Fetal Human Cytomegalovirus Transmission Correlates with Delayed Maternal Antibodies to gH/gL/pUL128-130-131 Complex during Primary Infection

Primary human cytomegalovirus (HCMV) infections during pregnancy are associated with a high risk of virus transmission to the fetus. To identify correlates of intrauterine HCMV transmission, serial serum samples from HCMV transmitter and non-transmitter pregnant women with primary HCMV infection were analyzed for the presence of neutralizing antibodies against different glycoproteins and glycoprotein complexes, which are known to mediate entry into distinct types of host cells. Neutralizing activity was detected in the sera early after primary infection; absorption with a soluble pentameric complex formed by gH/gL/pUL128-131, but not with gH/gL dimer or with gB, abolished the capacity of sera to neutralize infection of epithelial cells. Importantly, an early, high antibody response to pentamer antigenic sites was associated with a significantly reduced risk of HCMV transmission to the fetus. This association is consistent with the high in vitro inhibition of HCMV infection of epithelial/endothelial cells as well as cell-to-cell spreading and virus transfer to leukocytes by anti-pentamer antibodies. Taken together, these findings indicate that the HCMV pentamer complex is a major target of the antibody-mediated maternal immunity.     

Comparative Analysis of gO Isoforms Reveals that Strains of Human Cytomegalovirus Differ in the Ratio of gH/gL/gO and gH/gL/UL128-131 in the Virion Envelope

Herpesvirus glycoprotein complex gH/gL provides a core entry function through interactions with the fusion protein gB and can also influence tropism through receptor interactions. The Epstein-Barr virus gH/gL and gH/gL/gp42 serve both functions for entry into epithelial and B cells, respectively. Human cytomegalovirus (HCMV) gH/gL can be bound by the UL128-131 proteins or gO. The phenotypes of gO and UL128-131 mutants suggest that gO-gH/gL interactions are necessary for the core entry function on all cell types, whereas the binding of UL128-131 to gH/gL likely relates to a distinct receptor-binding function for entry into some specific cell types (e.g., epithelial) but not others (e.g., fibroblasts and neurons). There are at least eight isoforms of gO that differ by 10 to 30% of amino acids, and previous analysis of two HCMV strains suggested that some isoforms of gO function like chaperones, disassociating during assembly to leave unbound gH/gL in the virion envelope, while others remain bound to gH/gL. For the current report, we analyzed the gH/gL complexes present in the virion envelope of several HCMV strains, each of which encodes a distinct gO isoform. Results indicate that all strains of HCMV contain stable gH/gL/gO trimers and gH/gL/UL128-131 pentamers and little, if any, unbound gH/gL. TR, TB40/e, AD169, and PH virions contained vastly more gH/gL/gO than gH/gL/UL128-131, whereas Merlin virions contained mostly gH/gL/UL128-131, despite abundant unbound gO remaining in the infected cells. Suppression of UL128-131 expression during Merlin replication dramatically shifted the ratio toward gH/gL/gO. These data suggest that Merlin gO is less efficient than other gO isoforms at competing with UL128-131 for binding to gH/gL. Thus, gO diversity may influence the pathogenesis of HCMV through effects on the assembly of the core versus tropism gH/gL complexes.     

