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.

A virus-free cell fusion assay relying on the transient transfection of Epstein-Barr virus (EBV) glycoproteins into cells provides an efficient and quantitative assay for characterizing the viral requirements necessary for fusion of the viral envelope with the B cell membrane. Extensive cellular fusion occurred when Daudi cells were layered onto Chinese hamster ovary K1 cells transiently expressing EBV glycoproteins gp42, gH, gL, and gB. This is the first direct evidence that gB is involved in the process of EBV entry. Moreover, mutational analysis of gB indicates that the cytoplasmic tail contains two distinct domains that function differentially in the process of fusion. The region from amino acids 802 to 816 is necessary for productive membrane fusion, while amino acids 817 to 841 comprise a domain that negatively regulates membrane fusion.

Laminin-5 (LN5) is a matrix component of epithelial tissue basement membranes and plays an important role in the initiation and maintenance of epithelial cell anchorage to the underlying connective tissue. Here we show that two distinct LN5 function-inhibitory antibodies, both of which bind the globular domain of the ␣3 subunit, inhibit proliferation of epithelial cells. These same antibodies also induce a decrease in mitogenactivated protein kinase activity. Inhibition of proliferation by the function-perturbing LN5 antibodies is reversed upon removal of the antibodies and can be overcome by providing the antibody-treated cells with exogenous LN5 and rat tail collagen. Because epithelial cells use the integrin receptor ␣3␤1 to interact with both LN5 and rat tail collagen, we next investigated the possibility that integrin ␣3␤1 is involved in mediating the proliferative impact of LN5. Proliferation of human epithelial cells is significantly inhibited by a function-perturbing ␣3 integrin antibody. In addition, antibody activation of ␤1 integrin restores the proliferation of epithelial cells treated with LN5 functionperturbing antibodies. These data indicate that a complex comprising LN5 and ␣3␤1 integrin is multifunctional and contributes not only to epithelial cell adhesion but also to the regulation of cell growth via a signaling pathway involving mitogen-activated protein kinase. We discuss our study in light of recent evidence that LN5 expression is up-regulated at the leading tips of tumors, where it may play a role in tumor cell proliferation. INTRODUCTIONCell interaction with elements of the extracellular matrix impacts their adherence, motility, as well as protein and gene expression (for example, see Adams and Watt, 1993;Roskelly et al., 1995). In intact normal tissue epithelial cells bind to extracellular matrix molecules, which are organized into a complex multiprotein structure called the basement membrane. The major components of the latter include type IV collagen, proteoglycans, and laminins. One laminin isoform, laminin-5 (LN5), 1 in particular plays an important role in establishing firm adherence of epithelial cells to the basement membrane, because it is necessary for the assembly and maintenance of stable anchorage devices between epithelial cells and matrix called hemidesmosomes (Baker et al., 1996;Green and Jones, 1996). However, recent reports also indicate that LN5 is expressed at the budding tips of invading tumor cells, i.e., at sites where cancer cells are undergoing cell division but where there are most likely no hemidesmosomes (Pyke et al., 1994(Pyke et al., , 1995. This provides an indication § Corresponding author. E-mail address: j-jones3@nwu.edu. 1 Abbreviations used: BrdU, bromodeoxyuridine; DAPI, 4,6-diamidino-2-phenylindole; EGF, epidermal growth factor; FN, fibronectin; hLN5, human laminin-5; Ig, immunoglobulin; LN, laminin; MAP, mitogen-activated protein; MAPK, MAP kinase; RTC, rat tail collagen; rtLN5, rat laminin-5.© 1999 by The American Society for Cell Biology 259 that L...

