INTRODUCTION

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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 ₁ 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.

	MATERIALS AND METHODS

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Cells. Akata, a Burkitt lymphoma-derived cell line that carries and can be induced to make EBV (41) (a gift of John Sixbey, St. Jude Children's Research Hospital, Memphis, Tenn.); EBV-negative Akata cells (a gift of Jeffrey Sample, St. Jude Children's Research Hospital); Raji (33), an EBV genome-positive, nonproducing human B-cell line that expresses CR2; and P3HR1-C15 (15), an EBV-positive human B-cell line that does not express CR2 (a gift of George Miller, Yale University, New Haven, Conn.), were grown in RPMI 1640 (Sigma Chemical Co., St. Louis, Mo.) supplemented with 10% heat-inactivated fetal bovine serum (Gibco-BRL Life Technologies, Grand Island, N.Y.). Human leukocytes were obtained from heparinized adult peripheral blood or from cord blood by flotation on lymphocyte separation medium and were depleted of T cells by a double cycle of rosetting with sheep erythrocytes as previously described (26).

Virus. EBV was obtained from Akata cells which were resuspended at a concentration of 4 × 10⁶ per ml and induced with 100 µg of anti-human immunoglobulin G per ml for 6 days. Virus to be used for extraction of DNA was harvested by high-speed centrifugation from spent culture medium which had been clarified by centrifugation at 4,000 × g for 10 min and was purified twice by sedimentation in dextran (30). Virus to be used for cell infection was harvested from clarified culture medium that had been passed through a 0.8-µm-pore-size filter.

Preparation and transfection of constructs used for homologous recombination. Dextran-purified virus harvested from the spent medium of 8 × 10⁹ Akata cells was digested with proteinase K, and virion DNA was purified three times by centrifugation in cesium chloride. DNA that sedimented at a density of 1.718 g of cesium per ml was digested with EcoRI. The 22.6-kb EBV EcoRI C fragment, which begins at bp 95238 of the B95-8 sequence and ends at approximately bp 117800 and includes the BZLF2 ORF (42), was cloned into the multiple cloning site of pBluescript II (Stratagene, La Jolla, Calif.). A 5.7-kb SpeI/SalI fragment, corresponding to bp 99537 to 105296 of the B95-8 sequence (3), was subcloned into the same vector, producing a fragment that had a unique HpaI site in the BZLF2 ORF (bp 102116 to 101448) 182 bp from the initiation codon. A 1.5-kb XmnI/HincII fragment, containing the neomycin resistance (Neo^r) gene under control of the simian virus 40 promoter, was digested from pcDNA3 (Invitrogen, San Diego, Calif.), blunt ended, and cloned into this HpaI site at bp 101934. The SpeI/SalI fragment, now 7.2 kb by virtue of the insertion of the Neo^r gene, was purified and used to transfect Akata cells with DEAE-dextran (2). Twenty million cells were incubated with 10 µg of DNA and 0.4 mg of dextran for 90 min at 37°C, washed, resuspended in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (Gibco) at a concentration of 2 × 10⁶ cells per ml, and cultured for 2 days. Cells were then plated at 10⁴ per well in 96-well tissue culture plates in medium containing 700 µg of G418 (Gibco) per ml and fed weekly with fresh drug-containing medium. Resistant clones began to emerge after approximately 3 weeks.

Derivation of cells containing only recombinant episomes. Drug-resistant Akata cells that contained wild-type episomes and episomes that had undergone homologous recombination with the Neo^r gene-containing fragment were induced with anti-immunoglobulin. After 6 days, 1 ml of spent culture medium, diluted 1/15, was used to infect 5 × 10⁶ EBV-negative Akata cells. After 2 more days of culture, the cells were plated in 96-well plates at 10⁴ per well in medium containing 700 µg of G418. Resistant clones began to emerge after approximately 3 weeks.

