Epstein-Barr Virus Uses Different Complexes of Glycoproteins gH and gL To Infect B Lymphocytes and Epithelial Cells

The Epstein-Barr virus (EBV) gH-gL complex includes a third glycoprotein, gp42. gp42 binds to HLA class II on the surfaces of B lymphocytes, and this interaction is essential for infection of the B cell. We report here that, in contrast, gp42 is dispensable for infection of epithelial cell line SVKCR2. A soluble form of gp42, gp42.Fc, can, however, inhibit infection of both cell types. Soluble gp42 can interact with EBV gH and gL and can rescue the ability of virus lacking gp42 to transform B cells, suggesting that a gH-gL-gp42.Fc complex can be formed by extrinsic addition of the soluble protein. Truncated forms of gp42.Fc that retain the ability to bind HLA class II but that cannot interact with gH and gL still inhibit B-cell infection by wild-type virus but cannot inhibit infection of SVKCR2 cells or rescue the ability of recombinant gp42-negative virus to transform B cells. An analysis of wild-type virions indicates the presence of more gH and gL than gp42. To explain these results, we describe a model in which wild-type EBV virions are proposed to contain two types of gH-gL complexes, one that includes gp42 and one that does not. We further propose that these two forms of the complex have mutually exclusive abilities to mediate the infection of B cells and epithelial cells. Conversion of one to the other concurrently alters the ability of virus to infect each cell type. The model also suggests that epithelial cells may express a molecule that serves the same cofactor function for this cell type as HLA class II does for B cells and that the gH-gL complex interacts directly with this putative epithelial cofactor.All herpesviruses examined to date encode a complex of two glycoproteins, gH and gL, that appear to be necessary, if not sufficient, for virus penetration. Glycoprotein gH is generally thought to be the major player in virus cell fusion (5, 6, 8, 14, 20, 25, 26), while the role of gL is to serve as a chaperone, essential for folding and transport of functional gH (3, 11, 13, 20, 21, 28, 29). The Epstein-Barr virus (EBV) gH-gL complex follows this pattern. Glycoprotein gp85, the gH homolog, is retained in the endoplasmic reticulum in the absence of gp25, the EBV gL (38), and virosomes made from EBV proteins depleted of the gH-gL complex bind to cells but fail to fuse (9). The EBV gH-gL complex, however, includes a third glycoprotein, gp42, which is the product of the BZLF2 open reading frame (ORF) (18). This third component has also proven to be essential for penetration of the major target cell of EBV, the B lymphocyte. Several lines of evidence indicate that gp42 is a ligand for HLA class II and, further, that HLA class II functions as a cell surface cofactor for EBV entry into this cell type. Glycoprotein gp42 interacts with the β₁ domain of HLA class II protein HLA-DR (30), and a monoclonal antibody (MAb) to gp42 called F-2-1 interferes with this interaction (17). MAb F-2-1 has no effect on EBV attachment via glycoprotein gp350/220 to its primary receptor, complement receptor type 2 (CR2; CD21) but inhibits the fusion of the virus with the B-cell membrane (22). Similarly, a MAb to HLA-DR or a soluble form of gp42 blocks B-cell transformation. Finally, B-cell lines which lack expression of HLA class II are not susceptible to superinfection with EBV unless expression of class II is restored (17). Most recently, we derived a recombinant virus with gp42 expression deleted and confirmed that loss of the glycoprotein resulted in a virus that attached to the B-cell surface but that failed to penetrate unless it was treated with the fusogenic agent polyethylene glycol (36).Although most is known about the early interactions of EBV with B lymphocytes in vitro since these cells are readily available and easy to culture, infection is not restricted to this cell type in vivo. During our initial analysis of the biology of gp42 we had therefore examined its potential role in infection of a then newly derived model epithelial cell line, SVKCR2. SVKCR2 cells are transformed with simian virus 40 and stably transfected with B-cell receptor CR2 (19). They are poorly infectable with many strains of EBV, but in excess of 30% of the cells can be infected with the Akata strain of virus as judged by the expression of EBV latent protein EBNA 1 (18, 19). We found that MAb F-2-1 had no effect on the infection of SVKCR2 cells. At the same time, a second MAb, E1D1, which reacts with an epitope that can be formed by the coexpression of gH and gL in the absence of gp42, neutralized infection of SVKCR2 cells, but had no effect on the infection of lymphocytes. These data strongly suggested that the involvement of the gH-gL complex in the internalization of virus into the two cell types was different. We hypothesized that just as EBV has evolved a glycoprotein, gp350/220, which is uniquely adapted for attachment to B lymphocytes, so it has evolved a second glycoprotein, gp42, uniquely adapted for penetration into the same cell type (18). The implication was that gp42 might be dispensable for infection of epithelial cells.Since we made our initial observations with SVKCR2 cells, several novel reagents, including the Akata strain virus with the expression of gp42 deleted, have become available. The recent insights into the role of HLA class II in B-cell infection also provided new impetus to reexamine the involvement of the gH-gL complex in epithelial cell infection. We report here that gp42 is not required for infection of SVKCR2 cells despite the fact that the soluble form of the protein that inhibits B-cell infection can also neutralize infection of SVKCR2 cells. To explain these apparently anomalous results, we describe a model which proposes that wild-type EBV virions contain two types of gH-gL complexes, one that includes gp42 and one that does not. We further propose that the tripartite “B-cell complexes” are not functional for infection of epithelial cells, just as the bipartite “epithelial cell complexes” are unable to mediate infection of the B lymphocyte.     

