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.     

Human cytomegalovirus glycoprotein gO complexes with gH/gL, promoting interference with viral entry into human fibroblasts but not entry into epithelial cells

A complex of five human cytomegalovirus virus (HCMV) proteins, gH, gL, UL128, UL130, and UL131 (gH/gL/UL128-131), is essential for virus entry into epithelial cells. We previously showed that gH/gL/UL128-131 expressed in epithelial cells interferes with subsequent HCMV entry into cells. There was no interference with only gH/gL or gB. We concluded that the expression of gH/gL/UL128-131 causes a mislocalization or downregulation of epithelial cell proteins that HCMV requires for entry. In contrast, gH/gL/UL128-131 expression in fibroblasts did not produce interference, suggesting a different mechanism for entry. Here, we show that the coexpression of another HCMV glycoprotein, gO, with gH/gL in human fibroblasts interferes with HCMV entry into fibroblasts but not epithelial cells. However, the coexpression of gO with gH/gL did not increase the cell surface expression level of gH/gL and did not enhance cell-cell fusion, a process that depends upon cell surface gH/gL. Instead, gO promoted the export of gH/gL from the endoplasmic reticulum (ER) and the accumulation of gH/gL in the trans-Golgi network. Thus, interference with gH/gL or gH/gL/gO, i.e., the mislocalization or blocking of entry mediators, occurs in cytoplasmic membranes and not in cell surface membranes of fibroblasts. Together, the results provide additional support for our hypotheses that epithelial cells express putative gH/gL/UL128-1331 receptors important for HCMV entry and that fibroblasts express distinct gH/gL receptors.     

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.     

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.     

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.     

UL74 of human cytomegalovirus contributes to virus release by promoting secondary envelopment of virions

The glycoprotein (g) complex gH/gL represents an essential part of the herpesvirus fusion machinery mediating entry of cell-free virions and cell-associated viral spread. In some herpesviruses additional proteins are associated with gH/gL contributing to the cell tropism of the respective virus. Human cytomegalovirus (HCMV) gH/gL forms complexes with either gO (UL74) or proteins of the UL128-131A gene locus. While a contribution of UL128-131A to endothelial cell tropism is known, the role of gO is less clear. We studied the role of gH/gL-associated proteins in HCMV replication in human foreskin fibroblasts (HFF) and human umbilical vein endothelial cells (HUVEC). Deletions of UL74 alone or in combination with mutations of the UL128-131A gene region were introduced into bacterial artificial chromosome vectors derived from the endotheliotropic strain TB40/E. Deletion of UL74 caused a profound defect regarding virus release from infected HFF and HUVEC. Large numbers of capsids accumulated in the cytoplasm of infected HFF but failed to acquire an envelope. Clear cell type differences were observed in the cell-associated spread of the UL74-defective virus. In HFF, focal growth was severely impaired, whereas it was normal in HUVEC. Deletion of UL131A abolished focal growth in endothelial cells. UL74/UL128-131A dual mutants showed severely impaired reconstitution efficiency. Our data suggest that gO plays a critical role in secondary envelopment and release of cell-free virions independent of the cell type but affects cell-associated growth specifically in HFF, whereas UL128-131A contributes to cell-associated spread in HFF and HUVEC.     

PDGF Receptor-α Does Not Promote HCMV Entry into Epithelial and Endothelial Cells but Increased Quantities Stimulate Entry by an Abnormal Pathway

Epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor-α (PDGFRα) were reported to mediate entry of HCMV, including HCMV lab strain AD169. AD169 cannot assemble gH/gL/UL128–131, a glycoprotein complex that is essential for HCMV entry into biologically important epithelial cells, endothelial cells, and monocyte-macrophages. Given this, it appeared incongruous that EGFR and PDGFRα play widespread roles in HCMV entry. Thus, we investigated whether PDGFRα and EGFR could promote entry of wild type HCMV strain TR. EGFR did not promote HCMV entry into any cell type. PDGFRα–transduction of epithelial and endothelial cells and several non-permissive cells markedly enhanced HCMV TR entry and surprisingly, promoted entry of HCMV mutants lacking gH/gL/UL128–131 into epithelial and endothelial cells. Entry of HCMV was not blocked by a panel of PDGFRα antibodies or the PDGFR ligand in fibroblasts, epithelial, or endothelial cells or by shRNA silencing of PDGFRα in epithelial cells. Moreover, HCMV glycoprotein induced cell-cell fusion was not increased when PDGFRα was expressed in cells. Together these results suggested that HCMV does not interact directly with PDGFRα. Instead, the enhanced entry produced by PDGFRα resulted from a novel entry pathway involving clathrin-independent, dynamin-dependent endocytosis of HCMV followed by low pH-independent fusion. When PDGFRα was expressed in cells, an HCMV lab strain escaped endosomes and tegument proteins reached the nucleus, but without PDGFRα virions were degraded. By contrast, wild type HCMV uses another pathway to enter epithelial cells involving macropinocytosis and low pH-dependent fusion, a pathway that lab strains (lacking gH/gL/UL128–131) cannot follow. Thus, PDGFRα does not act as a receptor for HCMV but increased PDGFRα alters cells, facilitating virus entry by an abnormal pathway. Given that PDGFRα increased infection of some cells to 90%, PDGFRα may be very useful in overcoming inefficient HCMV entry (even of lab strains) into the many difficult-to-infect cell types.     