Human Cytomegalovirus Vaccine Based on the Envelope gH/gL Pentamer Complex

Human Cytomegalovirus (HCMV) utilizes two different pathways for host cell entry. HCMV entry into fibroblasts requires glycoproteins gB and gH/gL, whereas HCMV entry into epithelial and endothelial cells (EC) requires an additional complex composed of gH, gL, UL128, UL130, and UL131A, referred to as the gH/gL-pentamer complex (gH/gL-PC). While there are no established correlates of protection against HCMV, antibodies are thought to be important in controlling infection. Neutralizing antibodies (NAb) that prevent gH/gL-PC mediated entry into EC are candidates to be assessed for in vivo protective function. However, these potent NAb are predominantly directed against conformational epitopes derived from the assembled gH/gL-PC. To address these concerns, we constructed Modified Vaccinia Ankara (MVA) viruses co-expressing all five gH/gL-PC subunits (MVA-gH/gL-PC), subsets of gH/gL-PC subunits (gH/gL or UL128/UL130/UL131A), or the gB subunit from HCMV strain TB40/E. We provide evidence for cell surface expression and assembly of complexes expressing full-length gH or gB, or their secretion when the corresponding transmembrane domains are deleted. Mice or rhesus macaques (RM) were vaccinated three times with MVA recombinants and serum NAb titers that prevented 50% infection of human EC or fibroblasts by HCMV TB40/E were determined. NAb responses induced by MVA-gH/gL-PC blocked HCMV infection of EC with potencies that were two orders of magnitude greater than those induced by MVA expressing gH/gL, UL128-UL131A, or gB. In addition, MVA-gH/gL-PC induced NAb responses that were durable and efficacious to prevent HCMV infection of Hofbauer macrophages, a fetal-derived cell localized within the placenta. NAb were also detectable in saliva of vaccinated RM and reached serum peak levels comparable to NAb titers found in HCMV hyperimmune globulins. This vaccine based on a translational poxvirus platform co-delivers all five HCMV gH/gL-PC subunits to achieve robust humoral responses that neutralize HCMV infection of EC, placental macrophages and fibroblasts, properties of potential value in a prophylactic vaccine.     

The HCMV gH/gL/UL128-131 Complex Triggers the Specific Cellular Activation Required for Efficient Viral Internalization into Target Monocytes

We have established that HCMV acts as a specific ligand engaging and activating cellular integrins on monocytes. As a result, integrin signaling via Src activation leads to the functional activation of paxillin required for efficient viral entry and for the biological changes in monocytes needed for viral dissemination. These biological/molecular changes allow HCMV to use monocytes as “vehicles” for systemic spread and the establishment of lifelong persistence. However, it remains unresolved how HCMV specifically induces this observed monocyte activation. It was previously demonstrated that the HCMV gH/gL/UL128-131 glycoprotein complex facilitates viral entry into biologically relevant cell types. Nevertheless, the mechanism by which the gH/gL/UL128-131 complex promotes this process is unknown. We now show that only HCMV virions possessing the gH/gL/UL128-131 complex are capable of activating integrin/Src/paxillin-signaling in monocytes. In fibroblasts, this signaling is reversed, such that virus lacking the gH/gL/UL128-131 complex is the only virus able to induce the paxillin activation cascade. The presence of the gH/gL/UL128-131 complex also may have an inhibitory effect on integrin-mediated signaling pathway in fibroblasts. Furthermore, we demonstrate that the presence of the gH/gL/UL128-131 complex on the viral envelope, through its activation of the integrin/Src/paxillin pathway, is necessary for efficient HCMV internalization into monocytes and that appropriate actin and dynamin regulation is critical for this entry process. Importantly, productive infection in monocyte-derived macrophages was seen only in cells exposed to HCMV expressing the gH/gL/UL128-131 complex. From our data, the HCMV gH/gL/U128-131 complex emerges as the specific ligand driving the activation of the receptor-mediated signaling required for the regulation of the actin cytoskeleton and, consequently, for efficient and productive internalization of HCMV into monocytes. To our knowledge, our studies demonstrate a possible molecular mechanism for why the gH/gL/UL128-131 complex dictates HCMV tropism and why the complex is lost as clinical isolates are passaged in the laboratory.     

Characterization of the human cytomegalovirus gH/gL/UL128-131 complex that mediates entry into epithelial and endothelial cells

The entry of human cytomegalovirus (HCMV) into biologically relevant epithelial and endothelial cells involves endocytosis followed by low-pH-dependent fusion. This entry pathway is facilitated by the HCMV UL128, UL130, and UL131 proteins, which form one or more complexes with the virion envelope glycoprotein gH/gL. gH/gL/UL128-131 complexes appear to be distinct from the gH/gL/gO complex, which likely facilitates entry into fibroblasts. In order to better understand the assembly and protein-protein interactions of gH/gL/UL128-131 complexes, we generated HCMV mutants lacking UL128-131 proteins and nonreplicating adenovirus vectors expressing gH, gL, UL128, UL130, and UL131. Our results demonstrate that UL128, UL130, and UL131 can each independently assemble onto gH/gL scaffolds. However, the binding of individual UL128-131 proteins onto gH/gL can significantly affect the binding of other proteins; for example, UL128 increased the binding of both UL130 and UL131 to gH/gL. Direct interactions between gH/UL130, UL130/UL131, gL/UL128, and UL128/UL130 were also observed. The export of gH/gL complexes from the endoplasmic reticulum (ER) to the Golgi apparatus and cell surface was dramatically increased when all of UL128, UL130, and UL131 were coexpressed with gH/gL (with or without gO expression). Incorporation of gH/gL complexes into the virion envelope requires transport beyond the ER. Thus, we concluded that UL128, UL130, and UL131 must all bind simultaneously onto gH/gL for the production of complexes that can function in entry into epithelial and endothelial cells.     

