Structural Basis for the Recognition of Human Cytomegalovirus Glycoprotein B by a Neutralizing Human Antibody

Human cytomegalovirus (HCMV) infections are life-threating to people with a compromised or immature immune system. Upon adhesion, fusion of the virus envelope with the host cell is initiated. In this step, the viral glycoprotein gB is considered to represent the major fusogen. Here, we present for the first time structural data on the binding of an anti-herpes virus antibody and describe the atomic interactions between the antigenic domain Dom-II of HCMV gB and the Fab fragment of the human antibody SM5-1. The crystal structure shows that SM5-1 binds Dom-II almost exclusively via only two CDRs, namely light chain CDR L1 and a 22-residue-long heavy chain CDR H3. Two contiguous segments of Dom-II are targeted by SM5-1, and the combining site includes a hydrophobic pocket on the Dom-II surface that is only partially filled by CDR H3 residues. SM5-1 belongs to a series of sequence-homologous anti-HCMV gB monoclonal antibodies that were isolated from the same donor at a single time point and that represent different maturation states. Analysis of amino acid substitutions in these antibodies in combination with molecular dynamics simulations show that key contributors to the picomolar affinity of SM5-1 do not directly interact with the antigen but significantly reduce the flexibility of CDR H3 in the bound and unbound state of SM5-1 through intramolecular side chain interactions. Thus, these residues most likely alleviate unfavorable binding entropies associated with extra-long CDR H3s, and this might represent a common strategy during antibody maturation. Models of entire HCMV gB in different conformational states hint that SM5-1 neutralizes HCMV either by blocking the pre- to postfusion transition of gB or by precluding the interaction with additional effectors such as the gH/gL complex.     

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.     

Receptor-Binding Properties of a Soluble Form of Human Cytomegalovirus Glycoprotein B