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.     

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.     

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.     

Coexpression of UL20p and gK inhibits cell-cell fusion mediated by herpes simplex virus glycoproteins gD, gH-gL, and wild-type gB or an endocytosis-defective gB mutant and downmodulates their cell surface expression

Syncytium formation in cells that express herpes simplex virus glycoprotein B (gB), gD, gH, and gL is blocked by gK (E. Avitabile, G. Lombardi, and G. Campadelli-Fiume, J. Virol. 77:6836-6844, 2003). Here, we report the results of two series of experiments. First, UL20 protein (UL20p) expression weakly inhibited cell-cell fusion. Coexpression of UL20p and gK drastically reduced fusion in a cell-line-dependent manner, with the highest inhibition in BHK cells. Singly expressed UL20p and gK localized at the endoplasmic reticulum and nuclear membranes. When they were coexpressed, both proteins relocalized to the Golgi apparatus. Remarkably, in cells that coexpressed UL20p and gK, the antifusion activity correlated with a downmodulation of gD, gB, gH, and gL cell surface expression. Second, gB(Delta867) has a partial deletion in the cytoplasmic tail that removed endocytosis motifs. Whereas wild-type (wt) gB was internalized in vesicles lined with the endosomal marker Rab5, gB(delta867) was not internalized, exhibited enhanced cell surface expression, and was more efficient in mediating cell-cell fusion than wt gB. The antifusion activity of UL20p and gK was also exerted when gB(delta867) replaced wt gB in the cell fusion assay. These studies show that the gB C tail carries a functional endocytosis motif(s) and that the removal of the motif correlated with increased gB surface expression and increased fusion activity. We conclude that cell-cell fusion in wt-virus-infected cells is negatively controlled by at least two mechanisms. The novel mechanism described here involves the concerted action of UL20p and gK and correlates with a moderate but consistent reduction in the cell surface expression of the fusion glycoproteins. This mechanism is independent of the one exerted through endocytosis-mediated downmodulation of gB from the plasma membrane.     

Coiled-coil domains in glycoproteins B and H are involved in human cytomegalovirus membrane fusion

Human cytomegalovirus (CMV) utilizes a complex route of entry into cells that involves multiple interactions between viral envelope proteins and cellular receptors. Three conserved viral glycoproteins, gB, gH, and gL, are required for CMV-mediated membrane fusion, but little is known of how these proteins cooperate during entry (E. R. Kinzler and T. Compton, submitted for publication). The goal of this study was to begin defining the molecular mechanisms that underlie membrane fusion mediated by herpesviruses. We identified heptad repeat sequences predicted to form alpha-helical coiled coils in two glycoproteins required for fusion, gB and gH. Peptides derived from gB and gH containing the heptad repeat sequences inhibited virus entry when introduced coincident with virus inoculation onto cells or when mixed with virus prior to inoculation. Neither peptide affected binding of CMV to fibroblasts, suggesting that the peptides inhibit membrane fusion. Both gB and gH coiled-coil peptides blocked entry of several laboratory-adapted and clinical strains of human CMV, but neither peptide affected entry of murine CMV or herpes simplex virus type 1 (HSV-1). Although murine CMV and HSV-1 gB and gH have heptad repeat regions, the ability of human CMV gB and gH peptides to inhibit virus entry correlates with the specific residues that comprise the heptad repeat region. The ability of gB and gH coiled-coil peptides to inhibit virus entry independently of cell contact suggests that the coiled-coil regions of gB and gH function differently from those of class I, single-component fusion proteins. Taken together, these data support a critical role for alpha-helical coiled coils in gB and gH in the entry pathway of CMV.     

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.     

