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.     

A monoclonal antibody to glycoprotein gp85 inhibits fusion but not attachment of Epstein-Barr virus.

Epstein-Barr virus (EBV) codes for at least three glycoproteins, gp350, gp220, and gp85. The two largest glycoproteins are thought to be involved in the attachment of the virus to its receptor on B cells, but despite the fact that gp85 induces neutralizing antibody, no function has been attributed to it. As an indirect approach to understanding the role of gp85 in the initiation of infection, we determined the point at which a neutralizing, monoclonal antibody that reacted with the glycoprotein interfered with virus replication. The antibody had no effect on virus binding. To examine the effect of the antibody on later stages of infection, the fusion assay of Hoekstra and colleagues (D. Hoekstra, T. de Boer, K. Klappe, and J. Wilshaut, Biochemistry 23:5675-5681, 1984) was adapted for use with EBV. The virus was labeled with a fluorescent amphiphile that was self-quenched at the high concentration obtained in the virus membrane. When the virus and cell membrane fused, there was a measurable relief of self-quenching that could be monitored kinetically. Labeling had no effect on virus binding or infectivity. The assay could be used to monitor virus fusion with lymphoblastoid lines or normal B cells, and its validity was confirmed by the use of fixed cells and the Molt 4 cell line, which binds but does not internalize the virus. The monoclonal antibody to gp85 that neutralized virus infectivity, but not a second nonneutralizing antibody to the same molecule, inhibited the relief of self-quenching in a dose-dependent manner. This finding suggests that gp85 may play an active role in the fusion of EBV with B-cell membranes.     

The Epstein-Barr virus (EBV) BZLF2 gene product associates with the gH and gL homologs of EBV and carries an epitope critical to infection of B cells but not of epithelial cells.

Glycoprotein gp85, the product of the BXLF2 open reading frame (ORF), is the gH homolog of Epstein-Barr virus (EBV) and has been implicated in penetration of virus into B cells. Like its counterparts in other herpesviruses, it associates with a gL homolog, gp25, which is the product of the BKRF2 ORF. Unlike the gH homologs of other herpesviruses, however, gp85 also complexes with two additional glycoproteins of 42 and 38 kDa. Glycoproteins gp42 and gp38 were determined to be alternatively processed forms of the BZLF2 gene product. Coexpression of EBV gH and gL facilitated transport of gH to the cell surface and resulted in formation of a stable complex of gH and gL. It also restored expression of an epitope recognized by monoclonal antibody E1D1, which immunoprecipitates the native gH complex but not recombinant gH expressed in isolation. Coexpression of gH, gL, and the BZLF2 ORF restored expression of an epitope recognized by a second monoclonal antibody, F-2-1, which immunoprecipitates the native gH-gL-gp42/38 complex but not the complex of recombinant gH and gL alone. The epitope recognized by antibody F-2-1 was mapped to the BZLF2 gene product itself. Antibody F-2-1 inhibited the ability of EBV to infect B lymphocytes but had no effect on the ability of the virus to infect the epithelial cell line SVK-CR2. In contrast, antibody E1D1 had no effect on infection of the B-cell line but inhibited infection of the epithelial cell line. These results indicate that penetration of the two cell types by EBV involves differential use of the gH-gL-gp42/38 complex and suggest the hypothesis that the BZLF2 gene product has evolved as a unique adaptation to infection of B lymphocytes by EBV.     

Binding to cells of virosomes containing herpes simplex virus type 1 glycoproteins and evidence for fusion