The human cytomegalovirus (HCMV) glycoprotein B (gB) (also known as gpUL55) homolog is an important mediator of virus entry and cell-to-cell dissemination of infection. To examine the potential ligand-binding properties of gB, a soluble form of gB (gB-S) was radiolabeled, purified, and tested in cell-binding experiments. Binding of gB-S to human fibroblast cells was found to occur in a dose-dependent, saturable, and specific manner. Scatchard analysis demonstrated a biphasic plot with the following estimated dissociation constants (K_d): K_d1, 4.96 × 10⁻⁶ M; K_d2, 3.07 × 10⁻⁷ M. Cell surface heparan sulfate proteoglycans (HSPGs) were determined to serve as one class of receptors able to facilitate gB-S binding. Both HSPG-deficient Chinese hamster ovary (CHO) cells and fibroblast cells with enzymatically removed HSPGs had 40% reductions in gB-S binding, whereas removal of chondroitin sulfate had no effect. However, a significant proportion of gB-S was able to associate with the cell surface in the absence of HSPGs via an undefined nonheparin component. Binding affinity analysis of gB-S binding to wild-type CHO-K1 cells demonstrated biphasic binding kinetics (K_d1, 9.85 × 10⁻⁶ M; K_d2, 4.03 × 10⁻⁸ M), whereas gB-S binding to HSPG-deficient CHO-677 cells exhibited single-component binding kinetics (K_d, 7.46 × 10⁻⁶ M). Together, these data suggest that gB-S associates with two classes of cellular receptors. The interaction of gB with its receptors is physiologically relevant, as evidenced by an inhibitory effect on HCMV entry when cells were pretreated with purified gB-S. This inhibition was determined to be manifested at the level of virus attachment. We conclude that gB is a ligand for HCMV that mediates an interaction with a cellular receptor(s) during HCMV infection.Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that is present in approximately 80% of the adult population, as demonstrated by seroreactivity (3, 23). Primary HCMV infection of persons with intact immune systems often results in a self-limiting asymptomatic disease, while HCMV is a significant human pathogen for immunocompromised individuals that is often manifested as severe and debilitating sequelae (2). Despite its importance as a pathogen, limited antiviral therapies exist, due in part to a lack of detailed knowledge of the virus lifecycle.HCMV infection requires that a viral envelope glycoprotein(s) and the respective cellular receptor(s) engage in a synchronized series of interactions, ultimately resulting in fusion of the viral envelope with the plasma membrane. Initial attachment of HCMV to permissive host cells is dependent upon the presence of cell surface heparan sulfate proteoglycans (HSPGs) (14, 43). Heparin affinity chromatography identified two HCMV glycoprotein complexes that possess the ability to bind immobilized heparin (14, 26). The HCMV glycoprotein complex II (gC-II) was described to be the major HCMV envelope protein complex retained on the heparin matrix, while a lesser proportion of glycoprotein B (gB) (also known as gpUL55) was bound (26). Due to the lack of a manipulable genetic system for HCMV, to date there has been no effective manner by which to evaluate independently the functional relevance of heparin binding for gB or gC-II. This initial heparin-dissociable binding state is rapidly converted to a stable attachment, suggesting that HCMV absorption involves a sequential association with multiple cellular receptors (14). After stable attachment to the cell surface, a direct pH-independent fusion event occurs between the viral envelope and the plasma membrane (13). Two HCMV envelope glycoprotein complexes, gB and gH-gL (also known as gpUL75-gpUL115), are crucial components in mediating fusion events required for subsequent virus entry. The identity of cellular receptors for stable binding or of fusion facilitators is not known, although a number of candidates have been proposed (1, 28, 29, 52, 53).HCMV gB is a 906-amino-acid protein encoded by the UL55 open reading frame (12, 16). The gB precursor is synthesized as a 105-kDa protein, which matures into a 130- to 160-kDa glycoprotein by acquiring N-linked glycosylation modifications in the endoplasmic reticulum and Golgi network (6, 7). The cellular protease furin cleaves the mature gB into two components, a 93- to 116-kDa amino-terminal fragment and a 55-kDa carboxy-terminal fragment (60). These two fragments have been shown to associate as a disulfide-linked monomer (53, 54) which is presented on the viral envelope as well as on the surface of virus-infected cells as a covalently associated homodimer (9). gB is the most abundant constituent of the viral envelope and is a potent immunogenic HCMV protein (8, 35).gB has the potential to be a multifunctional regulator of HCMV entry. As described above, HCMV gB is a putative viral ligand in that it possesses heparin-binding capacity (perhaps critical in the initial attachment phase) and is involved in virus penetration and cell-to-cell spread. Neutralizing anti-gB monoclonal antibodies significantly blocked viral fusion events, including penetration and cell-to-cell transmission, while viral attachment remained unaffected (41). Similarly, U373 glioblastoma cells constitutively expressing gB formed multinucleated syncytia, a process which was effectively precluded by the addition of neutralizing anti-gB antibodies (59). In an effort to address the receptor-binding properties of gB, we tested a recombinant soluble form of gB (gB-S) in cellular binding experiments. Previously, we showed that the gB-S protein retained features attributable to the viral protein in that it was dimeric, properly folded, and bound to a heparin affinity matrix (11). Our results presented here demonstrate that gB-S does exhibit conventional ligand properties and may engage more than one class of receptors on the surfaces of both fibroblast and wild-type Chinese hamster ovary (CHO) cells. Cell surface HSPGs were determined to be one receptor for the recombinant gB molecule, since gB-S binding was reduced when these molecules were absent; however, a second HSPG-independent binding site was also implicated. Treatment of cells with gB-S inhibited virus entry and infection, supporting a physiological relevance for the interaction of gB with its cellular receptor(s).     

The Human Cytomegalovirus UL74 Gene Encodes the Third Component of the Glycoprotein H-Glycoprotein L-Containing Envelope Complex

The human cytomegalovirus (HCMV) gCIII envelope complex is composed of glycoprotein H (gH; gpUL75), glycoprotein L (gL; gpUL115), and a third, 125-kDa protein not related to gH or gL (M. T. Huber and T. Compton, J. Virol. 71:5391–5398, 1997; L. Li, J. A. Nelson, and W. J. Britt, J. Virol. 71:3090–3097, 1997). Glycosidase digestion analysis demonstrated that the 125-kDa protein was a glycoprotein containing ca. 60 kDa of N-linked oligosaccharides on a peptide backbone of 65 kDa or less. Based on these biochemical characteristics, two HCMV open reading frames, UL74 and TRL/IRL12, were identified as candidate genes for the 125-kDa glycoprotein. To identify the gene encoding the 125-kDa glycoprotein, we purified the gCIII complex, separated the components by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and subjected gH and the 125-kDa glycoprotein to amino acid microsequence analysis. Microsequencing of an internal peptide derived from purified 125-kDa glycoprotein yielded the amino acid sequence LYVGPTK. A FASTA search revealed an exact match of this sequence to amino acids 188 to 195 of the predicted product of the candidate gene UL74, which we have designated glycoprotein O (gO). Anti-gO antibodies reacted in immunoblots with a protein species migrating at ca. 100 to 125 kDa in lysates of HCMV-infected cells and with 100- and 125-kDa protein species in purified virions. Anti-gO antibodies also immunoprecipitated the gCIII complex and recognized the 125-kDa glycoprotein component of the gCIII complex. Positional homologs of the UL74 gene were found in other betaherpesviruses, and comparisons of the predicted products of the UL74 homolog genes demonstrated a number of conserved biochemical features.     

