Structure of a Neutralizing Antibody Bound Monovalently to Human Rhinovirus 2

The structure of a complex between human rhinovirus 2 (HRV2) and the Fab fragment of neutralizing monoclonal antibody (MAb) 3B10 has been determined to 25-Å resolution by cryoelectron microscopy and three-dimensional reconstruction techniques. The footprint of 3B10 on HRV2 is very similar to that of neutralizing MAb 8F5, which binds bivalently across the icosahedral twofold axis. However, the 3B10 Fab fragment (Fab-3B10) is bound in an orientation, inclined at approximately 45° to the surface of the virus capsid, which is compatible only with monovalent binding of the antibody. The canyon around the fivefold axis is not directly obstructed by the bound Fab. The X-ray structures of a closely related HRV (HRV1A) and a Fab fragment were fitted to the density maps of the HRV2–Fab-3B10 complex obtained by cryoelectron microscope techniques. The footprint of 3B10 on the viral surface is largely on VP2 but also covers the VP3 loop centered on residue 3064 and the VP1 loop centered on residue 1267. MAb 3B10 can interact directly with VP2 residue 2164, the site of an escape mutation on VP2, and with VP1 residues 1264 to 1267, the site of a deletion escape mutation. Deletion of these residues shortens the VP1 loop, moving it away from the MAb binding site. All structural and biochemical evidence indicates that MAb 3B10 binds to a conformation epitope on HRV2.     

Crystal Structure of the Hendra Virus Attachment G Glycoprotein Bound to a Potent Cross-Reactive Neutralizing Human Monoclonal Antibody

The henipaviruses, represented by Hendra (HeV) and Nipah (NiV) viruses are highly pathogenic zoonotic paramyxoviruses with uniquely broad host tropisms responsible for repeated outbreaks in Australia, Southeast Asia, India and Bangladesh. The high morbidity and mortality rates associated with infection and lack of licensed antiviral therapies make the henipaviruses a potential biological threat to humans and livestock. Henipavirus entry is initiated by the attachment of the G envelope glycoprotein to host cell membrane receptors. Previously, henipavirus-neutralizing human monoclonal antibodies (hmAb) have been isolated using the HeV-G glycoprotein and a human naïve antibody library. One cross-reactive and receptor-blocking hmAb (m102.4) was recently demonstrated to be an effective post-exposure therapy in two animal models of NiV and HeV infection, has been used in several people on a compassionate use basis, and is currently in development for use in humans. Here, we report the crystal structure of the complex of HeV-G with m102.3, an m102.4 derivative, and describe NiV and HeV escape mutants. This structure provides detailed insight into the mechanism of HeV and NiV neutralization by m102.4, and serves as a blueprint for further optimization of m102.4 as a therapeutic agent and for the development of entry inhibitors and vaccines.     

Crystal Structure Analysis of DNA Uridine Endonuclease Mth212 Bound to DNA

The reliable repair of pre-mutagenic U/G mismatches that originated from hydrolytic cytosine deamination is crucial for the maintenance of the correct genomic information. In most organisms, any uracil base in DNA is attacked by uracil DNA glycosylases (UDGs), but at least in Methanothermobacter thermautotrophicus ΔH, an alternative strategy has evolved. The exonuclease III homologue Mth212 from the thermophilic archaeon M. thermautotrophicus ΔH exhibits a DNA uridine endonuclease activity in addition to the apyrimidinic/apurinic site endonuclease and 3′ → 5′exonuclease functions. Mth212 alone compensates for the lack of a UDG in a single-step reaction thus substituting the two-step pathway that requires the consecutive action of UDG and apyrimidinic/apurinic site endonuclease.In order to gain deeper insight into the structural basis required for the specific uridine recognition by Mth212, we have characterized the enzyme by means of X-ray crystallography. Structures of Mth212 wild-type or mutant proteins either alone or in complex with DNA substrates and products have been determined to a resolution of up to 1.2 Å, suggesting key residues for the uridine endonuclease activity. The insertion of the side chain of Arg209 into the DNA helical base stack resembles interactions observed in human UDG and seems to be crucial for the uridine recognition. In addition, Ser171, Asn153, and Lys125 in the substrate binding pocket appear to have important functions in the discrimination of aberrant uridine against naturally occurring thymidine and cytosine residues in double-stranded DNA.     

