Mutational analyses of Epstein-Barr virus glycoprotein 42 reveal functional domains not involved in receptor binding but required for membrane fusion

Epstein-Barr virus (EBV) is a human gammaherpesvirus associated with malignancies of both epithelial and lymphoid origin. Efficient infection of the latent host reservoir B lymphocytes involves the binding of glycoproteins gp350/220 for initial attachment, followed by the concerted action of gH, gL, gB, and gp42 for membrane fusion. The type II membrane protein gp42 is required for infection of B cells and assembles into a complex with gH and gL. The cellular host receptor for gp42, class II human leukocyte antigen (HLA), has been structurally verified by crystallization analyses of gp42 bound to HLA-DR1. Interestingly, the crystal structure revealed a hydrophobic pocket consisting of many aromatic and aliphatic residues from the predicted C-type lectin domain of gp42 that in other members of the C-type lectin family binds major histocompatibility complex class I or other diverse ligands. Although the hydrophobic pocket does not bind HLA class II, mutational analyses presented here indicate that this domain is essential for EBV-induced membrane fusion. In addition, mutational analysis of the region of gp42 contacting HLA class II in the gp42-HLA-DR1 cocrystal confirms that this region interacts with HLA class II and that this interaction is also important for EBV-induced membrane fusion.     

Structure of the Epstein-Barr virus gp42 protein bound to the MHC class II receptor HLA-DR1

Epstein-Barr virus (EBV) causes infectious mononucleosis, establishes long-term latent infections, and is associated with a variety of human tumors. The EBV gp42 glycoprotein binds MHC class II molecules, playing a critical role in infection of B lymphocytes. EBV gp42 belongs to the C-type lectin superfamily, with homology to NK receptors of the immune system. We report the crystal structure of gp42 bound to the human MHC class II molecule HLA-DR1. The gp42 binds HLA-DR1 using a surface site that is distinct from the canonical lectin and NK receptor ligand binding sites. At the canonical ligand binding site, gp42 forms a large hydrophobic groove, which could interact with other ligands necessary for EBV entry, providing a mechanism for coupling MHC recognition and membrane fusion.     

Epstein-Barr virus entry utilizing HLA-DP or HLA-DQ as a coreceptor

Epstein-Barr virus (EBV) glycoprotein gp350/gp220 association with cellular CD21 facilitates virion attachment to B lymphocytes. Membrane fusion requires the additional interaction between virion gp42 and cellular HLA-DR. This binding is thought to catalyze membrane fusion through a further association with the gp85-gp25 (gH-gL) complex. Cell lines expressing CD21 but lacking expression of HLA class II molecules are resistant to infection by a recombinant EBV expressing enhanced green fluorescent protein. Surface expression of HLA-DR, HLA-DP, or HLA-DQ confers susceptibility to EBV infection on resistant cells that express CD21. Therefore, HLA-DP or HLA-DQ can substitute for HLA-DR and serve as a coreceptor in EBV entry.     

Epstein-Barr virus gp42 is posttranslationally modified to produce soluble gp42 that mediates HLA class II immune evasion

Epstein-Barr virus (EBV) resides as a persistent infection in human leukocyte antigen (HLA) class II+ B lymphocytes and is associated with a number of malignancies. The EBV lytic-phase protein gp42 serves at least two functions: gp42 acts as the coreceptor for viral entry into B cells and hampers T-cell recognition via HLA class II molecules through steric hindrance of T-cell receptor-class II-peptide interactions. Here, we show that gp42 associates with class II molecules at their various stages of maturation, including immature alphabetaIi heterotrimers and mature alphabeta-peptide complexes. When analyzing the biosynthesis and maturation of gp42 in cells stably expressing the viral protein, we found that gp42 occurs in two forms: a full-length type II membrane protein and a truncated soluble form. Soluble gp42 is generated by proteolytic cleavage in the endoplasmic reticulum and is secreted. Soluble gp42 is sufficient to inhibit HLA class II-restricted antigen presentation to T cells. In an almost pure population of Burkitt's lymphoma cells in the EBV lytic cycle, both transmembrane and soluble forms of gp42 are detected. These results imply that soluble gp42 is generated during EBV lytic infection and could contribute to undetected virus production by mediating evasion from T-cell immunity.     