Antibodies against the gH/gL/UL128/UL130/UL131 complex comprise the majority of the anti-cytomegalovirus (anti-CMV) neutralizing antibody response in CMV hyperimmune globulin

Anti-cytomegalovirus (anti-CMV) hyperimmune globulin (HIG) has demonstrated efficacy in preventing CMV disease in solid-organ transplant patients as well as congenital disease when administered to pregnant women. To identify the neutralizing component of cytomegalovirus hyperimmune globulin (CMV-HIG), we performed serial depletions of CMV-HIG on cell-surface-expressed CMV antigens as well as purified antigens. Using this approach, we demonstrate that the major neutralizing antibody response is directed at the gH/gL/UL128/UL130/UL131 complex, suggesting little role for anti-gB antibodies in CMV-HIG neutralization.     

Antigenic Characterization of the HCMV gH/gL/gO and Pentamer Cell Entry Complexes Reveals Binding Sites for Potently Neutralizing Human Antibodies

Human Cytomegalovirus (HCMV) is a major cause of morbidity and mortality in transplant patients and in fetuses following congenital infection. The glycoprotein complexes gH/gL/gO and gH/gL/UL128/UL130/UL131A (Pentamer) are required for HCMV entry in fibroblasts and endothelial/epithelial cells, respectively, and are targeted by potently neutralizing antibodies in the infected host. Using purified soluble forms of gH/gL/gO and Pentamer as well as a panel of naturally elicited human monoclonal antibodies, we determined the location of key neutralizing epitopes on the gH/gL/gO and Pentamer surfaces. Mass Spectrometry (MS) coupled to Chemical Crosslinking or to Hydrogen Deuterium Exchange was used to define residues that are either in proximity or part of neutralizing epitopes on the glycoprotein complexes. We also determined the molecular architecture of the gH/gL/gO- and Pentamer-antibody complexes by Electron Microscopy (EM) and 3D reconstructions. The EM analysis revealed that the Pentamer specific neutralizing antibodies bind to two opposite surfaces of the complex, suggesting that they may neutralize infection by different mechanisms. Together, our data identify the location of neutralizing antibodies binding sites on the gH/gL/gO and Pentamer complexes and provide a framework for the development of antibodies and vaccines against HCMV.     

Human Anti-Varicella-Zoster Virus (VZV) Recombinant Monoclonal Antibody Produced after Zostavax Immunization Recognizes the gH/gL Complex and Neutralizes VZV Infection

Varicella-zoster virus (VZV) is a ubiquitous, highly cell-associated, and exclusively human neurotropic alphaherpesvirus. VZV infection is initiated by membrane fusion, an event dependent in part on VZV glycoproteins gH and gL. Consistent with its location on the virus envelope, the gH/gL complex is a target of neutralizing antibodies produced after virus infection. One week after immunizing a 59-year-old VZV-seropositive man with Zostavax, we sorted his circulating blood plasma blasts and amplified expressed immunoglobulin variable domain sequences by single-cell PCR. Sequence analysis identified two plasma blast clones, one of which was used to construct a recombinant monoclonal antibody (rec-RC IgG). The rec-RC IgG colocalized with VZV gE on the membranes of VZV-infected cells and neutralized VZV infection in tissue culture. Mass spectrometric analysis of proteins immunoprecipitated by rec-RC IgG identified both VZV gH and gL. Transfection experiments showed that rec-RC IgG recognized a VZV gH/gL protein complex but not individual gH or gL proteins. Overall, our recombinant monoclonal anti-VZV antibody effectively neutralizes VZV and recognizes a conformational epitope within the VZV gH/L protein complex. An unlimited supply of this antibody provides the opportunity to analyze membrane fusion events that follow virus attachment and to identify multiple epitopes on VZV-specific proteins.     