Crystal Structures of Human Orexin 2 Receptor Bound to the Subtype-Selective Antagonist EMPA

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Synthesis,Crystal Structure and Biological Activity of 2-Hydroxyethylammonium Salt of p-Aminobenzoic Acid

p-Aminobenzoic acid (pABA) plays important roles in a wide variety of metabolic processes. Herein we report the synthesis, theoretical calculations, crystallographic investigation, and in vitro determination of the biological activity and phytotoxicity of the pABA salt, 2-hydroxyethylammonium p-aminobenzoate (HEA-pABA). The ability of neutral and anionic forms of pABA to interact with TIR1 pocket was investigated by calculation of molecular electrostatic potential maps on the accessible surface area, docking experiments, Molecular Dynamics and Quantum Mechanics/Molecular Mechanics calculations. The docking study of the folate precursor pABA, its anionic form and natural auxin (indole-3-acetic acid, IAA) with the auxin receptor TIR1 revealed a similar binding mode in the active site. The phytotoxic evaluation of HEA-pABA, pABA and 2-hydroxyethylamine (HEA) was performed on the model plant Arabidopsis thaliana ecotype Col 0 at five different concentrations. HEA-pABA and pABA acted as potential auxin-like regulators of root development in Arabidopsis thaliana (0.1 and 0.2 mM) and displayed an agravitropic root response at high concentration (2 mM). This study suggests that HEA-pABA and pABA might be considered as potential new regulators of plant growth.     

In Vivo and In Vitro Synthesis of Human Rhinovirus Type 2 Ribonucleic Acid

HeLa cells infected with human rhinovirus type 2 synthesize a mixture of single-and double-stranded ribonucleic acid (RNA). The RNA synthesized by the membrane-bound RNA polymerase complex in vitro is also a mixture of single- and double-stranded RNA, whereas the deoxycholate-treated RNA polymerase complex synthesized only double-stranded RNA. Although twice as much cell-associated viral RNA is synthesized in vivo at 34 C than at 37 C, there is no difference in the rate of RNA synthesized in vitro at 34 C and 37 C by the polymerase complex. The RNA polymerase complex, after treatment with deoxycholate, sediments as a broad peak with an average sedimentation value of 120S.     

Structure of Human MDM4 N-Terminal Domain Bound to a Single-Domain Antibody

The N-terminal domain of MDM4 binds to the N-terminal transactivation domain of the tumor suppressor p53 and is an important negative regulator of its transactivation activity. As such, inhibition of the binding of MDM4 to p53 is a target for anticancer therapy. The protein has not been crystallized satisfactorily for structural studies without the addition of an N-terminal p53 peptide. We selected a single-domain antibody (VH9) that bound to the human domain with a dissociation constant of 44 nM. We solved the structure of the complex at 2.0-Å resolution. The asymmetric unit contained eight molecules of VH9 and four molecules of MDM4. A molecule of VH9 was located in each transactivation domain binding site, and the four non-MDM4-bound VH9 domains provided additional crystal contacts. There are differences between the structures of human MDM4 domain bound to VH9 and those of human and zebra fish MDM4 bound to a p53 peptide. Molecular dynamics simulations showed that the binding pocket in the three MDM4 structures converged to a common conformation after removal of the ligands, indicating that the differences are due to induced fit. The largest conformational changes were for the MDM4 molecules bound to p53. The simulated and observed structures should aid rational drug design. The use of single-domain antibodies to aid crystallization by creating a molecular scaffold may have a wider range of applications.     

Crystal Structure of a Bioactive Pactamycin Analog Bound to the 30S Ribosomal Subunit

Biosynthetically and chemically derived analogs of the antibiotic pactamycin and de-6-methylsalicylyl (MSA)-pactamycin have attracted recent interest as potential antiprotozoal and antitumor drugs. Here, we report a 3.1-Å crystal structure of de-6-MSA-pactamycin bound to its target site on the Thermus thermophilus 30S ribosomal subunit. Although de-6-MSA-pactamycin lacks the MSA moiety, it shares the same binding site as pactamycin and induces a displacement of nucleic acid template bound at the E-site of the 30S. The structure highlights unique interactions between this pactamycin analog and the ribosome, which paves the way for therapeutic development of related compounds.     

