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

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.Human cytomegalovirus (HCMV) is a member of the β-herpesvirus subfamily with >60% seropositivity in adults worldwide (1). HCMV infection is typically asymptomatic, but can cause severe disease or death in immunocompromised solid organ and hematopoietic stem cell transplant recipients. In addition, HCMV can infect the placenta and cross this barrier to infect developing fetuses, causing severe birth defects (2). Given the severity and importance of this disease, obtaining an effective vaccine is considered a public health priority (3).The ability of HCMV to cause disease in a wide range of organs and tissue types is reflected at the cellular level by the virus infecting epithelial cells, endothelial cells, fibroblasts, dendritic cells, hepatocytes, neurons, macrophages, and leukocytes (4). Similar to other herpesviruses, the envelope glycoproteins gB and gH/gL form the conserved fusion machinery required for viral entry (5, 6). Recent structural and mutagenesis analysis suggested that gB is responsible for mediating virus and host membrane fusion during viral entry (7, 8). The role of gH/gL in fusion is less clear because crystal structures of herpes simplex virus 2 (HSV-2), pseudo-rabies virus (PrV), and Epstein–Barr virus (EBV) gH/gL did not reveal any similarity to known viral fusion proteins (9–11). It has been proposed that gH/gL is involved in the entry process through activation of gB (12). In addition to gB and gH/gL, most herpesviruses encode additional glycoproteins that are able to interact with gH/gL and are capable of either mediating binding to specific cellular receptors or regulating the activity of the gH/gL–gB complex (5, 6).HCMV entry into both epithelial and endothelial cells requires a pentameric glycoprotein complex (Pentamer) formed between gH/gL and the UL128, UL130, and UL131A proteins (13, 14). Mutations in the UL131A–UL128 gene locus are sufficient to eliminate epithelial/endothelial tropism and occur spontaneously within only a few passages of wild-type (WT) HCMV in fibroblasts (15, 16). In addition, Pentamer cell surface overexpression interferes with HCMV entry into epithelial cells, but not into fibroblasts, suggesting the presence of a cell-type-specific Pentamer receptor (17).HCMV entry into fibroblasts is mediated by the gH/gL/gO complex at the cell surface at neutral pH (18–21). gO is a highly glycosylated protein and has been shown to covalently interact with gH/gL (22, 23). It has been proposed that gO might function as a molecular chaperone to promote gH/gL incorporation, but not gH/gL/gO, into the virion (21). However, it has been recently demonstrated that gH/gL/gO and Pentamer are much more abundant on the HCMV envelope than gH/gL alone (24).Highly potent HCMV-neutralizing monoclonal antibodies were isolated from the memory B-cell repertoire of HCMV-immune donors and shown to bind the Pentamer. These antibodies were capable of neutralizing HCMV infection of epithelial/endothelial cells, but not fibroblasts (25, 26). In addition, several studies have demonstrated that the Pentamer is the main target of the neutralizing humoral response to HCMV infection in epithelial/endothelial cells (27–29). Consistent with these observations, immunization with the Pentamer has been shown to elicit a strong neutralizing antibody response in mouse, rabbit, and rhesus macaque models (30–32). Together these data indicate that the Pentamer represents a key antigenic target for vaccine development against HCMV infection.Here we report the purification and biochemical characterization of HCMV gH/gL, gH/gL/gO, and Pentamer. In addition, we describe the architecture of these complexes by electron microscopy (EM) and characterize their interaction with MSL-109, a previously described HCMV-neutralizing antibody isolated from the spleen of a HCMV-seropositive individual (33, 34). Our data provide new insights into the structure and function of the HCMV gH/gL/gO and Pentamer complexes.