The Viral Chemokine MCK-2 of Murine Cytomegalovirus Promotes Infection as Part of a gH/gL/MCK-2 Complex

Human cytomegalovirus (HCMV) forms two gH/gL glycoprotein complexes, gH/gL/gO and gH/gL/pUL(128,130,131A), which determine the tropism, the entry pathways and the mode of spread of the virus. For murine cytomegalovirus (MCMV), which serves as a model for HCMV, a gH/gL/gO complex functionally homologous to the HCMV gH/gL/gO complex has been described. Knock-out of MCMV gO does impair, but not abolish, virus spread indicating that also MCMV might form an alternative gH/gL complex. Here, we show that the MCMV CC chemokine MCK-2 forms a complex with the glycoprotein gH, a complex which is incorporated into the virion. We could additionally show that mutants lacking both, gO and MCK-2 are not able to produce infectious virus. Trans-complementation of these double mutants with either gO or MCK-2 showed that both proteins can promote infection of host cells, although through different entry pathways. MCK-2 has been extensively studied in vivo by others. It has been shown to be involved in attracting cells for virus dissemination and in regulating antiviral host responses. We now show that MCK-2, by forming a complex with gH, strongly promotes infection of macrophages in vitro and in vivo. Thus, MCK-2 may play a dual role in MCMV infection, as a chemokine regulating the host response and attracting specific target cells and as part of a glycoprotein complex promoting entry into cells crucial for virus dissemination.     

A Combination of Broadly Neutralizing HIV-1 Monoclonal Antibodies Targeting Distinct Epitopes Effectively Neutralizes Variants Found in Early Infection

Neutralizing antibody protection against HIV-1 may require broad and potent antibodies targeting multiple epitopes. We tested 7 monoclonal antibodies (MAbs) against 45 viruses of diverse subtypes from early infection. The CD4 binding site MAb NIH45-46W was most broad and potent (91% coverage; geometric mean 50% inhibitory concentration [IC(50)], 0.09 μg/ml). Combining NIH45-46W and a V3-specific MAb, PGT128, neutralized 96% of viruses, while PGT121, another V3-specific MAb, neutralized the remainder. Thus, 2 or 3 antibody specificities may prevent infection by most HIV-1 variants.     

Enhancement and Neutralization of Feline Infectious Peritonitis Virus Infection in Feline Macrophages by Neutralizing Monoclonal Antibodies Recognizing Different Epitopes

The interaction between the enhancing and neutralizing activities of three monoclonal antibodies (MAbs) (5-6-2, 6-4-2 and 7-4-1) to the spike protein of feline infectious peritonitis virus (FIPV) strain 79-1146 was determined using feline macrophages. At a high MAb concentration, all of the three MAbs completely inhibited the FIPV infection at 37 C. However, two of them (6-4-2 and 7-4-1) enhanced FIPV infection when either the MAb concentration or reaction temperature was lowered. These MAbs also exerted an immediate infectivity-enhancing activity for up to 10 min of reaction and by 20 min, neutralizing activities were observed. Only MAb 5-6-2 consistently showed neutralizing activity regardless of the reaction conditions. Competition with sera from cats experimentally infected with FIPV strain 79-1146 or feline enteric coronavirus strain 79-1683 showed that the two epitopes recognized by MAb 5-6-2 and MAb 6-4-2, respectively, are also recognized by the natural host.     

A Human Cytomegalovirus gO-Null Mutant Fails To Incorporate gH/gL into the Virion Envelope and Is Unable To Enter Fibroblasts and Epithelial and Endothelial Cells