Envelope proteins and lipids were extracted from purified herpes simplex virus type 1 virions with octyl glucoside and mixed with phosphatidylcholine for preparation of virosomes by removal of the detergent. Greater than 85% of the extracted envelope proteins, including all the glycoproteins and the nonglycosylated protein designated VP16, were associated with virosomes, which ranged in density from ca. 1.07 to 1.13 g/cm3. All the glycoproteins except gC were as susceptible to degradation by added protease in virosomes as in virions, indicating similar orientations in both. Approximately 30 to 40% of radiolabel incorporated into virosomes bound to HEp-2 cells within 1.5 h at either 4 or 37 degrees C. The cell-bound virosomes were enriched for gB and deficient in other glycoproteins, in comparison with unbound or total virosomes. Binding of virosomes to HEp-2 cells could be inhibited by purified virus, heparin, and monospecific antiviral antibodies. Polyclonal and monoclonal anti-gB antibodies were more effective at inhibiting virosome binding than were anti-gD or anti-gC antibodies. Virosomes depleted of gB or gD did not bind to cells as efficiently as did virosomes containing all the extracted enveloped components; this loss of binding activity was especially pronounced on depletion of gB. The binding of herpes simplex virus type 1 virosomes to cells is discussed in relation to possible heterogeneity of the virosomes and comparisons with binding of virions to cells. We also present electron microscopic evidence that bound virosomes can fuse with the cell surface.     

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.     

Interactions of Semliki Forest virus spike glycoprotein rosettes and vesicles with cultured cells

Semliki Forest virus (SFV)-derived spike glycoprotein rosettes (soluble octameric complexes), virosomes (lipid vesicles with viral spike glycoproteins), and liposomes (protein-free lipid vesicles) have been used to investigate the interaction of subviral particles with BHK-21 cells. Cell surface binding, internalization, degradation, and low pH- dependent membrane fusion were quantitatively determined. Electron microscopy was used to visualize the interactions. Virosomes and rosettes, but not liposomes, bound to cells. Binding occurred preferentially to microvilli and was inhibited by added SFV; it increased with decreasing pH but was, in all cases, less efficient than intact virus. At 37 degrees C the cell surface-bound rosettes and virosomes were internalized via coated pits and coated vesicles. After a lag period of 45 min the protein components of the internalized ligands were degraded and appeared, as acid-soluble activity, in the medium. The uptake of rosettes and virosomes was found to be similar to the adsorptive endocytosis of SFV except that their average residence times on the cell surface were longer. The rosettes and the liposomes did not show low pH-induced membrane fusion activity. The virosomes, however, irrespective of the lipid compositions used, displayed hemolytic activity at mildly acidic pH and were able to fuse with the plasma membrane of cells with an efficiency of 0.25 that observed with intact viruses. Cell-cell fusion activity was not observed with any of the subviral components. The results indicated that subviral components possess some of the entry properties of the intact virus.     

Induction of interleukin-6 after stimulation of human B-cell CD21 by Epstein-Barr virus glycoproteins gp350 and gp220.

The cellular receptor for Epstein-Barr virus (EBV) is the type 2 complement receptor, CD21. At initial infection, EBV virion glycoproteins gp350 and gp220 bind to CD21. We report here that the cross-linking of CD21 by gp350/220 results in increased amounts of interleukin 6 (IL-6) RNA and IL-6 protein. This effect could be blocked with anti-gp350/220 and anti-CD21 monoclonal antibodies. Induction of IL-6 in B cells by EBV could be mimicked by treatment with the protein kinase C (PKC) activator phorbol 12,13-dibutyrate but not with the calcium ionophore ionomycin. IL-6 induction by EBV was inhibited with the PKC-specific inhibitor bisindolylmaleimide or the protein tyrosine kinase inhibitors methyl 2,5-dihydroxycinnamate and herbimycin A, indicating that the induction of IL-6 following CD21 cross-linking is mediated through PKC- and protein tyrosine kinase-dependent pathways.     

Epstein-Barr virus enters B cells and epithelial cells by different routes.