Characterization of a Discontinuous Neutralizing Epitope on Glycoprotein B of Human Cytomegalovirus

Human cytomegalovirus (HCMV) is a ubiquitously distributed pathogen that causes severe disease in immunosuppressed patients and newborn infants infected in utero. The viral envelope glycoprotein B (gB) is an attractive molecule for active vaccination and passive immunoprophylaxis and therapy. Using human monoclonal antibodies (MAbs), we have recently identified antigenic region 4 (AD-4) on gB as an important target for neutralizing antibodies. AD-4 is formed by a discontinuous sequence comprising amino acids 121 to 132 and 344 to 438 of gB of HCMV strain AD169. To map epitopes for human antibodies on this protein domain, we used a three-dimensional (3D) model of HCMV gB to identify surface-exposed amino acids on AD-4 and selected juxtaposed residues for alanine scans. A tyrosine (Y) at position 364 and a lysine (K) at position 379 (the YK epitope), which are immediate neighbors on the AD-4 surface, were found to be essential for binding of the human MAbs. Recognition of AD-4 by sera from HCMV-infected individuals also was largely dependent on these two residues, indicating a general importance for the antibody response against AD-4. A panel of AD-4 recombinant viruses harboring mutations at the crucial antibody binding sites was generated. The viruses showed significantly reduced susceptibility to neutralization by AD-4-specific MAbs or polyclonal AD-4-specific antibodies, indicating that the YK epitope is dominant for the AD-4-specific neutralizing antibody response during infection. To our knowledge, this is the first molecular identification of a functional discontinuous epitope on HCMV gB. Induction of antibodies specific for this epitope may be a desirable goal following vaccination with gB.     

Glycoprotein N of Human Cytomegalovirus Protects the Virus from Neutralizing Antibodies

Herpes viruses persist in the infected host and are transmitted between hosts in the presence of a fully functional humoral immune response, suggesting that they can evade neutralization by antiviral antibodies. Human cytomegalovirus (HCMV) encodes a number of polymorphic highly glycosylated virion glycoproteins (g), including the essential envelope glycoprotein, gN. We have tested the hypothesis that glycosylation of gN contributes to resistance of the virus to neutralizing antibodies. Recombinant viruses carrying deletions in serine/threonine rich sequences within the glycosylated surface domain of gN were constructed in the genetic background of HCMV strain AD169. The deletions had no influence on the formation of the gM/gN complex and in vitro replication of the respective viruses compared to the parent virus. The gN-truncated viruses were significantly more susceptible to neutralization by a gN-specific monoclonal antibody and in addition by a number of gB- and gH-specific monoclonal antibodies. Sera from individuals previously infected with HCMV also more efficiently neutralized gN-truncated viruses. Immunization of mice with viruses that expressed the truncated forms of gN resulted in significantly higher serum neutralizing antibody titers against the homologous strain that was accompanied by increased antibody titers against known neutralizing epitopes on gB and gH. Importantly, neutralization activity of sera from animals immunized with gN-truncated virus did not exhibit enhanced neutralizing activity against the parental wild type virus carrying the fully glycosylated wild type gN. Our results indicate that the extensive glycosylation of gN could represent a potentially important mechanism by which HCMV neutralization by a number of different antibody reactivities can be inhibited.     

Structural Insights into the Human Metapneumovirus Glycoprotein Ectodomain

Human metapneumovirus is a major cause of respiratory tract infections worldwide. Previous reports have shown that the viral attachment glycoprotein (G) modulates innate and adaptive immune responses, leading to incomplete immunity and promoting reinfection. Using bioinformatics analyses, static light scattering, and small-angle X-ray scattering, we show that the extracellular region of G behaves as a heavily glycosylated, intrinsically disordered polymer. We discuss potential implications of these findings for the modulation of immune responses by G.     