Human cytomegalovirus (HCMV) depends upon a five-protein complex, gH/gL/UL128-131, to enter epithelial and endothelial cells. A separate HCMV gH/gL-containing complex, gH/gL/gO, has been described. Our prevailing model is that gH/gL/UL128-131 is required for entry into biologically important epithelial and endothelial cells and that gH/gL/gO is required for infection of fibroblasts. Genes encoding UL128-131 are rapidly mutated during laboratory propagation of HCMV on fibroblasts, apparently related to selective pressure for the fibroblast entry pathway. Arguing against this model in the accompanying paper by B. J. Ryckman et al. (J. Virol., 84:2597-2609, 2010), we describe evidence that clinical HCMV strain TR expresses a gO molecule that acts to promote endoplasmic reticulum (ER) export of gH/gL and that gO is not stably incorporated into the virus envelope. This was different from results involving fibroblast-adapted HCMV strain AD169, which incorporates gO into the virion envelope. Here, we constructed a TR gO-null mutant, TRΔgO, that replicated to low titers, spread poorly among fibroblasts, but produced normal quantities of extracellular virus particles. TRΔgO particles released from fibroblasts failed to infect fibroblasts and epithelial and endothelial cells, but the chemical fusogen polyethylene glycol (PEG) could partially overcome defects in infection. Therefore, TRΔgO is defective for entry into all three cell types. Defects in entry were explained by observations showing that TRΔgO incorporated about 5% of the quantities of gH/gL in extracellular virus particles compared with that in wild-type virions. Although TRΔgO particles could not enter cells, cell-to-cell spread involving epithelial and endothelial cells was increased relative to TR, apparently resulting from increased quantities of gH/gL/UL128-131 in virions. Together, our data suggest that TR gO acts as a chaperone to promote ER export and the incorporation of gH/gL complexes into the HCMV envelope. Moreover, these data suggest that it is gH/gL, and not gH/gL/gO, that is present in virions and is required for infection of fibroblasts and epithelial and endothelial cells. Our observations that both gH/gL and gH/gL/UL128-131 are required for entry into epithelial/endothelial cells differ from models for other beta- and gammaherpesviruses that use one of two different gH/gL complexes to enter different cells.Human cytomegalovirus (HCMV) infects a broad spectrum of cell types in vivo, including epithelial and endothelial cells, fibroblasts, monocyte-macrophages, dendritic cells, hepatocytes, neurons, glial cells, and leukocytes (6, 28, 36). Infection of this diverse spectrum of cell types contributes to the multiplicity of CMV-associated disease. HCMV infection of hepatocytes and epithelial cells in the gut and lungs following transplant immunosuppression is directly associated with CMV disease (3, 44). HCMV can be transported in the blood by monocyte-macrophages, and virus produced in these cells can infect endothelial cells, leading to virus spread into solid tissues such as the brain, liver, and lungs, etc. (16). Despite the broad spectrum of cells infected in vivo, propagation of HCMV in the laboratory is largely limited to normal human fibroblasts because other cells produce little virus. HCMV rapidly adapts to laboratory propagation in fibroblasts, losing the capacity to infect other cell types, i.e., epithelial and endothelial cells and monocyte-macrophages (9, 16, 18, 43). This adaptation to fibroblasts involves mutations in the unique long b′ (ULb′) region of the HCMV genome, which includes 22 genes (9). Targeted mutation of three of the ULb′ genes, UL128, UL130, and UL131, abolished HCMV infection of endothelial cells, transmission to leukocytes, and infection of dendritic cells (17, 18). Restoration of UL128-131 genes in HCMV laboratory strain AD169 (which cannot infect epithelial and endothelial cells) produced viruses capable of infecting these cells (18, 48). There is also evidence that the UL128-131 proteins are deleterious to HCMV replication in fibroblasts, resulting in rapid loss or mutation of one or more of the UL128-131 genes during passage in fibroblasts (2).A major step forward in understanding how the UL128-131 genes promote HCMV infection of epithelial and endothelial cells involved observations that the UL128-131 proteins assemble onto the extracellular domain of the membrane-anchored HCMV glycoprotein heterodimer gH/gL (1, 49). Antibodies to UL128, UL130, and UL131 each neutralized HCMV for infection of endothelial or epithelial cells (1, 49). All herpesviruses express gH/gL homologues and, where this has been tested, all depend upon gH/gL for replication and, more specifically, for entry into cells (14, 15, 31, 38). Indeed, we showed that the gH/gL/UL128-131 complex mediated entry into epithelial and endothelial cells (40). All five members of the gH/gL/UL128-131 complex were required for proper assembly and export from the endoplasmic reticulum (ER) and for function (39, 41). In addition, the expression of gH/gL/UL128-131, but not gH/gL or gB, in epithelial cells interfered with HCMV entry into these cells (39). This interference suggested that there are saturable gH/gL/UL128-131 receptors present on epithelial cells, molecules that HCMV uses for entry. There was no interference in fibroblasts expressing gH/gL/UL128-131, although some interference was observed with gH/gL (39). As noted above, gH/gL/UL128-131 plays no obvious role in entry into fibroblasts and, in fact, appears to be deleterious in this respect (2, 18, 40).HCMV also expresses a second gH/gL complex, as follows: gH/gL/gO (20, 21, 22, 30, 48). Fibroblast-adapted HCMV strain AD169 expresses a gO protein that is a 110- to 125-kDa glycoprotein (21). Pulse-chase studies suggest that gH/gL assembles first in the ER before binding and