Binding-site interactions between Epstein-Barr virus fusion proteins gp42 and gH/gL reveal a peptide that inhibits both epithelial and B-cell membrane fusion

Herpesviruses require membrane-associated glycoproteins gB, gH, and gL for entry into host cells. Epstein-Barr virus (EBV) gp42 is a unique protein also required for viral entry into B cells. Key interactions between EBV gp42 and the EBV gH/gL complex were investigated to further elucidate their roles in membrane fusion. Deletion and point mutants within the N-terminal region of gp42 revealed residues important for gH/gL binding and membrane fusion. Many five-residue deletion mutants in the N-terminal region of gp42 that exhibit reduced membrane fusion activity retain binding with gH/gL but map out two functional stretches between residues 36 and 96. Synthetic peptides derived from the gp42 N-terminal region were studied in in vitro binding experiments with purified gH/gL and in cell-cell fusion assays. A peptide spanning gp42 residues 36 to 81 (peptide 36-81) binds gH/gL with nanomolar affinity, comparable to full-length gp42. Peptide 36-81 efficiently inhibits epithelial cell membrane fusion and competes with soluble gp42 to inhibit B-cell fusion. Additionally, this peptide at low nanomolar concentrations inhibits epithelial cell infection by intact virus. Shorter gp42 peptides spanning the two functional regions identified by deletion mutagenesis had little or no binding to soluble gH/gL and were also unable to inhibit epithelial cell fusion, nor could they complement gp42 deletion mutants in B-cell fusion. These studies identify key residues of gp42 that are essential for gH/gL binding and membrane fusion activation, providing a nanomolar inhibitor of EBV-mediated membrane fusion.     

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.     

Cleavage and Secretion of Epstein-Barr Virus Glycoprotein 42 Promote Membrane Fusion with B Lymphocytes