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 (EBV) infection is prevalent in all humanpopulations and is linked to a variety of human diseases. EBV causes infectious mononucleosis and is associated with a variety of hemopoietic malignancies, such as Burkitt's lymphoma, some forms of Hodgkin's lymphoma, and adult T-cell leukemia (17,24,29). EBV is also etiologically associated with two diseases of epithelial cell origin, nasopharyngeal carcinoma and oral hairy leukoplakia (17,22,29). In vitro, the EBV host range is largely restricted to B cells. The entry of EBV into a B cell occurs through a cascade of events that requires the association of multiple cellular and viral factors. The initial event required for entry is the interaction of the major viral envelope glycoprotein, gp350, with complement receptor type 2 molecule CD21, previously referred to as CR2 (27,33). Virion penetration of the B-cell membrane requires the additional interaction of the ternary EBV glycoprotein gp85-gp25-gp42 complex with its cellular ligand (20, 35). gp85 and gp25 are the EBV homologs of herpes simplex virus gH and gL, respectively. gp42 can interact with the HLA class II protein HLA-DR (32). The ability of EBV to utilize HLA-DR as a coreceptor for entry is demonstrated by the observation that certain B-cell lines lacking HLA-DR expression are not susceptible to superinfection unless the expression of HLA-DR is restored (19).Studies of EBV infection have been limited by the lack of a recombinant EBV bearing a reporter gene. Such studies typically utilize cellular transformation, immortalization, or the detection of EBV antigens as indicators of viral infection. To investigate the dependence of EBV infection on cellular receptors, a recombinant EBV reporter virus expressing enhanced green fluorescent protein (EGFP), designated EBfaV-GFP, has been constructed (30). Two CD21-positive, HLA class II-negative cell lines, HPB-ALL and 721.174, were used to determine if the introduction of HLA-DP and/or HLA-DQ molecules to the surfaces of HLA class II-deficient cells could substitute for HLA-DR and mediate EBV entry. Here we show that surface expression of HLA-DP or HLA-DQ is sufficient to render CD21-expressing cells susceptible to infection by EBfaV-GFP.EBfaV-GFP was used to screen a panel of CD21-expressing cell lines. A Burkitt's lymphoma cell li...

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus that causes infectious mononucleosis and is etiologically associated with malignancies of multiple origins. EBV enters cells through a cascade of interactions between its envelope glycoprotein gp350 and the gp42-gH-gL complex with cellular receptors. Membrane fusion is catalyzed by the binding of gp42, a member of the C type lectin family, to HLA class II molecule HLA-DR, -DP, or -DQ. Here we demonstrate that only a subset of HLA-DQ alleles mediates EBV entry, indicating that individuals expressing these alleles may offer unique sites for EBV infection and subsequent sequelae. Additionally, the specific site within HLA-DQ determined to be essential for EBV entry is homologous to a site within MHC class I shown by structural studies to bind to the C type-lectin-like natural killer receptor, providing insight into the biochemical nature of the gp42-HLA class II interaction.

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Entry of Epstein-Barr virus (EBV) into B lymphocytes requires the binding of viral glycoprotein 42 (gp42), a C-type lectin family member, to HLA class II. Recently, the structure of the gp42:HLA-DR1 complex was determined. In order to confirm the interaction as determined in the structural study and to identify other potential interactive residues, a mutational analysis of HLA class II was performed. A secreted form of gp42 (sgp42) reacted with a conformation-specific monoclonal antibody and blocked EBV infection. The binding of sgp42 and EBV entry to two sets of HLA class II mutants were tested. The first set of mutants were based on the known interaction of the C-type lectin Ly49A with HLA class I, and the second set of mutants were based on the identified interface in the gp42:HLA-DR1 complex. As expected, none of the mutants that would be predicted to interfere with the interaction of Ly49A with class I affected the interaction of gp42 with HLA class II, whereas mutants in amino acids identified in the gp42:HLA-DR1 structure inhibited sg42 binding to class II. In general, sgp42 binding correlated with efficient entry of EBV, as demonstrated by the necessity of glutamic acid 46 or arginine 72 in class II molecules. Furthermore, other HLA class II residues buried within the interface of gp42 and HLA class II when mutated had either no effect or a decrease in both binding and entry and implicate a region of class II important in stabilizing the interaction with gp42. These studies provide insight into the entry and fusion processes of the critical interaction between gp42 and HLA class II.Epstein-Barr virus (EBV), a gammaherpesvirus, causes infectious mononucleosis and is associated with a variety of malignancies, such as Burkitt's lymphoma and nasopharyngeal carcinoma (18). The host range of EBV in vitro is largely restricted to B cells. Entry of the virus occurs through multiple interactions of viral glycoproteins with receptors found on B cells. The initial entry step is the interaction of the major viral glycoprotein 350/220 (gp350/220) with the complement receptor type 2 molecule CD21 (CR2/CD21) (15,25). This interaction is thought to bring the virus closer to the B-cell membrane, allowing for attachment of the viral glycoprotein gp42 with B-cell surface HLA class II molecules. Fusion of the viral envelope with the B-cell membrane requires gp42 along with the concerted action of the EBV-encoded glycoproteins gB, gH, and gL (8). Furthermore, the association of gp42 and HLA class II seems to be the key signal for triggering virus entry and fusion into B cells as well as tropism. A virus lacking gp42 is not able to infect B cells, and the amount of gp42 present on the virion determines the cell type that EBV infects (2, 28).The HLA class II molecule, HLA-DR, was first shown to interact with gp42 in an expression library screen for proteins that bound a soluble gp42-Fc construct (23). HLA class II molecules are comprised of two distinct gene products, ␣ and ␤, which noncovalently heterodimerize to form mature ...