Dimeric Structure of Pseudokinase RNase L Bound to 2-5A Reveals a Basis for Interferon-Induced Antiviral Activity

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Crystal Structure of Human Myosin 1c—The Motor in GLUT4 Exocytosis: Implications for Ca Regulation and 14-3-3 Binding

Myosin 1c (Myo1c) plays a key role in supporting motile events that underlie cell migration, vesicle trafficking, insulin-stimulated glucose uptake and hearing. Here, we present the crystal structure of the human Myo1c motor in complex with its light chain calmodulin. Our structure reveals tight interactions of the motor domain with calmodulin bound to the first IQ motif in the neck region. Several of the calmodulin residues contributing to this interaction are also involved in Ca^2 + binding. Contact residues in the motor domain are linked to the central β-sheet and the HO helix, suggesting a mechanism for communicating changes in Ca^2 + binding in the neck region to the actin and nucleotide binding regions of the motor domain. The structural context and the chemical environment of Myo1c mutations that are involved in sensorineural hearing loss in humans are described and their impact on motor function is discussed. We show that a construct consisting of the motor domain of Myo1c and the first IQ motif is sufficient to establish a tight interaction with 14-3-3β (K_D = 0.9 μM) and present the model of a double-headed Myo1c–14-3-3 complex. This complex has been implicated in the exocytosis of glucose transporter 4 storage vesicles during insulin-stimulated glucose uptake.     

Production and single-step purification of EGFP and a biotinylated version of the Human Rhinovirus 14 3C protease

The fluorescent reporter enhanced Green Fluorescent Protein (EGFP) has been used for assaying a wide range of biological activities ranging from gene expression, or localization of target proteins through to intermolecular interactions. However, over-production of this protein in Escherichia coli has resulted in the presence of inclusion bodies, which complicates recovery of the protein in significant quantities. In this paper, we describe a single-step method for isolating the protein from a Glutathione-S-Transferase (GST) fusion protein, release of the EGFP protein from the fusion was demonstrated using a biotinylated variant of Human Rhinovirus 14 3C protease that we have also constructed. We also suggest the potential uses of the biotinylated protease for bionanotechnology and synthetic biology.     

The Crystal Structure of Human Cyclin B

Cyclin B is the key regulatory protein controlling mitosis in all eukaryotes, where it binds cyclin-dependent kinase, cdk1, forming a complex which initiates the mitotic program through phosphorylation of select proteins. Cyclin B regulates the activation, subcellular localization, and substrate specificity of cdk1, and destruction of cyclin B is necessary for mitotic exit. Overexpression of human cyclin B1 has been found in numerous cancers and has been associated with tumor aggressiveness. Here we report the crystal structure of human cyclin B1 to 2.9 Å. Comparison of the structure with cyclin A and cyclin E reveals remarkably similar N-terminal cyclin box motifs but significant differences among the C-terminal cyclin box lobes. Divergence in sequence gives rise to unique interaction surfaces at the proposed cyclin B/ cdk1 interface as well as the ‘RxL’ motif substrate binding site on cyclin B. Examination of the structure provides insight into the molecular basis for differential affinities of protein based cyclin/cdk inhibitors such as p27, substrate recognition, and cdk interaction.     

Crystal Structure of Human Carbonic Anhydrase C

The three dimensional structure of human carbonic anhydrase C has been determined at 2.0 Å resolution. The active site has been identified by the binding of inhibitors and the location of the zinc ion.     

Antibody-Mediated Neutralization of Human Rhinovirus 14 Explored by Means of Cryoelectron Microscopy and X-Ray Crystallography of Virus-Fab Complexes