应用噬菌体展示技术筛选人巨细胞病毒糖蛋白M新的中和抗原表位

人巨细胞病毒(HCMV)糖蛋白复合物Ⅱ包括两种蛋白,即糖蛋白M(gM)和糖蛋白N(gN)．尽管来自于HCMV阳性病人血清中的糖蛋白复合物Ⅱ的IgG抗体能够中和HCMV粒子,但迄今为止,还没有gM中和性抗原表位的相关研究．应用消减杂交技术,通过噬菌体肽库筛选获得gM抗原的一个表位,即MAD．MAD氨基酸序列与gM第32～38位序列高度同源．将MAD与钥孔血蓝蛋白偶联免疫小鼠可产生抗MAD多抗,该多抗不仅结合天然HCMV病毒粒子,而且特异结合重组表达的gM30～78多肽．ELISA结果表明MAD能够特异结合HCMV阳性的病人血清．病毒中和实验结果进一步证明抗MAD多抗能够抑制HCMV AD169株病毒感染人胚肺细胞．总之,MAD表位有可能成为HCMV病毒疫苗潜在的保护性抗原.     

TRAM1 Participates in Human Cytomegalovirus US2- and US11-mediated Dislocation of an Endoplasmic Reticulum Membrane Glycoprotein

The human cytomegalovirus proteins US2 and US11 have co-opted endoplasmic reticulum (ER) quality control to facilitate the destruction of major histocompatibility complex class I heavy chains. The class I heavy chains are dislocated from the ER to the cytosol, where they are deglycosylated and subsequently degraded by the proteasome. We examined the role of TRAM1 (translocating chain-associated membrane protein-1) in the dislocation of class I molecules using US2- and US11-expressing cells. TRAM1 is an ER protein initially characterized for its role in processing nascent polypeptides. Co-immunoprecipitation studies demonstrated that TRAM1 can complex with the wild type US2 and US11 proteins as well as deglycosylated and polyubiquitinated class I degradation intermediates. In studies using US2- and US11-TRAM1 knockdown cells, we observed an increase in levels of class I heavy chains. Strikingly, increased levels of glycosylated heavy chains were observed in TRAM1 knockdown cells when compared with control cells in a pulse-chase experiment. In fact, US11-mediated class I dislocation was more sensitive to the lack of TRAM1 than US2. These results provide further evidence that these viral proteins may utilize distinct complexes to facilitate class I dislocation. For example, US11-mediated class I heavy chain degradation requires Derlin-1 and SEL1L, whereas signal peptide peptidase is critical for US2-induced class I destabilization. In addition, TRAM1 can complex with the dislocation factors Derlin-1 and signal peptide peptidase. Collectively, the data support a model in which TRAM1 functions as a cofactor to promote efficient US2- and US11-dependent dislocation of major histocompatibility complex class I heavy chains.HCMV² can down-regulate cell surface expression of the immunologically important molecule major histocompatibility complex class I to avoid immune detection by cytotoxic T cells (1, 2). More specifically, the HCMV US2 and US11 gene products alone can target the ER-localized major histocompatibility complex class I heavy chains for extraction across the ER membrane by a process referred to as dislocation or retrograde translocation. The N-linked glycan is then removed upon exposure to the cytosol by N-glycanase (3), followed by proteasomal destruction (4, 5). The HCMV US2 and US11 proteins utilize the ER quality control process to eliminate class I heavy cells in a similar manner as misfolded or damaged ER proteins (e.g. genetic mutants of α₁-antitrypsin (6) and the cystic fibrosis transmembrane conductance regulator protein (7)) are targeted for degradation (8). Hence, analysis of US2- and US11-mediated destruction of class I heavy chains provides an excellent system to delineate viral protein function as well as the ER quality control process.ER and cytosolic proteins are required for US2- and US11-mediated dislocation/degradation of class I heavy chains. Some of these proteins have also been identified in the processing of aberrant ER polypeptides. The ER chaperones calnexin, calreticulin, and BiP have been implicated in US2-mediated class I destruction (9) as well as in the removal of some misfolded ER proteins (10). The ubiquitination machinery also participates in the extraction of class I heavy chains as ubiquitinated heavy chains are observed prior to dislocation (11, 12). For misfolded ER degradation substrates, ubiquitin conjugation enzymes (e.g. Ubc6p and Ubc7p/Cue1p) and ubiquitin ligases Hrd1p/Der3p, Doa10p, and Ubc1p have been implicated in the dislocation reaction (8). Interestingly, the ER membrane protein Derlin-1 along with SEL1L are involved in US11-mediated class I heavy chain degradation (13-15), whereas SPP is critical for US2-induced class I destabilization (16). The ubiquitinated substrates are dislocated by the AAA-ATPase complex composed of p97-Ufd1-Npl4 (17) while docked to the ER through its interaction with VIMP (14) followed by proteasome destruction. The inhibition of the proteasome causes the accumulation of deglycosylated class I heavy chain intermediate in US2 and US11 cells, allowing the dislocation and degradation reactions to be studied as separate processes (4, 5).Despite the identification of some cellular proteins that assist US2- and US11-mediated class I dislocation, the dislocation pore and accessory factors that mediate the efficient extraction of class I through the bilayer have yet to be completely defined. The current study explores the role of TRAM1 (translocating chain-associated membrane protein-1) in US2- and US11-mediated class I dislocation. TRAM1 is an ER-resident multispanning membrane protein that can mediate the lateral movement of select signal peptides and transmembrane segments from the translocon into the membrane bilayer (18), a property that makes it uniquely qualified to participate in the dislocation of a membrane protein. TRAM1 has been cross-linked to signal peptides as well as transmembrane domains of nascent polypeptides during the early stages of protein processing (19-25). Interestingly, unlike the Sec61 complex and the signal recognition particle receptor, TRAM1 is not essential for the translocation of all membrane proteins into the ER (20, 21). Hence, TRAM1 may utilize its ability to engage hydrophobic domains to assist in the efficient dislocation of membrane proteins. In fact, association and TRAM1 knockdown studies demonstrate that TRAM1 participates in US2- and US11-mediated dislocation of class I heavy chains. Collectively, our data suggest for the first time that TRAM1 plays a role in the dislocation of a membrane glycoprotein.     

