The murid herpesvirus-4 gH/gL binds to glycosaminoglycans

The first contact a virus makes with cells is an important determinant of its tropism. Murid Herpesvirus-4 (MuHV-4) is highly dependent on glycosaminoglycans (GAGs) for cell binding. Its first contact is therefore likely to involve a GAG-binding virion glycoprotein. We have previously identified two such proteins, gp70 and gp150. Gp70 binds strongly to GAGs. However, deleting it makes little difference to MuHV-4 cell binding or GAG-dependence. Deleting gp150, by contrast, frees MuHV-4 from GAG dependence. This implies that GAGs normally displace gp150 to allow GAG-independent cell binding. But the gp150 GAG interaction is weak, and so would seem unlikely to make an effective first contact. Since neither gp70 nor gp150 matches the expected profile of a first contact glycoprotein, our understanding of MuHV-4 GAG interactions must be incomplete. Here we relate the seemingly disconnected gp70 and gp150 GAG interactions by showing that the MuHV-4 gH/gL also binds to GAGs. gH/gL-blocking and gp70-blocking antibodies individually had little effect on cell binding, but together were strongly inhibitory. Thus, there was redundancy in GAG binding between gp70 and gH/gL. Gp150-deficient MuHV-4 largely resisted blocks to gp70 and gH/gL binding, consistent with its GAG independence. The failure of wild-type MuHV-4 to do the same argues that gp150 is normally engaged only down-stream of gp70 or gH/gL. MuHV-4 GAG dependence is consequently two-fold: gp70 or gH/gL binding provides virions with a vital first foothold, and gp150 is then engaged to reveal GAG-independent binding.     

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.     

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.     

Kaposi's sarcoma-associated herpesvirus gH/gL: glycoprotein export and interaction with cellular receptors

The attachment, entry, and fusion of Kaposi's sarcoma-associated herpesvirus (KSHV) with target cells are mediated by complex machinery containing, among others, viral glycoprotein H (gH) and its alleged chaperone, gL. We observed that KSHV gH, in contrast to its homologues in several other herpesviruses, is transported to the cytoplasm membrane independently from gL, but not vice versa. Mutational analysis revealed that the N terminus of gH is sufficient for gL interaction. However, the entire extracellular part of gH is required for efficient gL secretion. The soluble ectodomain of gH was sufficient to interact with the surfaces of potential target cells in a heparin-dependent manner, and binding was further enhanced by coexpression of gL. Surface plasmon resonance revealed a remarkably high affinity of gH for glycosaminoglycans. Heparan sulfate (HS) proteoglycans of the syndecan family act as cellular receptors for the gH/gL complex. They promoted KSHV infection, and expression of gH/gL on target cells inhibited subsequent KSHV infection. Whereas gH alone was able to bind to HS, we observed that only the gH/gL complex adhered to heparan sulfate-negative cells at lamellipodium-like structures.     

N-terminal mutants of herpes simplex virus type 2 gH are transported without gL but require gL for function

Glycoprotein H (gH) is conserved among all herpesviruses and is essential for virus entry and cell fusion along with gL, gB, and, in most alphaherpesviruses, gD. Within the gH/gL heterodimer, it is thought that gH accounts for the fusion function and gL acts as a chaperone for the folding and transport of gH. Here, we found that the N terminus of gH2 contains important elements involved in both its folding and its transport. Our conclusions are based on the phenotypes of a series of gH deletion mutants in which the signal sequence (residues 1 to 18) was retained and N-terminal residues were removed up to the number indicated. The first mutant, gH2Delta29 (deletion of residues 19 to 28), like wild-type (WT) gH, required gL for both transport and function. To our surprise, two other mutants (gH2Delta64 and gH2Delta72) were transported to the cell surface independent of gL but were nonfunctional, even when complexed with gL. Importantly, a fourth mutant (gH2Delta48) was transported independent of gL but was functional only when complexed with gL. Using a panel of monoclonal antibodies against gH2, we found that when gH2Delta48 was expressed alone, its antigenic structure differed from that of gH2Delta48/gL or gH2-WT/gL. Mutation of gH2 residue R39, Y41, W42, or D44 allowed gL-independent transport of gH. Our results also show that gL is not merely required for gH transport but is also necessary for the folding and function of the complex. Since gH2Delta64/gL and gH2Delta72/gL were nonfunctional, we hypothesized that residues critical for gH/gL function lie within this deleted region. Additional mutagenesis identified L66 and L72 as important for function. Together, our results highlight several key gH residues: R39, Y41, W42, and D44 for gH transport and L66 and L72 for gH/gL structure and function.     