Epstein-Barr virus (EBV) infects two cell types, B lymphocytes and epithelial cells. Electron microscopic studies have shown that the virus fuses with the lymphoblastoid cell line Raji but is endocytosed into thin-walled non-clathrin-coated vesicles in normal B cells before fusion takes place. To compare early interactions of EBV with epithelial cells and B cells, a fluorescence dequenching assay of fusion was employed, using virus labeled either with the pH-insensitive probe octadecyl rhodamine B chloride (R18) or with 5(N-octadecanoyl) aminofluorescein (AF), which loses emission intensity at a pH below 7.4. Fusion of virus labeled with R18 could be monitored with B cells, Raji cells, and epithelial cells. Lowering the extracellular pH or pretreatment of cells with ammonium chloride or methylamine had no effect on these measurements. In contrast, fusion of virus labeled with AF could be measured with Raji cells and epithelial cells, but not with normal B cells unless cells were previously treated with ammonium chloride. Fusion of virus with normal B cells was inhibited with chlorpromazine, chloroquine, and sodium azide, but none of these reagents had any effect on fusion with Raji or epithelial cells. These results suggest that entry of EBV into nonpolarized suspensions of epithelial cells occurs by fusion at the cell surface, that EBV may be incapable of fusing with normal B cells unless it has first been endocytosed, and that pH appears to be irrelevant to either event. A combination of the two probes, R18 and AF, may have general use for determining the sites of entry of enveloped viruses that fuse in a pH-independent manner.     

Antigenic cross-reactions among herpes simplex virus types 1 and 2, Epstein-Barr virus, and cytomegalovirus.

Polyvalent rabbit antisera against herpes simplex virus type 1 and 2 (HSV-1 and HSV-2), cytomegalovirus (CMV), and Epstein-Barr virus (EBV), monospecific antisera against affinity-purified HSV-2 glycoproteins gB and gG, and a panel of monoclonal antibodies against HSV and EBV proteins were used to analyze cross-reactive molecules in cells infected with the four herpesviruses. A combination of immunoprecipitation and Western blotting with these reagents was used to determine that all four viruses coded for a glycoprotein that cross-reacted with HSV-1 gB. CMV coded for proteins that cross-reacted with HSV-2 gC, gD, and gE. Both CMV and EBV coded for proteins that cross-reacted with HSV-2 gG. Antigenic counterparts to the p45 nucleocapsid protein of HSV-2 were present in HSV-1 and CMV, and counterparts of the major DNA-binding protein and the ribonucleotide reductase of HSV-1 were present in all the viruses. The EBV virion glycoprotein gp85 was immunoprecipitated by antisera to HSV-1, HSV-2, and CMV. Antisera to CMV and EBV neutralized the infectivity of both HSV-1 and HSV-2 at high concentrations. This suggests that cross-reactivity between these four human herpesviruses may have pathogenic as well as evolutionary significance.     

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.     

A cell surface protein that binds avian hepatitis B virus particles.

We have identified a 180-kDa cellular glycoprotein (gp180) that binds with high affinity to duck hepatitis B virus (DHBV) particles. The protein was detected by coprecipitating labeled duck hepatocyte proteins with virions or recombinant DHBV envelope proteins, using nonneutralizing monoclonal antibodies to the virion envelope. Binding of gp180 requires only the pre-S region of the viral large envelope protein, since recombinant fusion proteins bearing only this region efficiently coprecipitate gp180. The DHBV-gp180 interaction is blocked by two independent neutralizing monoclonal antibodies. The protein is found on both internal and surface membranes of the cell, and the species distribution of gp180 binding activity mirrors the known host range of DHBV infection. Functional gp180 is expressed in a wide variety of tissues in susceptible ducks.     

Identifying gp85-regions involved in Epstein-Barr virus binding to B-lymphocytes

Epstein-Barr virus lacking glycoprotein gp85 cannot infect B-cells and epithelial cells. The gp85 belongs to the molecular complex required for virus invasion of B-lymphocyte or epithelial cells. Moreover, there is evidence that gp85 is necessary for virus attachment to epithelial cells. Thirty-six peptides from the entire gp85-sequence were tested in epithelial and lymphoblastoid cell line binding assays to identify gp85-regions involved in virus-cell interaction. Five of these peptides presented high binding activity to Raji, Ramos, P3HR-1, and HeLa cells, but not to erythrocytes; Raji-cell affinity constants were between 80 and 140nM. Of these five peptides, 11435 ((181)TYKRVTEKGDEHVLSLVFGK(200)), 11436 ((201)TKDLPDLRGPFSYPSLTSAQ(220)), and 11438 ((241)YFVPNLKDMFSRAVTMTAAS(260)) bound to a 65kDa protein on Raji-cell surface. These peptides and antibodies induced by them (recognising live EBV-infected cells) inhibited Epstein-Barr virus interaction with cord blood lymphocytes. It is thus probable that gp85-regions defined by peptides 11435, 11436, and 11438 are involved in EBV invasion of B-lymphocytes.     