Antibodies. MAbs F-2-1 (40), which reacts with gp42 (26); E1D1 (31), which reacts with the EBV gH-gL complex (26); and 72A1, which reacts with gp350 (17), were obtained from spent culture medium of hybridoma cells grown in RPMI 1640 supplemented with 20% heat-inactivated fetal bovine serum. Two anti-peptide antibodies were made to synthetic peptides corresponding to residues 125 to 137 of gpL (45) and to residues 219 to 232 of gp150, respectively (23). All antibodies were purified by chromatography on protein A (Sigma) coupled to Affigel-15 (Bio-Rad, Richmond, Calif.).

Radiolabeling and immunoprecipitation. EBV proteins were labeled biosynthetically with [³H]glucosamine (20 Ci/mmol; Amersham Corp., Arlington Heights, Ill.) for 20 h at 6 h after induction with anti-human immunoglobulin G as previously described (45). Labeled cells were solubilized in radioimmunoprecipitation buffer (50 mM Tris-HCl [pH 7.2], 0.15 M NaCl, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 0.1 mM phenylmethylsulfonyl fluoride, and 100 U of aprotinin per ml) and immunoprecipitated with antibody and protein A-Sepharose CL4B (Sigma). Immunoprecipitated proteins were washed, dissociated by boiling in sample buffer containing 2-mercaptoethanol, and analyzed by SDS-polyacrylamide gel electrophoresis in acrylamide cross-linked with 0.28% N,N'-diallyltartardiamide, followed by fluorography.

Slot blot assays. The amount of EBV DNA in cells or virion particles was measured by hybridization with the BamHI W fragment of EBV DNA labeled with ³²P by means of a random-primed DNA labeling kit (Boehringer Mannheim, Indianapolis, Ind.). Serial dilutions of cells (8 × 10⁴ to 2.5 × 10³) in phosphate-buffered saline were applied to a charged nylon membrane (Magnacharge; Micron Separations Inc., Westborough, Mass.) with a slot blot filtration manifold. Membranes were treated as previously described (20) except that DNA was cross-linked by UV irradiation rather than by baking the membrane. Samples (150 µl) of culture supernatant containing virus were filtered through a 0.8-µm-pore-size filter to remove cells and digested for 10 min at room temperature with DNase I. Five microliters of 0.5 M EDTA was added, and virus particles were sedimented for 1 h at 16,000 × g. Sedimented virus was digested overnight at 56°C with proteinase K (12 mg per ml in 0.2 M EDTA), and serial dilutions were made in phosphate-buffered saline. The DNA in each sample was denatured by the addition of 1/10 volume of 5N NaOH, neutralized with 2 M ammonium acetate, applied to a nylon membrane, and cross-linked by UV irradiation. Hybridizations were carried out as described previously (20) and quantified by scanning with a Molecular Dynamics Storm PhosphorImager.

Southern blotting. Cells were digested overnight at 56°C with proteinase K (1 mg per ml in 100 mM NaCl, 10 mM Tris-HCl [pH 8.0], 25 mM EDTA, and 0.5% SDS), and DNA was purified by phenol-chloroform extraction and ethanol precipitation. Purified DNA was digested overnight with EcoRI and HindIII and separated by agarose gel electrophoresis in 0.7% agarose. Separated DNA was transferred to a nylon membrane (Magnacharge) by capillary action, cross-linked, and hybridized either with the 5.7-kb SpeI/SalI fragment corresponding to bp 99537 to 105296 of the B95-8 sequence or with the XmnI/HincII fragment of pcDNA3 containing the Neo^r gene. Both probes were labeled with ³²P.

Virus binding assays. Akata cells (140 million) containing wild-type or recombinant virus were induced with goat anti-human immunoglobulin in 8 ml of medium. Four hours later 500 µCi of [³H]thymidine (20 to 30 Ci per mmol; Amersham) was added, and 30 min later the volume of medium was increased to 80 ml. After 3 days of incubation virus was harvested from the culture supernatant by centrifugation and resuspended in 800 µl of fresh medium. EBV receptor-positive Raji and EBV receptor-negative P3HR1-C15 cells were fixed in ice-cold 0.1% paraformaldehyde, and the ability of the radiolabeled virus to bind to the cells in the presence or absence of antibody 72A1 to gp350 was determined as previously described (29).