Mutations in the Cytoplasmic Tail of Herpes Simplex Virus 1 gH Reduce the Fusogenicity of gB in Transfected Cells

Mutations within the cytoplasmic tail (cytotail) of herpes simplex virus 1 (HSV-1) gH were previously observed to suppress the syncytial phenotype of gB cytoplasmic domain mutant A855V in infected cells. Here, we examined the effects of gH cytotail mutations on virus-free cell-cell fusion in transfected cells to exclude the contributions of viral proteins other than gD, gH/gL, and gB. We show that a truncation at residue 832 coupled with the point mutation V831A within the cytotail of gH reduces fusion regardless of whether the wild type (WT) or a syn gB allele is present. We hypothesize that the gH cytotail mutations either reduce activation of gB by gH/gL or suppress the fusogenicity of gB through another, as yet unknown mechanism. The gB cytodomain and the gH cytotail do not interact in vitro, suggesting that mutations in the gH cytotail may instead affect the function of the gH/gL ectodomain. Nevertheless, we cannot exclude the possibility that the gB cytodomain and the gH cytotail interact in the context of full-length membrane-anchored proteins. The observed fusion suppression in transfected cells is less prominent than what was seen in infected cells, and we propose that gH cytotail mutations may additionally suppress syncytium formation in cells infected with syn HSV-1 by acting on other viral proteins, reinforcing the idea that fusion of HSV-infected cells is a complex phenomenon. Although fusion suppression by the gH cytotail mutant in transfected cells was evident when syncytia were visualized and counted, it was not detected by the luciferase assay, highlighting the differences between the two assays.     

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.     

Insertional Mutations in Herpes Simplex Virus Type 1 gL Identify Functional Domains for Association with gH and for Membrane Fusion