Identification of three gp350/220 regions involved in Epstein-Barr virus invasion of host cells

Epstein-Barr virus (EBV) invasion of B-lymphocytes involves EBV gp350/220 binding to B-lymphocyte CR2. The anti-gp350 monoclonal antibody (mAb)-72A1 Fab inhibits this binding and therefore blocks EBV invasion of target cells. However, gp350/220 regions interacting with mAb 72A1 and involved in EBV invasion of target cells have not yet been identified. This work reports three gp350/220 regions, defined by peptide 11382, 11389, and 11416 sequences, that are involved in EBV binding to B-lymphocytes. Peptides 11382, 11389, and 11416 bound to CR2(+) but not to CR2(-) cells, inhibited EBV invasion of cord blood lymphocytes (CBLs), were recognized by mAb 72A1, and inhibited mAb 72A1 binding to EBV. Peptides 11382 and 11416 binding to peripheral blood lymphocytes (PBLs) induced interleukin-6 protein synthesis in these cells, this phenomenon being inhibited by mAb 72A1. The same behavior has been reported for gp350/220 binding to PBLs. Anti-peptide 11382, 11389, and 11416 antibodies inhibited EBV binding and EBV invasion of PBLs and CBLs. Peptide 11382, 11389, and 11416 sequences presented homology with the C3dg regions coming into contact with CR2 (C3dg and gp350 bound to similar CR2 regions). These peptides could be used in designing strategies against EBV infection.     

Structure of the Epstein-Barr virus major envelope glycoprotein

Epstein-Barr virus (EBV) infection of B cells is associated with lymphoma and other human cancers. EBV infection is initiated by the binding of the viral envelope glycoprotein (gp350) to the cell surface receptor CR2. We determined the X-ray structure of the highly glycosylated gp350 and defined the CR2 binding site on gp350. Polyglycans shield all but one surface of the gp350 polypeptide, and we demonstrate that this glycan-free surface is the receptor-binding site. Deglycosylated gp350 bound CR2 similarly to the glycosylated form, suggesting that glycosylation is not important for receptor binding. Structure-guided mutagenesis of the glycan-free surface disrupted receptor binding as well as binding by a gp350 monoclonal antibody, a known inhibitor of virus-receptor interactions. These results provide structural information for developing drugs and vaccines to prevent infection by EBV and related viruses.     

Induction of antibodies to the Epstein-Barr virus glycoprotein gp85 with a synthetic peptide corresponding to a sequence in the BXLF2 open reading frame.

Epstein-Barr virus codes for at least three envelope glycoproteins, one of which, gp85, has not yet been mapped to the viral genome. The publication and analysis of the entire Epstein-Barr virus DNA sequence has allowed identification of open reading frames with potential for encoding membrane glycoproteins. To determine whether one of these candidate open reading frames, BXLF2, codes for gp85, an antibody was made to a 17-residue peptide derived from positions 518 to 533 of the predicted BXLF2 protein. The reactivity of the antipeptide antibody was then compared with that of the monoclonal antibody F-2-1, which was originally used to define and characterize gp85. Antipeptide antibody and F-2-1 immunoprecipitated glycosylated molecules with identical electrophoretic mobilities; digestion of the two immunoprecipitated proteins with V8 protease generated comparable peptides; and the antipeptide antibody reacted in Western immunoblots with the gp85 glycoprotein that had been immunoprecipitated by F-2-1 before transfer to nitrocellulose. In addition, a monospecific rabbit antibody, made against native gp85, reacted with the peptide used for immunization. These results are compatible with the hypothesis that the BXLF2 open reading frame codes for gp85.     