Assays for infection of B cells. Five million T-cell-depleted peripheral blood leukocytes were incubated at 37°C with 300 µl of dilutions of filtered culture supernatant from induced Akata cells. After 2 h, the volume was brought to 5 ml with medium containing serum and the cells were reincubated. Five hundred thousand EBV-negative Akata cells were incubated with 150 µl of virus for 2 h at 37°C, after which the volume was brought to 3 ml and the cells were reincubated. Five days later both leukocytes and Akata cells were harvested and analyzed by Western blotting for expression of EBNA 1. In addition, 600,000 T-cell-depleted peripheral blood leukocytes were incubated for 2 h at 37°C with 240 µl of virus, plated in quintuplicate at 10⁵ cells per well in 96-well tissue culture plates, and reincubated for 4 weeks, at which time the wells were examined for the presence of transforming foci.

Western blotting. Cells were lysed in immunoprecipitation buffer, electrophoresed in polyacrylamide, and then electrically transferred onto nitrocellulose membranes (0.45-µm pore size; Schleicher and Schuell, Inc., Keene, N.H.) at 20 mA for 18 h. The transferred sheets were treated for at least 3 h with blocking buffer (10 mM Tris-HCl [pH 7.2], 0.15 M NaCl, 5% skim milk, 0.05% sodium azide) and reacted for at least 3 h with blocking buffer containing a 1/500 dilution of EBNA 1-positive human serum. They were then washed five times with wash buffer (10 mM Tris-HCl [pH 7.2], 0.15% NaCl, 0.3% Tween 20) for 10 min each time followed by an overnight wash. The washed sheets were reacted with alkaline phosphatase-conjugated goat anti-human antibodies (Hyclone) for 3 h, and the bound anti-human antibodies were detected by reacting with substrate 5-bromo-4-chloro-3-indolylphosphate and Nitro Blue Tetrazolium (Sigma).

Polyethylene glycol-mediated infection. Samples of 5 × 10⁶ T-cell-depleted peripheral blood leukocytes were incubated for 1 h on ice with recombinant virus or growth medium. Cells were washed once and gently resuspended in 1 ml of 35% polyethylene glycol 1500 (Boehringer Mannheim) or serum-free medium for 5 min. Ten milliliters of medium was added, and cells were centrifuged at 400 × g, resuspended in fresh growth medium, and incubated for 14 days before being harvested and subjected to Western blot analysis. In some experiments cells were fed weekly and kept until outgrowth of cell lines occurred.

	RESULTS

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Generation of recombinant EBV with a selectable marker in the BZLF2 ORF. Several different strategies have been used to derive EBV with expression of individual proteins blocked. However, the Akata strain of EBV can be induced synchronously with anti-human immunoglobulin to make virus (41), and it is easier to label less abundant proteins like gp42 in Akata cells, which produce relatively high numbers of viruses, than in other EBV-producing cell lines. We therefore derived Akata virus with expression of gp42 blocked by transfection of Akata cells with a 7.2-kb fragment of Akata DNA that encompassed the BZLF2 ORF and contained the Neo^r gene 182 bp from its 5' end. Cells in which recombination had occurred were obtained by selection in the presence of G418.

View larger version (15K): [in this window] [in a new window]   FIG. 1.   (A) Diagram of the positions of the EcoRI and HindIII sites, numbered according to the B95-8 sequence, surrounding the SpeI/SalI fragment targeted for homologous recombination. The boxes above the line indicate the position of the SpeI/SalI fragment used as a probe and the insertion of the Neor gene at bp 101934. (B) Sizes of fragments expected from DNA from cells harboring wild-type episomes, a mixture of wild-type and recombinant episomes, or pure recombinant episomes after digestion with EcoRI and HindIII and probing with the 5.7-kb SpeI/SalI fragment.

View larger version (47K): [in this window] [in a new window]   FIG. 2.   Southern blot analysis of DNA extracted from Akata cells harboring wild-type episomes (Wt), a parental clone of Akata cells harboring a mixture of wild-type and recombinant episomes (Wt + Rc), and a clone derived from the parental clone which contains only recombinant episomes (Rc). DNA was digested with EcoRI and HindIII, and the two identical halves of the membrane were cut apart and probed either with the SpeI/SalI fragment of EBV or the XmnI/HincII fragment containing the Neor gene as indicated. Sizes in kilobases are indicated by the arrows.