Epstein-Barr virus (EBV) membrane glycoprotein 42 (gp42) is required for viral entry into B lymphocytes through binding to human leukocyte antigen (HLA) class II on the B-cell surface. EBV gp42 plays multiple roles during infection, including acting as a coreceptor for viral entry into B cells, binding to EBV glycoprotein H (gH) and gL during the process of membrane fusion, and blocking T-cell recognition of HLA class II-peptide complexes through steric hindrance. 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. Both the full-length and the secreted gp42 forms bind to gH/gL and HLA class II, and the functional significance of gp42 cleavage is currently unclear. We found that in a virus-free cell-cell fusion assay, enhanced secretion of gp42 promoted fusion with B lymphocytes, and mutation of the site of gp42 cleavage inhibited membrane fusion activity. The site of gp42 cleavage was found to be physically distinct from the residues of gp42 necessary for binding to gH/gL. These results suggest that cleavage and secretion of gp42 are necessary for the process of membrane fusion with B lymphocytes, providing the first indicated functional difference between full-length and cleaved, secreted gp42.Epstein-Barr virus (EBV) is a large DNA virus belonging to the human gammaherpesvirus subfamily. EBV is orally transmitted through saliva and persists for the lifetime of its human host, establishing a latency reservoir in B lymphocytes with intermittent viral reactivation (1, 27). More than 90% of the world''s adult population is infected with EBV, although in healthy individuals, viral reactivation from latency is quickly controlled by the immune system. During primary infection and viral reactivation from latency, EBV infects epithelial cells as well as B lymphocytes (27). Primary infection with EBV can lead to development of infectious mononucleosis, and EBV has also been strongly associated with a number of human malignancies of epithelial and B-cell origin, including Burkitt''s lymphoma and nasopharyngeal carcinoma (4, 9, 10, 33, 36).EBV encodes a number of membrane glycoproteins important in a variety of viral processes, including entry of the virus into target host cells and virus-induced cell-cell fusion. The membrane glycoproteins necessary for fusion with both epithelial and B cells are glycoprotein B (gB), gH, and gL, and together, they form the core virus fusion machinery (7, 20, 24, 29). In addition to these glycoproteins, glycoprotein 42 (gp42) has been shown to play an essential role in membrane fusion with B cells (7, 18, 20). Attachment of EBV virions to B cells occurs through binding of the main envelope protein gp350/220 to CD21 (also known as complement receptor type 2) (5, 23, 34). This interaction enhances the efficiency of EBV infection of B cells but is not required for viral entry (12, 30). Antibodies to gp350/220 inhibit EBV infection of B cells but enhance infection of epithelial cells, possibly by facilitating the access of other viral glycoproteins to the epithelial cell membrane (35). Virus-cell membrane fusion is subsequently triggered by binding of gp42 to human leukocyte antigen (HLA) class II on the B-cell surface (6, 8, 11, 17, 31). Interestingly, gp42 appears to function as a switch of cellular tropism between epithelial and B cells. The presence of gp42 in the viral envelope is necessary for infection of B lymphocytes, and virions that are low in gp42 are better able to infect HLA class II-negative epithelial cells (3). Aside from its role in membrane fusion, gp42 plays a significant role in evasion of the host immune system. Gp42 binds to HLA class II-peptide complexes in infected cells, sterically hindering T-cell recognition of the complex by the T-cell receptor (25). This inhibition may allow EBV to delay detection by the host immune system.Two different mature forms of gp42 are produced by EBV-positive B lymphocytes in the lytic cycle (26). The first form is a full-length type II membrane protein, and the second is a truncated soluble form (s-gp42) (26). s-gp42 is generated by posttranslational cleavage (most likely mediated by a cellular protease resident in the endoplasmic reticulum) and is secreted (26). Both forms of gp42 associate with HLA class II intracellularly, and both inhibit HLA class II-restricted antigen presentation to T cells (26). Both forms of gp42 produced by EBV-positive B cells in the lytic cycle were found to be present in gH-gL-gp42 complexes, indicating that s-gp42 retains the ability to bind gH/gL (26). The physiological significance of s-gp42 is currently unclear, but this form has been suggested to function in infection and immune evasion, blocking EBV entry receptors on lytically infected B cells to prevent reinfection and neutralizing gp42-specific antibodies following its secretion from infected cells (26).Both forms of gp42 have been examined for their functions in mediating evasion from T-cell immunity through binding to HLA class II complexes (26), but the functions of the two forms of the protein in membrane fusion are unknown. To examine how each form of gp42 functions during membrane fusion, we have assayed the effect of gp42 cleavage site mutation on this process. Also, to distinguish residues important for gp42 cleavage from those necessary for association with gH/gL, we have constructed a number of fully secreted gp42 truncation mutants and examined their interaction with gH/gL and their ability to mediate fusion. Mutation or deletion of the gp42 cleavage site inhibited or eliminated cleavage of the protein, which had a direct effect on gp42 function in membrane fusion. An assay of N-terminal truncations of gp42 indicated that the region of gp42 necessary for cleavage was physically distinct from the region of gp42 necessary for association with gH/gL. We show that membrane association of gp42 has an inhibitory effect on gp42 function in membrane fusion and that increased secretion of gp42 stimulates membrane fusion in vitro. Cleavage of gp42 may be necessary for EBV gp42 to assume a functional position, interaction, or conformation for participation in membrane fusion.     

Assembly and Architecture of the EBV B Cell Entry Triggering Complex

Epstein-Barr Virus (EBV) is an enveloped double-stranded DNA virus of the gammaherpesvirinae sub-family that predominantly infects humans through epithelial cells and B cells. Three EBV glycoproteins, gH, gL and gp42, form a complex that targets EBV infection of B cells. Human leukocyte antigen (HLA) class II molecules expressed on B cells serve as the receptor for gp42, triggering membrane fusion and virus entry. The mechanistic role of gHgL in herpesvirus entry has been largely unresolved, but it is thought to regulate the activation of the virally-encoded gB protein, which acts as the primary fusogen. Here we study the assembly and function of the reconstituted B cell entry complex comprised of gHgL, gp42 and HLA class II. The structure from negative-stain electron microscopy provides a detailed snapshot of an intermediate state in EBV entry and highlights the potential for the triggering complex to bring the two membrane bilayers into proximity. Furthermore, gHgL interacts with a previously identified, functionally important hydrophobic pocket on gp42, defining the overall architecture of the complex and playing a critical role in membrane fusion activation. We propose a macroscopic model of the initiating events in EBV B cell fusion centered on the formation of the triggering complex in the context of both viral and host membranes. This model suggests how the triggering complex may bridge the two membrane bilayers, orienting critical regions of the N- and C- terminal ends of gHgL to promote the activation of gB and efficient membrane fusion.     

Binding of the natural killer cell inhibitory receptor Ly49A to its major histocompatibility complex class I ligand. Crucial contacts include both H-2Dd AND beta 2-microglobulin.