The structures of three different human rhinovirus 14 (HRV14)-Fab complexes have been explored with X-ray crystallography and cryoelectron microscopy procedures. All three antibodies bind to the NIm-IA site of HRV14, which is the β-B–β-C loop of the viral capsid protein VP1. Two antibodies, Fab17-IA (Fab17) and Fab12-IA (Fab12), bind bivalently to the virion surface and strongly neutralize viral infectivity whereas Fab1-IA (Fab1) strongly aggregates and weakly neutralizes virions. The structures of the two classes of virion-Fab complexes clearly differ and correlate with observed binding neutralization differences. Fab17 and Fab12 bind in essentially identical, tangential orientations to the viral surface, which favors bidentate binding over icosahedral twofold axes. Fab1 binds in a more radial orientation that makes bidentate binding unlikely. Although the binding orientations of these two antibody groups differ, nearly identical charge interactions occur at all paratope-epitope interfaces. Nucleotide sequence comparisons suggest that Fab17 and Fab12 are from the same progenitor cell and that some of the differing residues contact the south wall of the receptor binding canyon that encircles each of the icosahedral fivefold vertices. All of the antibodies contact a significant proportion of the canyon region and directly overlap much of the receptor (intercellular adhesion molecule 1 [ICAM-1]) binding site. Fab1, however, does not contact the same residues on the upper south wall (the side facing away from fivefold axes) at the receptor binding region as do Fab12 and Fab17. All three antibodies cause some stabilization of HRV14 against pH-induced inactivation; thus, stabilization may be mediated by invariant contacts with the canyon.Picornaviruses are among the largest of animal virus families and include the well-known poliovirus, rhinovirus, foot-and-mouth disease virus (FMDV), coxsackievirus, and hepatitis A virus. The rhinoviruses, of which there are more than 100 serotypes subdivided into two groups, are major causative agents of the common cold in humans (42). The viruses are nonenveloped and have an ∼300-Å-diameter protein shell that encapsidates a single-stranded, plus-sense RNA genome of about 7,200 bases. The human rhinovirus 14 (HRV14) capsid exhibits a pseudo-T=3 (P=3) icosahedral symmetry and consists of 60 copies each of four viral proteins, VP1, VP2, VP3, and VP4, with VP4 at the RNA-capsid interface (40). An ∼20-Å deep canyon lies roughly at the junction of VP1 (forming the north rim) with VP2 and VP3 (forming the south rim) and surrounds each of the 12 icosahedral fivefold vertices. The canyon regions of HRV14 and HRV16, both major receptor group rhinoviruses, were shown to contain the binding site of the cellular receptor, intercellular adhesion molecule 1 (ICAM-1) (8, 24a, 37). Four major neutralizing immunogenic (NIm) sites, NIm-IA, NIm-IB, NIm-II, and NIm-III, were identified by studies of neutralization escape mutants with monoclonal antibodies (MAbs) (46, 47) and then mapped to four protruding regions on the viral surface (40).Several mechanisms of antibody-mediated neutralization have been proposed. Perhaps the simplest is based on aggregation of virions (5, 53, 54), which generally occurs over a narrow range of antibody/virus ratios. This limited range has raised questions about the role of aggregation in vivo. Alternative suggestions are that antibodies may neutralize virions by inducing extensive conformational changes in the capsid (15, 29), abrogate virus attachment to the host cell (8, 14), or prevent uncoating (57). There is no universal acceptance of a single neutralization mechanism, and the various MAbs may neutralize with different combinations of these mechanisms.Neutralizing MAbs against HRV14 have been divided into three groups: strong, intermediate, and weak neutralizers (26, 34). All strongly neutralizing antibodies bind to the NIm-IA site, which was defined by natural escape mutations at residues D1091 and E1095 of VP1 on the loop between the β-B and β-C strands of the VP1 β-barrel (the letter designates the amino acid, the first digit identifies the viral protein, and the remaining three digits specify the sequence number). Because strongly neutralizing antibodies form stable, monomeric virus-antibody complexes with a maximum stoichiometry of 30 antibodies per virion, it was concluded that they bind bivalently to the virions (26, 34). Weakly neutralizing antibodies form unstable, monomeric complexes with HRV14 and bind with a stoichiometry of ∼60 antibodies per virion (26, 52). The remaining antibodies, all of which precipitate the virions, are classified as intermediate neutralizers (26, 34).The structures of two complexes, the strongly neutralizing antibody MAb17-IA and its Fab fragment, Fab17, bound to HRV14, were determined by means of cryo-transmission electron microscopy (cryo-TEM) and three-dimensional image reconstruction (51, 52) and interpreted on the basis of model-building studies that used the atomic structures of HRV14 (40) and Fab17 (28). These studies showed that no observable conformational changes were induced in the viral capsid upon Fab or MAb binding. Modeling and site-directed mutagenesis studies demonstrated that electrostatic interactions play a key role in the binding of Fab17 to HRV14 (52). In the complex, the loop of the NIm-IA site on HRV14 sits clamped in the cleft between the heavy- and light-chain hypervariable regions and forms complementary electrostatic interactions with Lys58^H (on the heavy chain) and Arg91^L (on the light chain) of Fab17. In addition, a cluster of lysines on HRV14 (K1236, K1097, and K1085) interact with two acidic residues, Asp45^H and Asp54^H, in the CDR2 (CDR stands for complementarity-determining region) of the Fab heavy chain (49). Earlier modeling studies also suggested that bidentate binding of MAb17-IA to HRV14 is facilitated by rotation of the Fab constant domains about the elbow axes towards the viral twofold axes (51). This suggested that the flexibility of the elbow region (the junction between the variable and constant domains) plays a role in the bivalent binding process, which in turn increases antibody avidity. Finally, the 4-Å-resolution crystal structure of the Fab17-HRV14 complex clearly showed that the virion does not undergo conformational changes upon Fab binding (49). This crystal structure determination also revealed that the earlier docking of the HRV14 and Fab17 atomic structures into the 22-Å cryo-TEM density map (50) yielded a pseudo-atomic model that was very close to the real structure of the complex.We have expanded our complementary X-ray crystallography and cryo-TEM microscopy studies to examine the structures of two more Fab-virus complexes, using Fab fragments from two other NIm-IA antibodies, MAb1-IA (MAb1) and MAb12-IA (MAb12), bound to HRV14. MAb1 and MAb12 are weak and strong neutralizing antibodies, respectively. Image reconstructions of these two complexes are interpreted on the basis of pseudo-atomic models, which substantiate the previous hypothesis that neutralizing efficacy and binding valency are interrelated (34). Electrostatic interactions at the epitope-paratope interface are highly conserved and apparently important for the antibody binding to the virion surface. Like Fab17, Fab1 and Fab12 penetrate the canyon. There are, however, differences between the orientations of the strongly and weakly neutralizing antibodies and in the contacts made with the receptor binding region of the canyon. Finally, data suggesting that antibody binding to HRV14 is alone sufficient for neutralization and that other possible mechanisms are not required are presented.     