Glycoprotein L (gL) is one of four glycoproteins required for the entry of herpes simplex virus (HSV) into cells and for virus-induced cell fusion. This glycoprotein oligomerizes with gH to form a membrane-bound heterodimer but can be secreted when expressed without gH. Twelve unique gL linker-insertion mutants were generated to identify regions critical for gH binding and gH/gL processing and regions essential for cell fusion and viral entry. All gL mutants were detected on the cell surface in the absence of gH, suggesting incomplete cleavage of the signal peptide or the presence of a cell surface receptor for secreted gL. Coexpression with gH enhanced the levels of cell surface gL detected by antibodies for all gL mutants except those that were defective in their interactions with gH. Two insertions into a conserved region of gL abrogated the binding of gL to gH and prevented gH expression on the cell surface. Three other insertions reduced the cell surface expression of gH and/or altered the properties of gH/gL heterodimers. Altered or absent interaction of gL with gH was correlated with reduced or absent cell fusion activity and impaired complementation of virion infectivity. These results identify a conserved domain of gL that is critical for its binding to gH and two noncontiguous regions of gL, one of which contains the conserved domain, that are critical for the gH/gL complex to perform its role in membrane fusion.Glycoprotein L (gL) is one of the four glycoproteins required for the entry of herpes simplex virus (HSV) into cells and for virus-induced cell fusion (26, 33). The others are gB, gD, and gH (30). The functional unit containing gL is a heterodimer formed with gH (gH/gL) (15). Because mature gL has no membrane-spanning domain, other than a cleavable signal peptide, it is secreted unless it is coexpressed with gH, a type 1 glycoprotein that anchors gL to the cell membrane (2). Also, gH is not properly processed or transported out of the endoplasmic reticulum unless it is coexpressed with gL (15).Most, if not all, herpesviruses express orthologs of gB, gH, and gL, which are believed to form the core membrane-fusing machinery necessary for viral entry and cell fusion. For some herpesviruses, such as Epstein-Barr virus and human cytomegalovirus, the gH/gL oligomer may contain additional viral subunits that can influence binding of the complex to cell receptors and determine cell tropism (14, 34, 35). For HSV, however, only gD and gB have been shown to have receptor-binding activities that are required for entry (27, 31). Although HSV gH has an RGD motif and the gH/gL heterodimer can bind to certain integrins, this binding seems not to be necessary for viral entry (3, 22).The initial interaction of HSV with cells can be the reversible attachment of virus to cell surface heparan sulfate, mediated by viral glycoprotein gB and/or gC (29). Then, gD can bind to one of its receptors, including herpesvirus entry mediator (HVEM), a member of the tumor necrosis factor receptor family; nectin-1 or nectin-2, cell adhesion molecules belonging to the immunoglobulin superfamily; or specific sites in heparan sulfate generated by 3-O-sulfotransferases (31). In addition to binding to heparan sulfate, gB can also bind to other cell surface receptors, including paired immunoglobulin-like receptor alpha (PILRα) (27). Binding of both gD and gB to one of their respective receptors appears to be required for triggering the membrane-fusing activity of gB and/or gH/gL, which leads to viral entry.A recent X-ray structure of HSV type 1 (HSV-1) gB suggests that it is a class III viral fusogen similar in domain organization, but not primary sequence, to the G protein of vesicular stomatitis virus (13). It has been proposed that HSV-1 gH has features characteristic of class I viral fusogens, such as putative heptad repeats and fusion peptides (6, 9-11). Also, peptides matching the sequence of gH can interact with lipids and/or induce the fusion of lipid vesicles (4, 5, 8). Hemifusion between cells and between virus and cell can be induced by gH/gL and gD in the absence of gB (32). Many questions remain about the respective roles of gH/gL and gB in inducing membrane fusion.The four conserved cysteines in gL were found to be essential for gL-gH association and function (1). Mutational analyses of gL by C-terminal deletions showed that the first 147 amino acids of gL are sufficient for association with gH but that the first 161 amino acids are necessary for cotransport of gH and gL to the cell surface (17, 23) and for gL activity in cell fusion and viral entry (17). Lastly, certain anti-gL monoclonal antibodies (MAbs) can inhibit cell fusion but not viral entry, despite demonstrable binding of the MAbs to virus, suggesting that gL may play a different role in each process (21). These MAbs were mapped to the C-terminal region of gL (21, 23). The diagram at the bottom of Fig. ​Fig.11 shows the locations of the gL features mentioned above and of the signal peptide.Open in a separate windowFIG. 1.Effects of insertional mutations on HSV-1 gL and gH cell surface expression. CHO cells were transfected with plasmids expressing gH and WT gL or a gL mutant. Cell surface expression of gL and gH was quantified by CELISA using polyclonal R88 antiserum (filled circles) and MAb 52S (open triangles), respectively. A linear representation of the gL polypeptide is shown below the graph, with coded bars identifying features of gL. The bars represent the signal peptide (uncolored hatched), the N-terminal 161-amino-acid fragment necessary for the formation of functional gH/gL complexes (dark gray), highly conserved residues within this fragment (cross-hatched dark- gray bar), and epitopes recognized by a panel of anti-gL MAbs (light-gray and uncolored vertically striped bars). The values presented for cell surface expression of each mutant gL and of cotransfected WT gH are means from three independent experiments expressed as percentages of WT gL (or of gH cotransfected with WT gL) values, after subtraction of background values obtained in the absence of gL expression and as a function of the position of the insertion. Standard deviations are presented in Fig. ​Fig.22 and ​and33 for similar experiments.The interactions between gL and gH required for proper intracellular transport, processing, and cell surface expression make it difficult to investigate the functional role of one of these glycoproteins in cell fusion and viral entry independently of the other. We generated a panel of gL linker-insertion mutants to identify regions critical for gH binding and transport and regions essential for cell fusion and viral entry. One aim was to determine whether these roles of gL could be dissociated or were linked. Characterization of 12 unique gL linker-insertion mutants showed that (i) a conserved domain of gL is critical for the physical interaction of gL with gH and for the normal processing of gH, (ii) two noncontiguous regions of gL, one of which contains the highly conserved domain, are critical for the normal conformation and function of gH/gL heterodimers, and (iii) wild-type (WT) and mutant gLs can be detected on the cell surface in the absence of gH, suggesting the possibility of an independent role for uncomplexed gL. These results support and extend previous studies suggesting that gL has a larger role in membrane fusion than serving as a chaperone for gH and that specific mutations in gL can influence the function of the gH/gL heterodimer.     