Ly49A, an inhibitory C-type lectin-like mouse natural killer cell receptor, functions through interaction with the major histocompatibility complex class I molecule, H-2D(d). The x-ray crystal structure of the Ly49A.H-2D(d) complex revealed that homodimeric Ly49A interacts at two distinct sites of H-2D(d): Site 1, spanning one side of the alpha1 and alpha2 helices, and Site 2, involving the alpha1, alpha2, alpha3, and beta(2)m domains. Mutants of Ly49A, H-2D(d), and beta(2)-microglobulin at intermolecular contacts and the Ly49A dimer interface were examined for binding affinity and kinetics. Although mutations at Site 1 had little affect, several at Site 2 and at the dimer interface hampered the Ly49A.H-2D(d) interaction, with no effect on gross structure or T cell receptor interaction. The region surrounding the most critical residues (in H-2D(d), Asp(122); in Ly49A, Asp(229), Ser(236), Thr(238), Arg(239), and Asp(241); and in beta(2)-microglobulin, Gln(29) and Lys(58)) of the Ly49A.H-2D(d) interface at Site 2 includes a network of water molecules, suggesting a molecular basis for allelic specificity in natural killer cell recognition.     

Alternate replication in B cells and epithelial cells switches tropism of Epstein-Barr virus

Epstein-Barr virus is ubiquitous and is causally implicated in lymphoid and epithelial malignancies. Virus invades oropharyngeal mucosa and establishes latency in B lymphocytes. Reactivating lymphocytes shed virus into saliva for spread to new hosts. A complex of three virus glycoproteins, gH, gL and gp42, is essential for entry. B-cell entry requires binding of gp42 to human leukocyte antigen (HLA) class II whereas entry into epithelial cells lacking HLA class II requires complexes without gp42. To accommodate infection of each, the virus carries both three-part and two-part complexes. We show here that HLA class II in the virus-producing cell alters the ratio of three-part to two-part complexes. As a consequence, virus originating in epithelial cells efficiently infects B cells whereas B-cell derived virus better infects epithelial cells. This molecular switch is a novel strategy that could alter tropism of virus from epithelium to B cells and then back to epithelium in a new host.     

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.     

Mutational analysis of the interaction between CD4 and class II MHC: class II antigens contact CD4 on a surface opposite the gp120-binding site

Using functional and adhesion assays, we have studied the ability of 30 human CD4 mutants to interact with class II major histocompatibility complex (MHC) molecules and also with gp120 from human immunodeficiency virus. The mutants cover the four domains (D1-D4) of CD4 and include several single-site substitutions. Analysis of the results, in the context of the CD4 crystal structure, shows that mutations that affect the interaction with class II MHC molecules are located on three exposed loops from CD4 domains 1 and 2. The specifically implicated residues, 19, 89, and 165, are separated from one another by 9 A, 24 A, and 24 A on one face of the CD4 molecule. Moreover, the class II binding site does not include residues 43 to 49 of the CD4 molecule, a region on an opposite face known to be involved in the binding of gp120.     

Epstein-Barr Virus Lacking Glycoprotein gp42 Can Bind to B Cells but Is Not Able To Infect

