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138; and Department of Cell Biology, Harvard Medical School, Boston, MA 02115.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Cianfrocco
authors.affiliation: Novartis Vaccines, Cambridge, MA 02139;
authors.forename: Rachel
authors.initials: R
authors.lastname: Gerrein
authors.affiliation: Novartis Institutes for Biomedical Research, Emeryville, CA 94608;
authors.forename: Adam L
authors.initials: AL
authors.lastname: Feire
authors.affiliation: Novartis Vaccines, Cambridge, MA 02139;
authors.forename: Susan W
authors.initials: SW
authors.lastname: Barnett
authors.affiliation: Novartis Vaccines, Cambridge, MA 02139;
authors.forename: Anders E
authors.initials: AE
authors.lastname: Lilja
authors.affiliation: Novartis Vaccines, 53100 Siena, Italy; rino.rappuoli@novartis.com andrea.carfi@novartis.com.
authors.forename: Rino
authors.initials: R
authors.lastname: Rappuoli
authors.affiliation: Novartis Vaccines, 53100 Siena, Italy;
authors.forename: Nathalie
authors.initials: N
authors.lastname: Norais
authors.affiliation: Novartis Vaccines, Cambridge, MA 02139;
authors.forename: Ethan C
authors.initials: EC
authors.lastname: Settembre
authors.affiliation: Novartis Vaccines, Cambridge, MA 02139; rino.rappuoli@novartis.com andrea.carfi@novartis.com.
authors.forename: Andrea
authors.initials: A
authors.lastname: Carfi
mesh_terms: D057134:Antibodies, Neutralizing / Q000276:immunology; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D002846:Chromatography, Affinity; D017124:Conserved Sequence / Q000235:genetics; D003587:Cytomegalovirus / Q000276:immunology* / Q000378:metabolism / Q000502:physiology*; D004220:Disulfides / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D007091:Image Processing, Computer-Assisted; D013058:Mass Spectrometry; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D008854:Microscopy, Electron; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism*; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000378:metabolism*; D053586:Virus Internalization
chemical_list: D057134:Antibodies, Neutralizing; D004220:Disulfides; D008562:Membrane Glycoproteins; D046912:Multiprotein Complexes; C077406:UL115 protein, Human herpesvirus 5; C532797:UL128 protein, human cytomegalovirus; C502890:UL130 protein, human cytomegalovirus; D014759:Viral Envelope Proteins; C071440:glycoprotein H, Cytomegalovirus; C439068:glycoprotein O, cytomegalovirus
keywords: HCMV; Pentamer complex; gH/gL/gO; human cytomegalovirus; virus entry