Phenotypic Responses of Differentiated Asthmatic Human Airway Epithelial Cultures to Rhinovirus

ObjectivesHuman airway epithelial cells are the principal target of human rhinovirus (HRV), a common cold pathogen that triggers the majority of asthma exacerbations. The objectives of this study were 1) to evaluate an in vitro air liquid interface cultured human airway epithelial cell model for HRV infection, and 2) to identify gene expression patterns associated with asthma intrinsically and/or after HRV infection using this model.MethodsAir-liquid interface (ALI) human airway epithelial cell cultures were prepared from 6 asthmatic and 6 non-asthmatic donors. The effects of rhinovirus RV-A16 on ALI cultures were compared. Genome-wide gene expression changes in ALI cultures following HRV infection at 24 hours post exposure were further analyzed using RNA-seq technology. Cellular gene expression and cytokine/chemokine secretion were further evaluated by qPCR and a Luminex-based protein assay, respectively.ConclusionsALI-cultured human airway epithelial cells challenged with HRV are a useful translational model for the study of HRV-induced responses in airway epithelial cells, given that gene expression profile using this model largely recapitulates some important patterns of gene responses in patients during clinical HRV infection. Furthermore, our data emphasize that both abnormal airway epithelial structure and inflammatory signaling are two important asthma signatures, which can be further exacerbated by HRV infection.     

Human Rhinovirus 14 Enters Rhabdomyosarcoma Cells Expressing ICAM-1 by a Clathrin-, Caveolin-, and Flotillin-Independent Pathway