Improved Recovery of a Fusion Protein Containing the Antigenic Domain 1 of the Human Cytomegalovirus Glycoprotein B

Heterologous expression in Escherichia coli often leads to production of the expressed proteins as insoluble and inactive inclusion bodies. The general strategy for protein recovery includes isolation and washing of inclusion bodies, solubilization of aggregated protein and refolding of solubilized protein. The process of refolding, as well as the other steps involved in inclusion body recovery, must be optimized according to the characteristics of each protein. For the development of reliable and inexpensive serodiagnostic tests, the antigenic domain 1 (AD-1) of human cytomegalovirus glycoprotein B was expressed in E. coli and a process was developed to increase recovery of the fusion protein containing AD-1. A comparison of disruption methods and different conditions involved in recovery of this fusion protein from inclusion bodies is presented. The developed method gives a high yield of the fusion protein with a purity sufficient for use in diagnostic tests.     

Proteolytic Processing of Human Cytomegalovirus Glycoprotein B Is Dispensable for Viral Growth in Culture

Glycoprotein B (gB) of human cytomegalovirus (HCMV), which is considered essential for the viral life cycle, is proteolytically processed during maturation. Since gB homologues of several other herpesviruses remain uncleaved, the relevance of this property of HCMV gB for viral infectivity is unclear. Here we report on the construction of a viral mutant in which the recognition site of gB for the cellular endoprotease furin was destroyed. Because mutagenesis of essential proteins may result in a lethal phenotype, a replication-deficient HCMV gB-null genome encoding enhanced green fluorescent protein was constructed, and complementation by mutant gBs was initially evaluated in transient-cotransfection assays. Cotransfection of plasmids expressing authentic gB or gB with a mutated cleavage site (gB-DeltaFur) led to the formation of green fluorescent miniplaques which were considered to result from one cycle of phenotypic complementation of the gB-null genome. To verify these results, two recombinant HCMV genomes were constructed: HCMV-BAC-DeltaMhdI, with a deletion of hydrophobic domain 1 of gB that appeared to be essential for viral growth in the cotransfection experiments, and HCMV-BACDeltaFur, in which the gB cleavage site was mutated by amino acid substitution. Consistent with the results of the cotransfection assays, only the DeltaFur mutant replicated in human fibroblasts, showing growth kinetics comparable to that of wild-type virus. gB in mutant-infected cells was uncleaved, whereas glycosylation and transport to the cell surface were not impaired. Extracellular mutant virus contained exclusively uncleaved gB, indicating that proteolytic processing of gB is dispensable for viral replication in cell culture.     