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.     

Male gametic nucleus-specific H2B and H3 histones,designated gH2B and gH3, in Lilium longiflorum

Two proteins that resemble core histones and might be specific to the male gametic (generative) nucleus within the pollen of Lilium longiflorum Thumb, (originally designated p22.5 and p18.5; K. Ueda and I. Tanaka, 1994, Planta, 192, 446–452) were characterized biochemically and immunochemically. Patterns of digestion of p22.5 and p18.5 by Staphylococcus aureus V8 protease closely resembled those of somatic histones H2B and H3, respectively. However, peptide fragments that were unique to p22.5 or p18.5 were also detected. Antibodies raised against these proteins did not cross-react with any somatic histones. These results indicate that p22.5 and p18.5 are different from somatic histones in terms of primary structure. Analysis of their amino-acid compositions revealed that p22.5 is a moderately lysine-rich protein while p18.5 is an arginine-rich protein. From these results, we conclude that p22.5 is a variant of histone H2B and p18.5 is a variant of histone H3. Immunofluorescence staining of pollen grains using the specific antibodies revealed that both p22.5 and p18.5 are only present in the generative cell nucleus and are not to be found in the vegetative cell nucleus. This study demonstrates that (i) specific histone variants are present in the male gametic nucleus of a higher plant, as they are in the sperm nucleus of animals, and (ii) distinct differences in histone composition exist between the nuclei of generative and vegetative cells in pollen. These novel histones (p22.5 and p18.5), specific to male gametic nuclei, have been designated gH2B and gH3, respectively.Abbreviations DAPI 46-diamidino-2-phenylindole - FITC fluorescein isothiocyanate The authors thank Dr. Y. Sado (Shigei Medical Institute, Japan) for his helpful advice on immunization and Prof. T. Iguchi and Prof. K. Manabe (Yokohama City University, Japan) for providing facilities for experiments. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan.     

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.     

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.     

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.     

A novel herpes simplex virus glycoprotein, gL, forms a complex with glycoprotein H (gH) and affects normal folding and surface expression of gH.

A glycoprotein encoded by the UL1 gene of herpes simplex virus type 1 (HSV-1) was detected in infected cells with antipeptide sera. The UL1 gene has previously been implicated in virus-induced cell fusion (S. Little and P. A. Schaffer, Virology 112:686-697, 1981). Two protein species, a 30-kDa precursor form and a 40-kDa mature form of the glycoprotein, both of which were modified with N-linked oligosaccharides, were observed. This novel glycoprotein is the 10th HSV-1 glycoprotein to be described and was named glycoprotein L (gL). A complex was formed between gL and gH, a glycoprotein known to be essential for entry of HSV-1 into cells and for virus-induced cell fusion. Previously, it had been reported that gH expressed in the absence of other viral proteins was antigenically abnormal, not processed, and not expressed at the cell surface (U.A. Gompels and A. C. Minson, J. Gen. Virol. 63:4744-4755, 1989; A. J. Forrester, V. Sullivan, A. Simmons, B. A. Blacklaws, G. L. Smith, A. A. Nash, and A. C. Minson, J. Gen. Virol. 72:369-375, 1991). However, gH coexpressed with gL by using vaccinia virus recombinants was antigenically normal, processed normally, and transported to the cell surface. Similarly, gL was dependent on gH for proper posttranslational processing and cell surface expression. These results suggest that it is a hetero-oligomer of gH and gL which is incorporated into virions and transported to the cell surface and which acts during entry of virus into cells.     

Neutralising human recombinant antibodies to human cytomegalovirus glycoproteins gB and gH

A phage antibody display library of single chain fragment variable (scFv) was applied to develop anti-HCMV glycoprotein B (gB) and glycoprotein H (gH) neutralising libraries. To enrich for specific scFvs, the phage antibody was panned against cytomegalovirus epitopes derived from the N-terminal part of gB, the C-terminal part of gB and the N-terminal part of gH (NETIYNTTLKYGDV, VTSGSTKD and AASEALDPHAFHLLLNTYGR). A number of clones were differentiated by Bst N1 fingerprinting. After isolation of specific clones against each peptide, the neutralising effect of each clone was assessed by plaque reduction assay. This resulted in the isolation of eight neutralising scFv antibodies with 51-63% neutralising effects. Sequence analysis of three neutralising clones revealed the amino acids specificity changes in heavy and light chains of antibody molecules.