forming disulfide links with gO (21, 22). The 220-kDa immature gH/gL/gO complex is transported from the ER to the Golgi apparatus and increases in size to ∼280 to 300 kDa before incorporation into the virion envelope (21). gH/gL/gO complexes are apparently distinct from gH/gL/UL128-131 complexes because gO-specific antibodies do not detect complexes containing either UL128 or UL130 and UL128-specific antibodies do not precipitate gO (49). Towne and AD169 gO-null mutant laboratory strains can produce small plaques on fibroblasts, leading to the conclusion that gO is not essential. However, the AD169 and Towne mutants produced ∼1,000-fold less infectious virus than wild-type HCMV (14, 19), which might also be interpreted to mean that gO is very important or even essential for replication. Thus, the prevailing model has been that wild-type HCMV particles contain the following two gH/gL complexes: gH/gL/gO, which promotes infection of fibroblasts, and gH/gL/UL128-131, which promotes entry into epithelial and endothelial cells. Supporting this model, there are two different entry mechanisms, as follows: HCMV enters fibroblasts by fusion at the plasma membrane at neutral pH (12), whereas entry into epithelial and endothelial cells involves endocytosis and a low pH-dependent fusion with endosomes (40). This model of HCMV entry parallels models for Epstein-Barr virus (EBV) entry that use gH/gL to enter epithelial cells and gH/gL/gp42 to enter B cells (24). Similarly, HHV-6 uses gH/gL/gO and gH/gL/gQ, which bind to different receptors (33).Many of the studies of gH/gL/gO have involved the fibroblast-adapted HCMV strain AD169, which fails to express UL131 and assemble gH/gL/UL128-131 or AD169 recombinants in which UL131 expression was restored (20, 21, 22, 48, 49). It seemed possible that the adaptation of AD169 to long-term passage in fibroblasts might also involve alterations in gO. HCMV gO is unusually variable (15 to 25% amino acid differences) among different HCMV strains compared with other viral genes (13, 34, 35, 37, 46). In recent studies, Jiang et al. (26) described a gO-null mutant derived from the HCMV strain TB40/E, a strain that can infect endothelial cells following extensive passage on these cells. The TB40/E gO-null mutant spread poorly on fibroblasts compared with wild-type TB40/E, and there was little infectious virus detected in fibroblast culture supernatants. However, the few TB40/E gO-null mutant particles produced by fibroblasts that could initiate infection of endothelial cells were able to spread to form normal-sized plaques on endothelial cells. These results further supported the model for which gH/gL/gO is required for infection of fibroblasts but not for epithelial/endothelial cells. Those authors also concluded that gO is important for the assembly of enveloped particles in fibroblasts, based on observations of few infectious virus particles in supernatants and cytoplasmic accumulation of unenveloped capsids (26).Our studies of gH/gL/UL128-131 have involved the clinical HCMV strain TR (39, 40, 41, 47). HCMV TR was originally an ocular isolate from an AIDS patient (45) and was passaged only a few times on fibroblasts before being genetically frozen in the form of a bacterial artificial chromosome (BAC) (34, 40). HCMV TR infects epithelial and endothelial cells (40) and monocyte-macrophages (D. Streblow and J. Nelson, unpublished results) well. In the accompanying paper (42), we characterized the biochemistry and intracellular trafficking of TR gO. TR gO expressed either in TR-infected cells or by using adenovirus vectors (expressed without other HCMV proteins) was largely retained in the ER. Coexpression of gO with gH/gL promoted transport of gH/gL beyond the ER. Importantly, TR gO was not found in extracellular virions. In contrast, AD169 gO was present in extracellular virus particles, as described previously (20, 21). We concluded that TR gO is a chaperone that promotes ER export of the gH/gL complex, but gO dissociates prior to incorporation into the virus envelope. Moreover, these differences highlight major differences between gO molecules expressed by fibroblast-adapted strain AD169 and low-passage TR.To extend these results and characterize how TR gO functions, whether in virus entry or virus assembly/egress, we constructed a TR gO-null mutant. TRΔgO exhibited major defects in entering fibroblasts, as evidenced by increased virus infection following treatment with the chemical fusogen polyethylene glycol (PEG). Unexpectedly, the mutant also failed to enter epithelial and endothelial cells, and again, PEG partially restored entry. Relatively normal numbers of TRΔgO particles were produced and released into cell culture supernatants, although even with PEG treatment, most of these virus particles remained defective in initiating immediate-early HCMV protein synthesis. Western blot analyses of TRΔgO extracellular particles demonstrated very low levels of gH/gL incorporated into virions, which likely explains the reduced entry of TRΔgO. However, the small amounts of gH/gL complexes that were present in TRΔgO virions were associated with increased quantities of UL130, and these TRΔgO particles spread better than wild-type HCMV on epithelial cell monolayers. Together with the results shown in the accompanying paper (42), we concluded that HCMV TR gO functions as a chaperone to promote ER export of gH/gL to HCMV assembly compartments and the incorporation of gH/gL into the virion envelope. The highly reduced quantities of gH/gL in virions are apparently responsible for the inability of HCMV to enter fibroblasts and epithelial and endothelial cells. These results suggest a modified version of our model, in which gH/gL, not gH/gL/gO, mediates entry into fibroblasts and both gH/gL and gH/gL/UL128-131 are required for entry into epithelial and endothelial cells.     