Human Cytomegalovirus TR Strain Glycoprotein O Acts as a Chaperone Promoting gH/gL Incorporation into Virions but Is Not Present in Virions

Human cytomegalovirus (HCMV) produces the following two gH/gL complexes: gH/gL/gO and gH/gL/UL128-131. Entry into epithelial and endothelial cells requires gH/gL/UL128-131, and we have provided evidence that gH/gL/UL128-131 binds saturable epithelial cell receptors to mediate entry. HCMV does not require gH/gL/UL128-131 to enter fibroblasts, and laboratory adaptation to fibroblasts results in mutations in the UL128-131 genes, abolishing infection of epithelial and endothelial cells. HCMV gO-null mutants produce very small plaques on fibroblasts yet can spread on endothelial cells. Thus, one prevailing model suggests that gH/gL/gO mediates infection of fibroblasts, while gH/gL/UL128-131 mediates entry into epithelial/endothelial cells. Most biochemical studies of gO have involved the HCMV lab strain AD169, which does not assemble gH/gL/UL128-131 complexes. We examined gO produced by the low-passage clinical HCMV strain TR. Surprisingly, TR gO was not detected in purified extracellular virus particles. In TR-infected cells, gO remained sensitive to endoglycosidase H, suggesting that the protein was not exported from the endoplasmic reticulum (ER). However, TR gO interacted with gH/gL in the ER and promoted export of gH/gL from the ER to the Golgi apparatus. Pulse-chase experiments showed that a fraction of gO remained bound to gH/gL for relatively long periods, but gO eventually dissociated or was degraded and was not found in extracellular virions or secreted from cells. The accompanying report by P. T. Wille et al. (J. Virol., 84:2585-2596, 2010) showed that a TR gO-null mutant failed to incorporate gH/gL into virions and that the mutant was unable to enter fibroblasts and epithelial and endothelial cells. We concluded that gO acts as a molecular chaperone, increasing gH/gL ER export and incorporation into virions. It appears that gO competes with UL128-131 for binding onto gH/gL but is released from gH/gL, so that gH/gL (lacking UL128-131) is incorporated into virions. Thus, our revised model suggests that both gH/gL and gH/gL/UL128-131 are required for entry into epithelial and endothelial cells.Human cytomegalovirus (HCMV) infects many different cell types in vivo, including epithelial and endothelial cells, fibroblasts, monocyte-macrophages, smooth muscle cells, dendritic cells, hepatocytes, neurons, glial cells, and leukocytes (reviewed in references 5, 30, 38, and 45). In the laboratory, HCMV is normally propagated in primary human fibroblasts because most other cell types yield low titers of virus. Commonly studied laboratory strains, such as AD169, were propagated extensively in fibroblasts, and this was accompanied by deletions or mutations in a cluster of 22 genes known as ULb′ (6). These mutations were correlated with the inability to infect other cell types, including endothelial and epithelial cells and monocyte-macrophages. Targeted mutagenesis of three of the ULb′ genes, UL128, UL130, and UL131, abolished infection of endothelial cells, transmission to leukocytes, and infection of dendritic cells (13, 15). Restoration of the UL128-131 genes in laboratory strains of HCMV strains restored the capacity to infect endothelial and epithelial cells and other cells (15, 52).The UL128, UL130, and UL131 proteins assemble onto the extracellular domain of HCMV gH/gL (1, 42, 53). For all herpesviruses, gH/gL complexes mediate entry into cells (12, 33, 39), suggesting that gH/gL/UL128-131 might participate in the entry mechanism. Indeed, we demonstrated that gH/gL/UL128-131 mediates entry into epithelial and endothelial cells by using the fusogenic agent polyethylene glycol to force entry of HCMV UL128-131 mutants into these cell types (41). This was consistent with reports that UL128-, UL130-, and UL131-specific antibodies blocked the capacity of HCMV to infect epithelial and endothelial cells but not fibroblasts (1, 53). Furthermore, expression of gH/gL/UL128-131, but not gH/gL or gB, in epithelial cells interfered with HCMV infection, consistent with saturable gH/gL/UL128-131 receptors (40). Expression of all five proteins was necessary so that the gH/gL/UL128-131 complexes were exported from the endoplasmic reticulum (ER) and could function (40-42, 53). Together, these data suggested that gH/gL/UL128-131 mediates entry into epithelial/endothelial cells but is not required for entry into fibroblasts. By extension, it was reasonable to propose that other forms of gH/gL might facilitate the entry into fibroblasts.The laboratory HCMV strain AD169 is known to express a second gH/gL complex containing glycoprotein O (gO) (21-23, 53). In cells infected with a recombinant AD169 in which the UL131 mutation was repaired, gH/gL/gO complexes were separate from gH/gL/UL128-131 complexes, i.e., gO was not detected following immunoprecipitation (IP) with UL128- and UL130-specifc antibodies, and gO-specific antibodies did not precipitate UL128 and UL130 (53). AD169 and Towne gO⁻ mutants produce small plaques on fibroblast monolayers and low titers of virus, supporting an important, although not essential, role for gH/gL/gO in virus replication in fibroblasts (11, 19). AD169 does not infect endothelial and epithelial cells, so AD169 gO⁻ mutants were not tested on these