Epstein-Barr virus (EBV) is a strict human pathogen for which no small animal models exist. Plasmids that contain the EBV plasmid origin of replication, oriP, and express EBV nuclear antigen 1 (EBNA1) are stably maintained extrachromosomally in human cells, whereas these plasmids replicate poorly in rodent cells. However, the ability of oriP and EBNA1 to maintain the entire EBV episome in proliferating rodent cells has not been determined. Expression of the two human B-cell receptors for EBV on the surfaces of murine B cells allows efficient viral entry that leads to the establishment of latent EBV infection and long-term persistence of the viral genome. Latent gene expression in these cells resembles the latency II profile in that EBNA1 and LMP1 can be detected whereas EBNA2 and the EBNA3s are not expressed.Epstein-Barr virus (EBV), also designated human herpesvirus 4, establishes latent infection in human hosts and is associated with a wide variety of human malignancies (29,41). Although humans are the exclusive natural host for EBV, several other Old World primates are infected with closely related herpesviruses of the same subgroup, lymphocryptovirus (3,14,23,37). Lymphocryptovirus genomes are colinear and homologous with the EBV genome (15), and the structural and nonstructural proteins are frequently well conserved (11,25,39). Despite these similarities, differences in disease progression and the complications arising from working on primates hinder the use of these viruses as a model for EBV infection (32). The ability to genetically manipulate and study mice in the laboratory makes a murine model for EBV infection preferable. Murine gammaherpesvirus 68 (MHV-68) establishes latent infection in lymphoid tissues, but the disease pattern caused by MHV-68 differs from that of EBV (43,45,46). In addition, MHV-68 does not encode the complement of EBV latency-associated and/or transforming proteins, including the nuclear antigens (EBNAs) and latent membrane proteins LMP1 and LMP2A/B, indicating fundamental differences between it and EBV (2, 48). To determine if mice may able to serve as a suitable model for EBV infection it is necessary to determine the viral and/or host restrictions that uniquely direct EBV susceptibility to humans.Murine cells lines are typically resistant to infection by EBV, but reports have indicated that introducing the expression of human CD21 (hCD21), the cellular receptor involved in binding the EBV envelope glycoprotein gp350 (36, 47), promotes EBV infection of murine L cells (1, 6). However, this may be a cell type-dependent phenomenon, since the requirements for viral entry into epithelial and fibroblast cell lines appear to be quite different than those for entry into B cells (16,27,33,54). In addition to the binding of gp350 with CD21, EBV entry into human B cells requires additional interaction with a second cellular receptor, HLA class II, with the ternary gH-gL-gp42 complex (26,27,50). Moreover, studies indicate that the expression of CD21 on human lymphocytes is not sufficient ...