The Epstein-Barr virus gH-gL complex includes a third glycoprotein, gp42, which is the product of the BZLF2 open reading frame (ORF). gp42 has been implicated as critical to infection of the B lymphocyte by virtue of its interaction with HLA class II on the B-cell surface. A neutralizing antibody that reacts with gp42 inhibits virus-cell fusion and blocks binding of gp42 to HLA class II; antibody to HLA class II can inhibit infection, and B cells that lack HLA class II can only be infected if HLA class II expression is restored. To confirm whether gp42 is an essential component of the virion, we derived a recombinant virus with a selectable marker inserted into the BZLF2 ORF to interrupt expression of the protein. A complex of gH and gL was expressed by the recombinant virus in the absence of gp42. Recombinant virus egressed from the cell normally and could bind to receptor-positive cells. It had, however, lost the ability to infect or transform B lymphocytes. Treatment with polyethylene glycol restored the infectivity of recombinant virus, confirming that gp42 is essential for penetration of the B-cell membrane.Entry of enveloped viruses into mammalian cells requires that the virion envelope fuse with the cell membrane after attachment to the cell surface. Herpesviruses require the functions of multiple protein species to mediate this event, and in keeping with the common origin and diverse habitats of these viruses, some of the proteins involved in penetration appear to be conserved throughout the family and some appear to be restricted to individual members or more closely related members with similar tropism. The two glycoproteins gH and gL fall into the first category of conserved proteins. Glycoprotein gH has been implicated as a major player in virus-cell fusion in many herpesviruses (8, 10, 11, 22, 28, 32, 34), and gL is an essential partner which is required for folding and transport of gH out of the endoplasmic reticulum (6, 19, 21, 27, 28, 35, 38, 45). The gH and gL homologs of Epstein-Barr virus (EBV) are gp85, the product of the BXLF2 open reading frame (ORF) (13, 31), and gp25, the product of the BKRF2 ORF (45), and these homologs appear to behave much as their counterparts in other herpesviruses do (45). However, a third glycoprotein, gp42, associates with the EBV gH-gL complex and falls into the second category of proteins, those with a more restricted distribution.Glycoprotein gp42 is the product of the BZLF2 ORF (26), and although there may be a functionally similar protein in cytomegalovirus (18, 24), it is not predicted to have a homolog in other human herpesviruses. It does, however, have a homolog in ORF51 of equine herpes virus 2 (43). Both EBV and equine herpes virus 2 infect B lymphocytes (1), and several lines of evidence suggest that, at least in the case of EBV, gp42 is critical to the infection of this cell type. A monoclonal antibody (MAb) called F-2-1 that reacts with gp42 has no affect on EBV attachment to its receptor, complement receptor type 2 (CR2) (CD21), but inhibits fusion of the virus with the B-cell membrane and neutralizes infection (29). Glycoprotein gp42 interacts with the β₁ domain of the HLA class II protein HLA-DR (39), and MAb F-2-1 interferes with this interaction (25). Like F-2-1, a MAb to HLA-DR or a soluble form of gp42 can block B-cell transformation, and B-cell lines which lack expression of HLA class II are not susceptible to superinfection with EBV unless expression of HLA class II is restored (25). Collectively these observations suggest that gp42, probably by virtue of its interaction with HLA class II, is essential to infection of the B lymphocyte. To answer directly the question of whether gp42 is an indispensable glycoprotein, we derived a virus that could be definitively shown to lack expression of the molecule and examined its ability to infect normal resting B lymphocytes. We report here that virus with expression of gp42 blocked can exit cells normally and can bind to receptor-positive target cells. However, it is unable to penetrate into cells and initiate infection.     

Epstein-Barr virus gH is essential for penetration of B cells but also plays a role in attachment of virus to epithelial cells

Entry of Epstein-Barr virus (EBV) into B cells is initiated by attachment of glycoprotein gp350 to the complement receptor type 2 (CR2). A complex of three glycoproteins, gH, gL, and gp42, is subsequently required for penetration. Gp42 binds to HLA class II, which functions as an entry mediator or coreceptor and, by analogy with other herpesviruses, gH is then thought to be involved virus-cell fusion. However, entry of virus into epithelial cells is thought to be different. It can be initiated by attachment by an unknown glycoprotein in the absence of CR2. There is no interaction between gp42 and HLA class II and instead a distinct complex of only the two glycoproteins gH and gL interacts with a novel entry mediator. Again, by analogy with other viruses gH is thought to be critical to fusion. To investigate further the different roles of gH in infection of the two cell types and to examine its influence on the assembly of the gH-gL-gp42 complex, we constructed two viruses, one in which the gH open reading frame was interrupted by a cassette expressing a neomycin resistance gene and the gene for green fluorescent protein and one as a control in which the neighboring nonessential thymidine kinase gene was interrupted with the same cassette. Virus lacking gH exited from cells normally, although loss of gH resulted in rapid turnover of gL and gp42 as well. The virus bound normally to B lymphocytes but could not infect them unless cells and bound virus were treated with polyethylene glycol to induce fusion. In contrast, virus that lacked the gH complex was impaired in attachment to epithelial cells and the effects of monoclonal antibodies to gH implied that this resulted from loss of gH rather than other members of the complex. These results suggest a role for gH in both attachment and penetration into epithelial cells.     