cells. Jiang et al. described a gO-null mutant derived from an endotheliotropic HCMV strain, TB40/E (27). The TB40/E gO-null mutant spread normally on endothelial cells, suggesting that gO or gH/gL/gO is less important for infection and spread in these cells. Given that the role of gH/gL in entry is highly conserved among the herpesviruses, it seemed likely that gH/gL/gO might be involved in entry into fibroblasts. Consistent with this notion, Paterson et al. showed that anti-gO antibodies decreased fusion from without caused by infection of cells with HCMV AD169 (37). These observations supported our working model in which gH/gL/UL128-131 mediates entry into epithelial and endothelial cells, while gH/gL/gO mediates entry into fibroblasts. There is also the possibility that gH/gL (lacking gO and UL128-131) might be incorporated into the virion envelope, although there is presently no direct evidence for this. Any gH/gL detected biochemically might result from dissociation of gO or UL128-131 during sample preparation and analysis. gH/gL expressed without other HCMV proteins was retained in the ER (42), arguing against incorporation into the virion.Other herpesviruses, e.g., Epstein-Barr virus, human herpesvirus 6 (HHV-6), and HHV-7, use different forms of gH/gL to enter different cell types via different pathways (25, 34, 43). Similarly, HCMV entry into fibroblasts occurs by fusion at the plasma membrane at a neutral pH and does not require gH/gL/UL128-131 (7), whereas entry into epithelial and endothelial cells involves endocytosis and low pH-dependent fusion and requires gH/gL/UL128-131 (41).All of the biochemical analyses of gO in terms of binding to gH/gL and intracellular transport have involved fibroblast-adapted strain AD169 (21-23, 31, 53). These studies indicated that gO is a 110- to 125-kDa glycoprotein encoded by the UL74 gene (22). Glycosidase digestion experiments demonstrated that the gO polypeptide chain is ∼62 to 65 kDa (21-23, 53). Pulse-chase studies showed that gH/gL assembles in the ER as a disulfide-linked heterodimer (28) that subsequently binds to, and establishes disulfides with, gO (22, 23). The 220-kDa immature gH/gL/gO trimer is initially sensitive to endoglycosidase H (endo H), which removes immature N-linked oligosaccharides from glycoproteins present in the ER (22, 23). Transport of gH/gL/gO to the Golgi apparatus is associated with processing of N-linked oligosaccharides to mature forms that resist endo H. Also associated with transport to the Golgi apparatus is the addition of O-linked oligosaccharides and phosphorylation, increasing the molecular weight of gO (after reduction) to 125 to 130 kDa and that of the gH/gL/gO complex to 240 to 260 kDa (22, 23, 29). It is the mature glycoprotein complex, previously known as gCIII, that is trafficked to HCMV assembly compartments for incorporation into the virion envelope (22, 23, 29).In addressing the function of gO, it is important to recognize that AD169 has adapted to replication in fibroblasts, losing expression of UL131 and failing to assemble gH/gL/UL128-131 complexes (6) (15). There seems to be strong pressure to mutate UL128-131, because clinical strain Merlin acquired a UL128 mutation within 5 passages on fibroblasts (2). It is also reasonable to suggest that fibroblast adaptation includes changes in gO. The gO genes (UL74) of several laboratory and clinical strains and clinical isolates are highly variable (up to 25% of amino acids) (10, 35, 37, 47). However, it is important to note that AD169-derived UL131-repair virus can infect epithelial and endothelial cells (52). Thus, if AD169 gO is important for infection of these cells, then gO must be functionally normal in this regard. These differences in laboratory versus clinical HCMV prompted us to characterize the gO molecule expressed by the HCMV strain TR. HCMV TR is a clinical isolate that was stabilized in the form of a bacterial artificial chromosome (BAC) after very limited passage in fibroblasts (35, 41). HCMV TR expresses gH/gL/UL128-131 (42) and infects epithelial and endothelial cells (41) and monocyte-macrophages well (D. Streblow and J. Nelson, unpublished results).Here, we report our biochemical and cell trafficking analyses of the TR gO protein. We were surprised to find that TR gO was not present in extracellular virus particles. In contrast, gO was detected in extracellular AD169 particles, consistent with previous findings (22). TR gO expressed either in HCMV-infected cells or by using nonreplicating Ad vectors (expressed without other HCMV proteins) was largely retained in the ER. Coexpression of TR gO with gH/gL promoted transport of gH/gL beyond the ER, and gO was slowly lost from gH/gL complexes but not secreted from cells and not observed in extracellular virus particles. Thus, TR gO acts as a chaperone. Consistent with this, in the accompanying paper by Wille et al. (54), a TR gO-null mutant was described that secreted extracellular particles containing markedly reduced quantities of gH and gL. The gO⁻ mutant failed to enter fibroblasts and also epithelial and endothelial cells. Together, these results suggest that it is gH/gL, not gH/gL/gO, which is incorporated into HCMV TR virions. It appears that gH/gL is required for entry into fibroblasts, and both gH/gL and gH/gL/UL128-131 are required for entry into epithelial and endothelial cells.     