Mapping the N-Terminal Residues of Epstein-Barr Virus gp42 That Bind gH/gL by Using Fluorescence Polarization and Cell-Based Fusion Assays

Epstein-Barr virus (EBV) requires at a minimum membrane-associated glycoproteins gB, gH, and gL for entry into host cells. B-cell entry additionally requires gp42, which binds to gH/gL and triggers viral entry into B cells. The presence of soluble gp42 inhibits membrane fusion with epithelial cells by forming a stable heterotrimer of gH/gL/gp42. The interaction of gp42 with gH/gL has been previously mapped to residues 36 to 81 at the N-terminal region of gp42. In this study, we further mapped this region to identify essential features for binding to gH/gL by use of synthetic peptides. Data from fluorescence polarization, cell-cell fusion, and viral infection assays demonstrated that 33 residues corresponding to 44 to 61 and 67 to 81 of gp42 were indispensable for maintaining low-nanomolar-concentration gH/gL binding affinity and inhibiting B-cell fusion and epithelial cell fusion as well as viral infection. Overall, specific, large hydrophobic side chain residues of gp42 appeared to provide critical interactions, determining the binding strength. Mutations of these residues also diminished the inhibition of B-cell and epithelial cell fusions as well as EBV infection. A linker region (residues 62 to 66) between two gH/gL binding regions served as an important spacer, but individual amino acids were not critical for gH/gL binding. Probing the binding site of gH/gL and gp42 with gp42 peptides is critical for a better understanding of the interaction of gH/gL with gp42 as well as for the design of novel entry inhibitors of EBV and related human herpesviruses.Epstein-Barr virus (EBV) is a large DNA virus belonging to the family of gammaherpesviruses. The virus is transmitted through saliva, and it can infect epithelial cells, as well as B cells, which provide the host latency reservoir (1, 22). Reactivation of the virus can occur intermittently, allowing virus infection of new hosts (1). Viral reactivation from latency is quickly controlled by the immune system. Primary infection with EBV can lead to the development of infectious mononucleosis. In addition, EBV infection is associated with a variety of human cancers, such as nasopharyngeal carcinoma, Hodgkin''s lymphoma, and Burkitt''s lymphoma (4, 8, 9, 27, 30). EBV is an enveloped virus which contains a number of membrane glycoproteins required for membrane fusion and viral entry into the host cell. EBV-mediated entry into epithelial cells requires the three viral glycoproteins gB, gH, and gL, which are conserved among herpesviruses, and entry into B cells additionally requires the viral glycoprotein gp42 (7, 16, 17). EBV lacking gp42 can attach to B cells but cannot enter them (29). However, EBV lacking gp42 can still efficiently infect epithelial cells. In fact, gp42 acts as an inhibitor of epithelial cell infection, and recent studies suggest that the level of gp42 in the virion regulates whether EBV preferentially infects epithelial cells or B cells (2). EBV gp42 has been shown to play an essential role in membrane fusion with B cells (7, 16, 17). It binds to human leukocyte antigen class II (HLA class II) proteins expressed on B cells to trigger virus-cell membrane fusion (6, 7, 10, 16, 25).Interestingly, EBV gp42 occurs in two forms in infected cells, a full-length membrane-bound form and a soluble form generated by proteolytic cleavage that is secreted from infected cells due to loss of the N-terminal transmembrane domain (21). Both the full-length form and the secreted gp42 form bind to gH/gL and HLA class II, and the functional significance of gp42 cleavage is not completely clear. In a virus-free