Lack of expression of gp42 in cells harboring only recombinant episomes. To determine whether expression of gp42 had been lost as a result of the insertion of the Neo^r gene into the BZLF2 ORF, cells were induced with anti-human immunoglobulin, labeled with [³H]glucosamine, and immunoprecipitated with antibodies to EBV glycoproteins. Glycoproteins gp350 (running at the top of the gel) and gp150 were immunoprecipitated from Akata cells harboring either wild-type or recombinant episomes (Fig. 3). The gH-gL-gp42 complex could be immunoprecipitated either by antibody F-2-1 or by an anti-peptide antibody to gp25 from cells harboring wild-type episomes. In contrast, however, no proteins were immunoprecipitated by antibody F-2-1 from cells harboring recombinant episomes and antibody to gL immunoprecipitated a complex that included only gH and gL. This indicated that the gH-gL complex was still intact but that it lacked its third member, gp42. To confirm that the gH-gL complex was transported to the cell surface normally, Akata cells harboring recombinant episomes were induced with anti-human immunoglobulin and 2 days later cells were briefly fixed in ice-cold 0.1% paraformaldehyde and incubated with MAb E1D1, which reacts with a complex of gH and gL. MAb E1D1, visualized by rabbit anti-mouse serum conjugated to fluorescein, stained the surfaces of cells carrying recombinant episomes as well as it did cells harboring wild-type virus (Fig. 4). Indirect immunofluorescence staining with MAb E1D1 of virus bound to Raji cells also indicated that the gH-gL complex was present in virions that lacked gp42 (data not shown).

View larger version (90K): [in this window] [in a new window]   FIG. 3.   Electrophoretic analysis of proteins immunoprecipitated by MAb 72A1 to gp350, MAb F-2-1 to gp42, or rabbit anti-peptide antibodies to gp150 or gp25 from Akata cells harboring wild-type or recombinant episomes. The cells were induced with anti-human immunoglobulin and labeled with [3H]glucosamine. The arrows and numbers at the right indicate the masses of the immunoprecipitated proteins in kilodaltons.

View larger version (39K): [in this window] [in a new window]   FIG. 4.   Indirect immunofluorescence staining of Akata cells harboring only recombinant episomes (A) or wild-type episomes (B). Cells were induced with anti-human immunoglobulin for 48 h, fixed in 0.1% paraformaldehyde, and reacted with MAb E1D1 to the gH-gL complex followed by anti-mouse immunoglobulin conjugated to fluorescein.

Recombinant virus can exit cells. Glycoproteins are involved in both entry and egress of viruses. To determine whether exit of virus lacking gp42 occurred normally, a slot blot assay was used to assess the total amount of virus DNA associated with induced cells and that of the DNase-resistant virion DNA that could be pelleted from spent culture medium after it had been filtered through a 0.8-µm-pore-size filter to remove the cells. The values obtained for each were then compared for wild-type and recombinant virus. The ratio of virus DNA in cells containing wild-type virus versus that in cells containing recombinant virus was approximately 1.3, and the ratio of released and encapsidated virus DNA in wild-type virus versus that in recombinant virus was approximately 1.1 (Table 1). This indicated that failure to express gp42 had no significant effect on release of virus from the cell.

Recombinant virus can bind to B cells. To determine whether virus derived from recombinant cells was able to bind to receptor-positive cells, virus was labeled intrinsically with [³H]thymidine and its ability to bind to receptor-negative P3HR1-C15 cells or receptor-positive Raji cells in the presence or absence of antibody to gp350 was evaluated. The amount of labeled virus that bound to receptor-positive Raji cells was slightly higher for recombinant than for wild-type virus in this experiment (Table 2). However, in both cases the amount of acid-precipitable radioactivity bound to the cells could be reduced by antibody to an amount similar to that associated with receptor-negative cells. This indicated that the initial binding of virus to cells via an interaction between CR2 and gp350 was not affected by the lack of expression of gp42.