The Murid Herpesvirus-4 gL regulates an entry-associated conformation change in gH

The glycoprotein H (gH)/gL heterodimer is crucial for herpesvirus membrane fusion. Yet how it functions is not well understood. The Murid Herpesvirus-4 gH, like that of other herpesviruses, adopts its normal virion conformation by associating with gL. However, gH switched back to a gL-independent conformation after virion endocytosis. This switch coincided with a conformation switch in gB and with capsid release. Virions lacking gL constitutively expressed the down-stream form of gH, prematurely switched gB to its down-stream form, and showed premature capsid release with poor infectivity. These data argue that gL plays a key role in regulating a gH and gB functional switch from cell binding to membrane fusion.     

Mutations in the Amino Terminus of Herpes Simplex Virus Type 1 gL Can Reduce Cell-Cell Fusion without Affecting gH/gL Trafficking

The gH/gL heterodimer represents two of the four herpes simplex virus glycoproteins necessary and sufficient for membrane fusion. We generated deletions and point mutations covering gL residues 24 to 43 to investigate that region''s role in gH/gL intracellular trafficking and in membrane fusion. Multiple mutants displayed a 40 to 60% reduction in cell fusion with no effect on gH/gL trafficking. The amino terminus of gL plays an important role in the gH/gL contribution to membrane fusion.     

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 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.     

Cascade of Events Governing Cell-Cell Fusion Induced by Herpes Simplex Virus Glycoproteins gD,gH/gL,and gB

Herpesviruses minimally require the envelope proteins gB and gH/gL for virus entry and cell-cell fusion; herpes simplex virus (HSV) additionally requires the receptor-binding protein gD. Although gB is a class III fusion protein, gH/gL does not resemble any documented viral fusion protein at a structural level. Based on those data, we proposed that gH/gL does not function as a cofusogen with gB but instead regulates the fusogenic activity of gB. Here, we present data to support that hypothesis. First, receptor-positive B78H1-C10 cells expressing gH/gL fused with receptor-negative B78H1 cells expressing gB and gD (fusion in trans). Second, fusion occurred when gH/gL-expressing C10 cells preexposed to soluble gD were subsequently cocultured with gB-expressing B78 cells. In contrast, prior exposure of gB-expressing C10 cells to soluble gD did not promote subsequent fusion with gH/gL-expressing B78 cells. These data suggest that fusion involves activation of gH/gL by receptor-bound gD. Most importantly, soluble gH/gL triggered a low level of fusion of C10 cells expressing gD and gB; a much higher level was achieved when gB-expressing C10 cells were exposed to a combination of soluble gH/gL and gD. These data clearly show that gB acts as the HSV fusogen following activation by gD and gH/gL. We suggest the following steps leading to fusion: (i) conformational changes to gD upon receptor binding, (ii) alteration of gH/gL by receptor-activated gD, and (iii) upregulation of the fusogenic potential of gB following its interaction with activated gH/gL. The third step may be common to other herpesviruses.Herpesviruses enter cells by fusing their envelopes with host cell membranes either by direct fusion at the plasma membrane or by pH-dependent or -independent endocytosis, depending on the target cell (27, 29, 39). Although the entry pathways of other enveloped viruses are similarly diverse (8), all systems for which molecular details have been obtained rely on a single fusion protein (43); herpesviruses are unique in their use of gB and the gH/gL heterodimer as their core fusion machinery (17, 37). Some herpesviruses employ additional receptor-binding glycoproteins, e.g., herpex simplex virus (HSV) gD, and others require gH/gL-associated proteins, e.g., UL128-131 of cytomegalovirus (CMV) (34) or gp42 of Epstein-Barr virus (EBV) (42). This complexity has made it difficult to unravel the mechanism of herpesvirus entry.Ultrastructural and biochemical studies have shown that for HSV entry, binding of gD to one of its receptors, either HVEM or nectin-1 (36), activates the downstream events that drive gB- and gH/gL-dependent fusion (17). The structure of the gB ectodomain (18) bears striking structural homology to the postfusion form of the single fusion protein G of vesicular stomatitis virus (VSV) (33). However, unlike the other class III viral fusion proteins, VSV G and baculovirus gp64 (5), gB requires gH/gL to function in virus-cell and cell-cell fusion (17). A number of investigations support the concept that gH/gL might also be fusogenic (13, 41). Some have suggested that a multiprotein complex comprised of gD, gH/gL, and gB might be assembled to cause fusion (14). Using bimolecular complementation (BiMC), we and others showed that interactions can occur between half enhanced yellow fluorescent protein (EYFP)-tagged gB (e.g., gBn) and tagged gD (e.g., gDc) or between tagged gD and tagged gH (1, 3). However, because these occur in the absence of one of the other essential components, e.g., a receptor, we could not assess their functional significance. Importantly, gH/gL and gB interact with each other only in response to receptor binding by gD (1-3, 12). We subsequently showed that this interaction precedes fusion and is required for it to occur (2). Thus, we were able to conclude that gH/gL must interact with gB, whether transiently or stably, in order for fusion to occur. Whether gD was indeed involved in a multiprotein complex was not clear, nor was the role of gH/gL in promoting fusion initiated by gD-receptor binding. The lack of structural data for gH/gL left its potential role as a fusogen unresolved.However, in 2010, the structure of gH/gL of HSV-2 was solved in collaboration with Chowdary et al. (12). Structurally, gH/gL does not resemble any known viral fusogen, thereby forcing a reconsideration of its function in promoting virus-cell and cell-cell fusion. We hypothesized that gH/gL does not likely act as a cofusogen with gB but rather regulates fusion by gB (12).In this report, we argue that as a regulator of fusion, gH/gL might not have to be in the same membrane as gB in order to regulate its activity, i.e., gH/gL on one cell might promote fusion of gB expressed by another cell, as long as gD and a gD receptor are also present. In support of this, it was recently shown that gH/gL and gB of human cytomegalovirus (HCMV) can cause cell-cell fusion when expressed by distinct cells (in trans) (41). We present evidence that HSV gB and gH/gL can cause cell-cell fusion when they are expressed in trans, a process that requires both gD and a gD receptor. Although the efficiency of fusion in trans is low compared with that of fusion when gB and gH/gL are in cis (as they would be when in the virus), separation of these proteins onto two different cells enabled us to dissect the order in which each protein acts along the pathway to fusion. Moreover, we found that a combination of soluble gD (not membrane bound) and soluble gH/gL (also not membrane bound) could trigger fusion of receptor-bearing cells that had been transfected with the gene for gB. Our data show that gD, gH/gL, and gB act in a series of steps whereby gD is first activated by binding its cell receptor. Previous studies showed that receptor binding causes gD to undergo conformational changes (17). Based on the data in this paper, we propose that these changes then enable gD to activate gH/gL into a form that in turn binds to and activates the fusogenic activity of gB. Although we do not know whether any of these reactions result in the formation of a stable complex, our data suggest that gB is the sole HSV fusogen and that gD and gH/gL act to upregulate cell-cell fusion and most likely virus-cell fusion, leading to HSV entry.     