Herpes Simplex Virus 1 Glycoprotein M and the Membrane-Associated Protein UL11 Are Required for Virus-Induced Cell Fusion and Efficient Virus Entry

Herpes simplex virus 1 (HSV-1) facilitates virus entry into cells and cell-to-cell spread by mediating fusion of the viral envelope with cellular membranes and fusion of adjacent cellular membranes. Although virus strains isolated from herpetic lesions cause limited cell fusion in cell culture, clinical herpetic lesions typically contain large syncytia, underscoring the importance of cell-to-cell fusion in virus spread in infected tissues. Certain mutations in glycoprotein B (gB), gK, UL20, and other viral genes drastically enhance virus-induced cell fusion in vitro and in vivo. Recent work has suggested that gB is the sole fusogenic glycoprotein, regulated by interactions with the viral glycoproteins gD, gH/gL, and gK, membrane protein UL20, and cellular receptors. Recombinant viruses were constructed to abolish either gM or UL11 expression in the presence of strong syncytial mutations in either gB or gK. Virus-induced cell fusion caused by deletion of the carboxyl-terminal 28 amino acids of gB or the dominant syncytial mutation in gK (Ala to Val at amino acid 40) was drastically reduced in the absence of gM. Similarly, syncytial mutations in either gB or gK did not cause cell fusion in the absence of UL11. Neither the gM nor UL11 gene deletion substantially affected gB, gC, gD, gE, and gH glycoprotein synthesis and expression on infected cell surfaces. Two-way immunoprecipitation experiments revealed that the membrane protein UL20, which is found as a protein complex with gK, interacted with gM while gM did not interact with other viral glycoproteins. Viruses produced in the absence of gM or UL11 entered into cells more slowly than their parental wild-type virus strain. Collectively, these results indicate that gM and UL11 are required for efficient membrane fusion events during virus entry and virus spread.     

Rational development of beta-peptide inhibitors of human cytomegalovirus entry

Human cytomegalovirus (HCMV) is a pervasive and significant pathogen. At present, there is no HCMV vaccine, and the available drugs target only replication events. Thus, new therapeutic strategies are needed. HCMV fusion appears to require interactions of alpha-helical regions in viral surface glycoproteins gB and gH. Oligomers of beta-amino acids ("beta-peptides") are attractive unnatural scaffolds for mimicry of specific protein surfaces, because beta-peptides adopt predictable helical conformations and resist proteolysis. Here, we report the development of beta-peptides designed to mimic the gB heptad repeat and block HCMV entry. The most potent beta-peptide inhibits HCMV infection in a cell based-assay with an IC50 of approximately 30 microm. Consistent with our structure-based design strategy, inhibition is highly specific for HCMV relative to other related viruses. Mechanistic studies indicate that inhibitory beta-peptides act by disrupting membrane fusion. Our findings raise the possibility that beta-peptides may provide a general platform for development of a new class of antiviral agents and that inhibitory beta-peptides will constitute new tools for elucidating viral entry mechanisms.     

Protein pUL128 of human cytomegalovirus is necessary for monocyte infection and blocking of migration

We have previously shown that only endotheliotropic strains of human cytomegalovirus (HCMV), such as TB40E, infect monocytes and impair their chemokine-driven migration. The proteins encoded by the UL128-131A region (UL128, UL130, and UL131A) of the HCMV genome, which assemble into a pentameric gH-gL-UL128-UL130-UL131A envelope complex, have been recognized as determinants for HCMV endothelial cell tropism. The genes for these proteins are typically inactivated by mutations in all fibroblast-adapted strains that have lost the diversified tropism of clinical isolates. By using mutant HCMV reconstituted from TB40E-derived bacterial artificial chromosomes (BAC) encoding a wild-type (wt) or mutated form of UL128, we show here that UL128-131A products are essential determinants of infection in monocytes and that pUL128, in particular, can block chemokine-driven motility. The virus BAC4, encoding wt UL128, established infection in monocytes, induced the intracellular retention of several chemokine receptors, and rendered monocytes unresponsive to different chemokines. In contrast, the virus BAC1, encoding a mutated UL128, failed to infect monocytes and to downregulate chemokine receptors. BAC1-exposed monocytes did not express immediate-early (IE) products, retained virions in cytoplasmic vesicles, and exhibited normal chemokine responsiveness. A potential role of second-site mutations in the observed phenotype was excluded by using the revertant viruses BAC1rep and BAC4mut. By incubating noninfected monocytes with soluble recombinant pUL128, we observed both the block of migration and the chemokine receptor internalization. We propose that among the gH-gL-UL128-UL130-UL131A complex subunits, the UL128 protein is the one that triggers monocyte paralysis.     