Intercellular adhesion molecule 1 (ICAM-1) mediates binding and entry of major group human rhinoviruses (HRVs). Whereas the entry pathway of minor group HRVs has been studied in detail and is comparatively well understood, the pathway taken by major group HRVs is largely unknown. Use of immunofluorescence microscopy, colocalization with specific endocytic markers, dominant negative mutants, and pharmacological inhibitors allowed us to demonstrate that the major group virus HRV14 enters rhabdomyosarcoma cells transfected to express human ICAM-1 in a clathrin-, caveolin-, and flotillin-independent manner. Electron microscopy revealed that many virions accumulated in long tubular structures, easily distinguishable from clathrin-coated pits and caveolae. Virus entry was strongly sensitive to the Na⁺/H⁺ ion exchange inhibitor amiloride and moderately sensitive to cytochalasin D. Thus, cellular uptake of HRV14 occurs via a pathway exhibiting some, but not all, characteristics of macropinocytosis and is similar to that recently described for adenovirus 3 entry via α_v integrin/CD46 in HeLa cells.Human rhinoviruses (HRVs), members of the family Picornaviridae that represent a major cause of the common cold, essentially utilize two different receptor types for host cell attachment. The 12 minor group HRVs, exemplified by HRV2, bind low-density lipoprotein receptor (LDLR), LDLR-related protein (LRP) (20), and very-LDLR (VLDLR) (29) and are internalized via the well-characterized clathrin-dependent endocytic pathway (44); however, these ligands, like others, can switch to different entry portals when the clathrin-dependent pathway is blocked (4). Once the virus arrives in endosomal carrier vesicles or late endosomes, uncoating (i.e., the release of the viral RNA genome) is triggered by the acidic pH (35, 39).The 87 major group HRVs, exemplified by HRV14, bind intercellular adhesion molecule-1 (ICAM-1). Following entry, uncoating is triggered by ICAM-1 itself (3), but the low endosomal pH facilitates this process (37). Based on inhibition of infection by the dominant negative (DN) dynamin-2 mutant dyn^K44A, it was proposed that HRV14 also follows a clathrin-dependent pathway in HeLa-H1 cells (9). However, ICAM-1 lacks a clathrin localization signal and even functions as a viral receptor when its cytoplasmic tail is replaced with a glycosylphosphatidylinositol (GPI) anchor (45). Furthermore, dynamin has also been shown to be involved in pathways other than clathrin-mediated endocytosis (CME), such as caveolae- and lipid raft-dependent entry, as a function of ligand and cell type (reviewed in references 30 and 34). Additionally, dynamin might play a role in formation and closure of circular pinocytic ruffles (31). More recently, a specific entry pathway for ICAM-1 ligands into human umbilical vein endothelial cells was identified and termed “cam-mediated endocytosis”; uptake was found to be triggered upon binding of multivalent ligands, such as immunoconjugates and immunobeads, and to occur independently from clathrin and caveolin. Inhibition by amiloride, actin depolymerization, and protein kinase C inhibitors pointed to macropinocytosis (33). So far, it is not known whether these findings are relevant to the entry pathway of HRVs via ICAM-1 as the uptake kinetics was significantly dependent on particle size. For all these reasons, involvement of clathrin in HRV14 uptake is questionable. Accordingly, we explored entry of HRV14 via ICAM-1 and compared the results with the well-characterized clathrin-dependent entry pathway of HRV2 (44). Employing pharmacological compounds, specific DN inhibitors, immunofluorescence, and electron microscopy, we demonstrate that HRV14 enters rhabdomyosarcoma ICAM-1-expressing (RD-ICAM) cells via a pathway independent of clathrin, caveolin, and flotillin.     

The Domain Structure of ICAM-1 and the Kinetics of Binding to Rhinovirus

Fragments of intercellular adhesion molecule 1 (ICAM-1) containing only the two most N terminal of its five immunoglobulin SF domains bind to rhinovirus 3 with the same affinity and kinetics as a fragment with the entire extracellular domain. The fully active two-domain fragments contain 5 or 14 more residues than a previously described fragment that is only partially active. Comparison of X-ray crystal structures show differences at the bottom of domain 2. Four different glycoforms of ICAM-1 bind with identical kinetics.     

Crystal Structure of the Human Cytomegalovirus Glycoprotein B

Human cytomegalovirus (HCMV), a dsDNA, enveloped virus, is a ubiquitous pathogen that establishes lifelong latent infections and caused disease in persons with compromised immune systems, e.g., organ transplant recipients or AIDS patients. HCMV is also a leading cause of congenital viral infections in newborns. Entry of HCMV into cells requires the conserved glycoprotein B (gB), thought to function as a fusogen and reported to bind signaling receptors. gB also elicits a strong immune response in humans and induces the production of neutralizing antibodies although most anti-gB Abs are non-neutralizing. Here, we report the crystal structure of the HCMV gB ectodomain determined to 3.6-Å resolution, which is the first atomic-level structure of any betaherpesvirus glycoprotein. The structure of HCMV gB resembles the postfusion structures of HSV-1 and EBV homologs, establishing it as a new member of the class III viral fusogens. Despite structural similarities, each gB has a unique domain arrangement, demonstrating structural plasticity of gB that may accommodate virus-specific functional requirements. The structure illustrates how extensive glycosylation of the gB ectodomain influences antibody recognition. Antigenic sites that elicit neutralizing antibodies are more heavily glycosylated than those that elicit non-neutralizing antibodies, which suggest that HCMV gB uses glycans to shield neutralizing epitopes while exposing non-neutralizing epitopes. This glycosylation pattern may have evolved to direct the immune response towards generation of non-neutralizing antibodies thus helping HCMV to avoid clearance. HCMV gB structure provides a starting point for elucidation of its antigenic and immunogenic properties and aid in the design of recombinant vaccines and monoclonal antibody therapies.