cell-cell fusion assay, enhanced secretion of gp42 promotes fusion with B lymphocytes. Cleavage and secretion of gp42 are necessary for membrane fusion with B lymphocytes (24). However, membrane fusion with epithelial cells is inhibited by the presence of gp42 for both virus infection and cell-cell fusion (14, 29). This is likely due to the formation of a heterotrimeric gH/gL/gp42 complex that is unable to mediate membrane fusion with epithelial cells, possibly due to steric hindrance of gH/gL receptor binding (3, 11).The interaction of gH/gL and gp42 plays a key role in membrane fusion, but it has not yet been fully understood. The crystal structures of a gH/gL/gp42 complex and gH/gL alone have not been available. Although the crystal structures of gp42 alone and gp42/HLA class II complex have been solved (15, 19), the N-terminal region of gp42 (bound to gH/gL) is not visible in the structures, most likely due to its flexibility. Previous studies have shown that the N-terminal region of gp42 contains multiple functional regions, including a cleavage site that results in the secretion of gp42, a potential homodimerization region, and two segments (15 residues each) required for gH/gL binding (Fig. ​(Fig.11 A) (13, 14). Extensive gp42 N-terminal deletion analysis demonstrated that residues 37 to 56 and 72 to 96 include functional regions of the N terminus of gp42 required to trigger fusion and suggested that some of the residues within residues 67 to 71 are also important. Additional experiments showed that amino acids within segments from residues 47 to 61 and 67 to 81 are critical for binding gH/gL (13). Those two segments are spaced by five residues, which appear to act as a linker. Previous studies also showed that a 46-mer peptide spanning residues 36 to 81, mimicking the full-length gp42, binds to gH/gL and inhibits the formation of gH/gL/gp42 complex, thus blocking membrane fusion with epithelial cells and fusion with B cells (13, 14).Open in a separate windowFIG. 1.Schematic representation of EBV gp42. (A) Representation of wild-type gp42 showing the relative locations of known functional domains. The transmembrane domain is predicted to span residues 9 to 22 and is shown as a gray box. The site of gp42 cleavage is between residues 40 and 42 and is indicated by a black bar. The two gH/gL binding regions, spanning residues 47 to 61 and 67 to 87, are indicted with hatched boxes, flanking the five-residue linker. The C-terminal C-type lectin domain, including the hydrophobic pocket and HLA class II-binding region, is indicated by cross-hatched boxes. The putative dimerization region is indicated by a dotted box. (B) Amino acid sequence of gp42 peptide spanning residues 36 to 81 of the gp42 protein.In order to obtain a better understanding of the interaction of gp42 with gH/gL, and the role of this interaction in membrane fusion, we probed the gH/gL binding site by using 27 synthetic peptide analogs spanning residues 36 to 81 of gp42. Peptides were tested for binding affinity to soluble gH/gL by using a fluorescence polarization (FP) assay, probed for inhibition of B-cell and epithelial cell fusion in cell-based assays, and finally investigated for their ability to block epithelial cell infection. The data from the FP assay agreed very well with cell-cell fusion data and infection data, providing correlative data for peptide binding affinity and inhibition of cell-cell fusion and infection in an apparent competitive manner. We have defined the minimal length requirements for high-affinity binding to gH/gL and obtained a more detailed map of the key amino acids of the gp42 N terminus that are necessary for optimal gH/gL binding and inhibition of epithelial cell and B-cell membrane fusion.