Palmitoylation Strengthens Cholesterol-dependent Multimerization and Fusion Activity of Human Cytomegalovirus Glycoprotein B (gB)

Herpesviruses are a large order of animal enveloped viruses displaying a virion fusion mechanism of unusual complexity. Their multipartite machinery has a conserved core made of the gH/gL ancillary complexes and the homo-trimeric fusion protein glycoprotein B (gB). Despite its essential role in starting the viral infection, gB interaction with membrane lipids is still poorly understood. Here, evidence is provided demonstrating that human cytomegalovirus (HCMV) gB depends on the S-palmitoylation of its endodomain for an efficient interaction with cholesterol-rich membrane patches. We found that, unique among herpesviral gB proteins, the HCMV fusion factor has a Cys residue in the C-terminal region that is palmitoylated and mediates methyl-β-cyclodextrin-sensitive self-association of purified gB. A cholesterol-dependent virus-like particle trap assay, based on co-expression of the HIV Gag protein, confirmed that this post-translational modification is functional in the context of cellular membranes. Mutation of the palmitoylated Cys residue to Ala or inhibition of protein palmitoylation decreased HCMV gB export via Gag particles. Moreover, purified gB_C777A showed an increased kinetic sensitivity in a cholesterol depletion test, demonstrating that palmitoyl-gB limits outward cholesterol diffusion. Finally, gB palmitoylation was required for full fusogenic activity in human epithelial cells. Altogether, these results uncover the palmitoylation of HCMV gB and its role in gB multimerization and activity.     

Localization of Human Cytomegalovirus Structural Proteins to the Nuclear Matrix of Infected Human Fibroblasts

The intranuclear assembly of herpesvirus subviral particles remains an incompletely understood process. Previous studies have described the nuclear localization of capsid and tegument proteins as well as intranuclear tegumentation of capsid-like particles. The temporally and spatially regulated replication of viral DNA suggests that assembly may also be regulated by compartmentalization of structural proteins. We have investigated the intranuclear location of several structural and nonstructural proteins of human cytomegalovirus (HCMV). Tegument components including pp65 (ppUL83) and ppUL69 and capsid components including the major capsid protein (pUL86) and the small capsid protein (pUL48/49) were retained within the nuclear matrix (NM), whereas the immediate-early regulatory proteins IE-1 and IE-2 were present in the soluble nuclear fraction. The association of pp65 with the NM resisted washes with 1 M guanidine hydrochloride, and direct binding to the NM could be demonstrated by far-Western blotting. Furthermore, pp65 exhibited accumulation along the nuclear periphery and in far-Western analysis bound to proteins which comigrated with proteins of the size of nuclear lamins. A direct interaction between pp65 and lamins was demonstrated by coprecipitation of lamins in immune complexes containing pp65. Together, our findings provide evidence that major virion structural proteins localized to a nuclear compartment, the NM, during permissive infection of human fibroblasts.