Lipid composition modulates the interaction of peptides deriving from herpes simplex virus type I glycoproteins B and H with biomembranes

Lipid membranes play a key role in the viral life cycle. Enveloped viruses particularly require a sequence of fusion and fission events between the viral envelope and the target membranes for entry into the cell and egress from it. These processes are controlled by one or more viral glycoproteins that undergo conformational changes favoring the necessary micro- and mesoscopic lipid re-arrangements. Multiple regions from these glycoproteins are thought to interact with the membranes, according to a concerted mechanism, in order to generate the distortion necessary for fusion. In this work, we perform an EPR study on the role played by the membrane composition in tuning the interaction between lipid bilayers and two peptides, gH626-644 and gB632-650, that are highly fusogenic fragments of the gH and gB glycoproteins of herpes simplex virus. Our results show that both peptides interact with lipid bilayers, perturbing the local lipid packing. gH626-644 localizes close to the hydrophilic bilayer surface, while gB632-650 penetrates deeply into the membrane. Chain perturbation by the peptides increases in the presence of charged phospholipids. Finally, cholesterol does not alter the ability of gB632-650 to penetrate deeply in the membrane, whereas it limits penetration of the gH626-644 peptide to the more external layer. The different modes of interaction result in a higher fusogenic ability of gB632-650 towards cholesterol-enriched membranes, as demonstrated by lipid mixing assays. These results suggest that the mechanism of action of the gH and gB glycoproteins is modulated by the properties and composition of the phospholipid bilayer.     

Soluble Human Cytomegalovirus gH/gL/pUL128–131 Pentameric Complex,but Not gH/gL,Inhibits Viral Entry to Epithelial Cells and Presents Dominant Native Neutralizing Epitopes

Congenital infection of human cytomegalovirus (HCMV) is one of the leading causes of nongenetic birth defects, and development of a prophylactic vaccine against HCMV is of high priority for public health. The gH/gL/pUL128–131 pentameric complex mediates HCMV entry into endothelial and epithelial cells, and it is a major target for neutralizing antibody responses. To better understand the mechanism by which antibodies interact with the epitopes of the gH/gL/pUL128–131 pentameric complex resulting in viral neutralization, we expressed and purified soluble gH/gL/pUL128–131 pentameric complex and gH/gL from Chinese hamster ovary cells to >95% purity. The soluble gH/gL, which exists predominantly as (gH/gL)₂ homodimer with a molecular mass of 220 kDa in solution, has a stoichiometry of 1:1 and a pI of 6.0–6.5. The pentameric complex has a molecular mass of 160 kDa, a stoichiometry of 1:1:1:1:1, and a pI of 7.4–8.1. The soluble pentameric complex, but not gH/gL, adsorbs 76% of neutralizing activities in HCMV human hyperimmune globulin, consistent with earlier reports that the most potent neutralizing epitopes for blocking epithelial infection are unique to the pentameric complex. Functionally, the soluble pentameric complex, but not gH/gL, blocks viral entry to epithelial cells in culture. Our results highlight the importance of the gH/gL/pUL128–131 pentameric complex in HCMV vaccine design and emphasize the necessity to monitor the integrity of the pentameric complex during the vaccine manufacturing process.     

Glycoproteins required for entry are not necessary for egress of pseudorabies virus

In the current perception of the herpesvirus replication cycle, two fusion processes are thought to occur during entry and nuclear egress. For penetration, glycoproteins gB and gH/gL have been shown to be essential, whereas a possible role of these glycoproteins in nuclear egress remains unclear. Viral envelope glycoproteins have been detected by immunolabeling in the nuclear membrane as well as in primary enveloped particles in several herpesviruses, indicating that they might be involved in the fusion process. Moreover, a herpes simplex virus type 1 mutant simultaneously lacking gB and gH was described to be deficient in nuclear egress (A. Farnsworth, T. W. Wisner, M. Webb, R. Roller, G. Cohen, R. Eisenberg, and D. C. Johnson, Proc. Natl. Acad. Sci. USA 104:10187-10192, 2007). To analyze the situation in the related alphaherpesvirus pseudorabies virus (PrV), mutants carrying single and double deletions of glycoproteins gB, gD, gH, and gL were constructed and characterized. We show here that the simultaneous deletion of gB and gD, gB and gH, gD and gH, or gH and gL has no detectable effect on PrV egress, implying that none of these glycoproteins either singly or in the tested combinations is required for nuclear egress. In addition, immunolabeling studies using different mono- or polyclonal sera raised against various PrV glycoproteins did not reveal the presence of viral glycoproteins in the inner nuclear membrane or in primary virions. Thus, our data strongly suggest that different fusion mechanisms are active during virus entry and egress.