The NK cell MHC class I receptor Ly49A detects mutations on H-2Dd inside and outside of the peptide binding groove

The NK cell inhibitory receptor Ly49A recognizes the mouse MHC class I molecule H-2D(d) and participates in the recognition of missing self. Previous studies indicated that the determinant recognized by Ly49A exists in alpha1/alpha2 domain of H-2D(d). Here we have substituted polymorphic as well as conserved residues of H-2D(d) alpha1/alpha2 domain (when compared with H-2K(d), which does not interact with Ly49A). We then tested the ability of the H-2D(d) mutants to interact with Ly49A by soluble Ly49A tetramer binding and NK cell cytotoxicity inhibition assays. Individual introduction of mutations converting the H-2D(d) residue into the corresponding H-2K(d) residue (N30D, D77S, or A99F) in H-2D(d) partially abrogated the interaction between Ly49A and H-2D(d). Introduction of the three mutations into H-2D(d) completely abolished Ly49A recognition. Individual introduction of D29N or R35A mutation into the residues of H-2D(d) that are conserved among murine MHC class I severely impaired the interaction. The crystal structure of H-2D(d) reveals that D77 and A99 are located in the peptide binding groove and that N30, D29, and R35 are in the interface of the three structural domains of MHC class I: alpha1/alpha2, alpha3, and beta(2)-microglobulin. These data suggest that Ly49A can monitor mutations in MHC class I inside and outside of the peptide binding groove and imply that inhibitory MHC class I-specific receptors are sensitive to mutations in MHC class I as well as global loss of MHC class I. Our results also provide insight into the molecular basis of Ly49A to distinguish MHC class I polymorphism.     

Structural and functional aspects of the Ly49 natural killer cell receptors

Natural killer cells are part of the first line of innate immune defence against virus-infected cells and cancer cells in the vertebrate immune system. They are called 'natural' killers because, unlike cytotoxic T cells, they do not require a previous challenge and preactivation to become active. The Ly49 NK receptors are type II transmembrane glycoproteins, structurally characterized as disulphide-linked homodimers. They share extensive homology with C-type lectins, and they are encoded by a multigene family that in mice maps on chromosome 6. A fine balance between inhibitory and activating signals regulates the function of NK cells. Inhibitory Ly49 molecules bind primarily MHC class I ligands, whereas the ligands for activating Ly49 molecules may include MHC class I, but also interestingly MHC class I-like molecules expressed by viruses, as is the case for Ly49H, which binds the m157 gene product of murine cytomegalovirus. In this study, we review the function and X-ray crystal structure of the Ly49 NK cell receptors hitherto determined (Ly49A, Ly49C and Ly49I), and the structural features of the Ly49/MHC class I interaction as revealed by the X-ray crystal structures of Ly49A/H-2Dd and the recently determined Ly49C/H-2Kb.     

Crystal structure of the Ly49I natural killer cell receptor reveals variability in dimerization mode within the Ly49 family

Natural killer (NK) cells play a crucial role in the detection and destruction of virally infected and tumor cells during innate immune responses. The cytolytic activity of NK cells is regulated through a balance of inhibitory and stimulatory signals delivered by NK receptors that recognize classical major histocompatabilty complex class I (MHC-I) molecules, or MHC-I homologs such as MICA, on target cells. The Ly49 family of NK receptors (Ly49A through W), which includes both inhibitory and activating receptors, are homodimeric type II transmembrane glycoproteins, with each subunit composed of a C-type lectin-like domain tethered to the membrane by a stalk region. We have determined the crystal structure, at 3.0 A resolution, of the murine inhibitory NK receptor Ly49I. The Ly49I monomer adopts a fold similar to that of other C-type lectin-like NK receptors, including Ly49A, NKG2D and CD69. However, the Ly49I monomers associate in a manner distinct from that of these other NK receptors, forming a more open dimer. As a result, the putative MHC-binding surfaces of the Ly49I dimer are spatially more distant than the corresponding surfaces of Ly49A or NKG2D. These structural differences probably reflect the fundamentally different ways in which Ly49 and NKG2D receptors recognize their respective ligands: whereas the single MICA binding site of NKG2D is formed by the precise juxtaposition of two monomers, each Ly49 monomer contains an independent binding site for MHC-I. Hence, the structural constraints on dimerization geometry may be relatively relaxed within the Ly49 family. Such variability may enable certain Ly49 receptors, like Ly49I, to bind MHC-I molecules bivalently, thereby stabilizing receptor-ligand interactions and enhancing signal transmission to the NK cell.     