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.     

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.     

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.     

Herpes Simplex Virus gD Forms Distinct Complexes with Fusion Executors gB and gH/gL in Part through the C-terminal Profusion Domain

Herpes simplex virus entry into cells requires a multipartite fusion apparatus made of glycoprotein D (gD), gB, and heterodimer gH/gL. gD serves as a receptor-binding glycoprotein and trigger of fusion; its ectodomain is organized in an N-terminal domain carrying the receptor-binding sites and a C-terminal domain carrying the profusion domain, required for fusion but not receptor binding. gB and gH/gL execute fusion. To understand how the four glycoproteins cross-talk to each other, we searched for biochemical defined complexes in infected and transfected cells and in virions. Previously, interactions were detected in transfected whole cells by split green fluorescent protein complementation (Atanasiu, D., Whitbeck, J. C., Cairns, T. M., Reilly, B., Cohen, G. H., and Eisenberg, R. J. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 18718–18723; Avitabile, E., Forghieri, C., and Campadelli-Fiume, G. (2007) J. Virol. 81, 11532–11537); it was not determined whether they led to biochemical complexes. Infected cells harbor a gD-gH complex (Perez-Romero, P., Perez, A., Capul, A., Montgomery, R., and Fuller, A. O. (2005) J. Virol. 79, 4540–4544). We report that gD formed complexes with gB in the absence of gH/gL and with gH/gL in the absence of gB. Complexes with similar composition were formed in infected and transfected cells. They were also present in virions prior to entry and did not increase at virus entry into the cell. A panel of gD mutants enabled the preliminary location of part of the binding site in gD to gB to the amino acids 240–260 portion and downstream with Thr³⁰⁴-Pro³⁰⁵ as critical residues and of the binding site to gH/gL at the amino acids 260–310 portion with Pro²⁹¹-Pro²⁹² as critical residues. The results indicate that gD carries composite-independent binding sites for gB and gH/gL, both of which are partly located in the profusion domain.