Functional reconstitution of influenza virus envelopes.

We have examined several procedures for the reconstitution of influenza virus envelopes, based on detergent removal from solubilized viral membranes. With octylglucoside, no functionally active virosomes are formed, irrespective of the rate of detergent removal: in the final preparation the viral spike proteins appear predominantly as rosettes. Protein incorporation in reconstituted vesicles is improved when a method based on reverse-phase evaporation of octylglucoside-solubilized viral membranes in an ether/water system is employed. However, the resulting vesicles do not fuse with biological membranes, but exhibit only a non-physiological fusion reaction with negatively charged liposomes. Functional reconstitution of viral envelopes is achieved after solubilization with octaethyleneglycol mono(n-dodecyl)ether (C12E8), and subsequent detergent removal with Bio-Beads SM-2. The spike protein molecules are quantitatively incorporated in a single population of virosomes of uniform buoyant density and appear on both sides of the membrane. The virosomes display hemagglutination activity and a strictly pH-dependent hemolytic activity. The virosomes fuse with erythrocyte ghosts, as revealed by a fluorescence resonance energy transfer assay. The rate and the pH dependence of fusion are essentially the same as those of the intact virus. The virosomes also fuse with cultured cells, either at the level of the endosomal membrane or directly with the cellular plasma membrane upon a brief exposure to low pH.     

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.     

Monoclonal antibody to the amino-terminal L sequence of murine leukemia virus glycosylated gag polyproteins demonstrates their unusual orientation in the cell membrane.

To analyze cell surface murine leukemia virus gag protein expression, we have prepared monoclonal antibodies against the spontaneous AKR T lymphoma KKT-2. One of these antibodies, 43-13, detects an AKR-specific viral p12 determinant. A second monoclonal antibody, 43-17, detects a novel murine leukemia virus-related antigen found on glycosylated gag polyproteins (gp95gag, gp85gag, and gp55gag) on the surface of cells infected with and producing ecotropic endogenous viruses, but does not detect antigens within these virions. The 43-17 antibody immunoprecipitates the precursor of the cell surface gag protein whether in its glycosylated or unglycosylated state, but does not detect the cytoplasmic precursor of the virion gag proteins (Pr65gag). Based on these findings, we have localized the 43-17 determinant to the unique amino-terminal part of the glycosylated gag polyprotein (the L domain). We have determined that gp95gag contains L-p15-p12-p30-p10 determinants, whereas gp85gag lacks the carboxyterminal p10 determinant, and gp55gag lacks both p30 and p10 carboxy terminal determinants. Analysis of cell surface gag expression with the 43-17 antibody leads us to propose that the L domain plays a crucial role in (i) the insertion and orientation of murine leukemia virus gag polyproteins in the cell membrane and (ii) the relative abundance of expression of AKR leukemia virus versus Moloney murine leukemia virus glycosylated gag polyproteins in infected cells.     

Early events of fusion between Epstein Barr virus and human lymphoblastoid cells (Raji) detected by R18 fluorescence dequenching measurements

Relief of fluorescence self-quenching was used to monitor fusion (14) of Epstein Barr virus (EBV) with Raji cells after exposure of the virus to a variety of experimental conditions such as neutral or low pH, enzymatic modification of the viral spike glycoproteins, or inhibition of the protein kinase C (PKC) activity. Incubation of the virus at pH 5.9 prior to the binding to the cell membrane led to a significant enhancement of fusion with the plasma membrane. Treatment of Raji cells with an agent known to elevate the endosomal and lysosomal pH (lysosomotropic agent) (3, 12) partially prevented fusion at neutral pH. Desialylation of EBV significantly reduced the extent of fusion with Raji cells. Protein kinase C inhibitor reduced EBV fusion with Raji cells, while treatment with the tumor promotor and the PKC activator TPA caused an increase in the final extent of fusion. Our results suggest that EBV fuses with lymphoblastoid cells in the endocytic vesicles after being rapidly internalized and that protein kinase C is involved in the process of viral entry into cells.