Mutations of Epstein-Barr virus gH that are differentially able to support fusion with B cells or epithelial cells

The core fusion machinery of all herpesviruses consists of three conserved glycoproteins, gB and gHgL, suggesting a common mechanism for virus cell fusion, but fusion of Epstein-Barr virus (EBV) with B cells and epithelial cells is initiated differently. Fusion with B cells requires a fourth protein, gp42, which complexes with gHgL and interacts with HLA class II, the B-cell coreceptor. Fusion with an epithelial cell does not require gp42 but requires interaction of gHgL with a novel epithelial cell coreceptor. Epithelial cell fusion can be inhibited by gp42 binding to gHgL and by antibodies to gH that fail to block B-cell fusion. This suggests that regions of gHgL initiating fusion with each cell are separable from each other and from regions involved in fusion itself. To address this possibility we mapped the region of gH recognized by a monoclonal antibody to gH that blocks EBV fusion with epithelial cells but not B cells by making a series of chimeras with the gH homolog of rhesus lymphocryptovirus. Proteins with mutations engineered within this region included those that preferentially mediate fusion with B cells, those that preferentially mediate fusion with epithelial cells, and those that mediate fusion with neither cell type. These results support the hypothesis that the core fusion function of gH is the same for B cells and epithelial cells and that it differs only in the way in which it is triggered into a functionally active state.     

Critical residues at the Ly49 natural killer receptor's homodimer interface determine functional recognition of m157, a mouse cytomegalovirus MHC class I-like protein

NK cell function is regulated by Ly49 receptors in mice and killer cell Ig-like receptors in humans. Although inhibitory Ly49 and killer cell Ig-like receptors predominantly ligate classical MHC class I molecules, recent studies suggest that their activating counterparts recognize infection. The quintessential example is resistance to the mouse CMV in C57BL/6 mice, which depends on the functional recognition of m157, a mouse CMV-encoded MHC class I-like molecule, by Ly49H, an activating NK cell receptor. We have taken advantage of the natural variation in closely related members of the Ly49C-like receptors and the availability of Ly49 crystal structures to understand the molecular determinants of the Ly49H-m157 interaction and to identify amino acid residues discriminating between m157 binding and nonbinding receptors. Using a site-directed mutagenesis approach, we have targeted residues conserved in receptors binding to m157 (Ly49H and Ly49I(129)) but different from receptors lacking m157 recognition (Ly49C, Ly49I(B6), and Ly49U). Wild-type and mutant receptors were transfected into reporter cells, and physical binding as well as functional activation by m157 was studied. Our findings suggested that the Ly49 MHC class I contact "site 2," I226, may not be involved in m157 binding. In contrast, residue Y146 and G151, mapping at the receptor homodimer interface, are likely critical for functional recognition of the m157 glycoprotein. Our combined functional and three-dimensional modeling approach suggested that the architecture of the Ly49H dimer is crucial to accessing m157, but not MHC class I. These results link Ly49 homodimerization variability to the direct recognition of pathogen products.     

MHC class I recognition by NK receptors in the Ly49 family is strongly influenced by the beta 2-microglobulin subunit

NK cell recognition of targets is strongly affected by MHC class I specific receptors. The recently published structure of the inhibitory receptor Ly49A in complex with H-2Dd revealed two distinct sites of interaction in the crystal. One of these involves the alpha1, alpha2, alpha3, and beta2-microglobulin (beta2m) domains of the MHC class I complex. The data from the structure, together with discrepancies in earlier studies using MHC class I tetramers, prompted us to study the role of the beta2m subunit in MHC class I-Ly49 interactions. Here we provide, to our knowledge, the first direct evidence that residues in the beta2m subunit affect binding of MHC class I molecules to Ly49 receptors. A change from murine beta2m to human beta2m in three different MHC class I molecules, H-2Db, H-2Kb, and H-2Dd, resulted in a loss of binding to the receptors Ly49A and Ly49C. Analysis of the amino acids involved in the binding of Ly49A to H-2Dd in the published crystal structure, and differing between the mouse and the human beta2m, suggests the cluster formed by residues Lys3, Thr4, Thr28, and Gln29, as a potentially important domain for the Ly49A-H-2Dd interaction. Another possibility is that the change of beta2m indirectly affects the conformation of distal parts of the MHC class I molecule, including the alpha1 and alpha2 domains of the heavy chain.