Recombinant virus cannot infect B cells. The ability of recombinant virus to infect B lymphocytes was then evaluated in terms of its ability to induce EBNA 1 in resting B cells or EBV-negative Akata cells and in terms of its ability to transform resting B cells. Slot blot analyses had indicated that the culture supernatant from Akata cells making wild-type virus contained 1.3 times as much virus as did supernatant from cells making recombinant virus. However, even undiluted recombinant virus was unable to induce EBNA 1 in peripheral blood leukocytes (Fig. 5A) or in EBV-negative Akata cells (Fig. 5B), whereas dilutions of 1/8 of wild-type virus induced EBNA 1 in both cell types. Wild-type virus at a dilution of 1/200 was able to transform peripheral blood leukocytes, whereas recombinant virus was unable to produce transformation foci at any dilution from 1/5 to 1/200 (Table 3).

View larger version (63K): [in this window] [in a new window]   FIG. 5.   Induction of EBNA 1 in T-cell-depleted peripheral blood leukocytes (A) or EBV-negative Akata cells (B) mock infected (0) or infected 5 days previously with wild-type (Wt) or recombinant (Rc) viruses at the dilutions indicated. Western blots were reacted with human serum containing antibody to EBNA 1 and with goat anti-human immunoglobulin conjugated to alkaline phosphatase.

Polyethylene glycol restores the infectivity of recombinant virus. Previous work has implicated gp42 in fusion of virus with the cell membrane (29). Since recombinant virus was able to bind to cells, we determined whether treatment of cells and bound virus with polyethylene glycol would restore the ability of recombinant virus to transform cells. Fourteen days after infection cells were harvested and examined for the presence of EBNA by Western blotting. Cells that had been infected with virus in the presence of polyethylene glycol, but not cells infected with recombinant virus alone, expressed EBNA (Fig. 6A). A B-cell line was also derived by the same protocol and is shown on the same Western blot. DNA from this line was extracted to confirm that it had been immortalized by recombinant virus rather than by any endogenous virus harbored by the donor of the cells. Southern blotting confirmed that recombinant virus carrying the Neo^r gene was the virus present in the immortalized line (Fig. 6B).

View larger version (54K): [in this window] [in a new window]   FIG. 6.   Infection and transformation of T-cell-depleted peripheral blood leukocytes with recombinant virus in the presence of polyethylene glycol. (A) Western blot analysis of Akata cells containing wild-type virus (Wt); a B-cell line derived from peripheral blood leukocytes by infection with recombinant virus in the presence of polyethylene glycol (PBL/Rc); or freshly isolated leukocytes harvested 14 days after mock infection (Mock), treatment with polyethylene glycol alone (PEG), infection with recombinant virus (Rc), or infection with recombinant virus in the presence of polyethylene glycol (PEG+Rc). Western blots were reacted with human serum containing antibody to EBNA 1 and with goat anti-human immunoglobulin conjugated to alkaline phosphatase. (B) Southern blot analysis of DNA extracted from Akata cells containing wild-type virus (Wt), a parental clone containing a mixture of recombinant and wild-type virus (Wt+Rc), Akata cells containing pure recombinant virus (Rc), and the B-cell line derived from peripheral blood leukocytes by infection with recombinant virus in the presence of polyethylene glycol (PBL/RC). DNA was digested with EcoRI and HindIII. The blot (left) was probed with the SpeI/SalI fragment of EBV, and then (right) stripped and reprobed with the 1.5-kb XmnI/SalI fragment containing the Neor gene. Sizes in kilobases are indicated by the arrows.

	DISCUSSION

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Indirect evidence has implicated the EBV glycoprotein gp42 as being essential to infection of B lymphocytes, and the work reported here confirms that B cells cannot be infected with virus that fails to express it. In addition, however, the development of strategies for isolating recombinant EBV genomes in virus strains that can be induced to make relatively large amounts of virus (37) has allowed us to evaluate the relative importance of the protein to several independent steps in the virus life cycle. As predicted by experiments with individually expressed recombinant proteins (25), gp42 is not required for the formation of an SDS-stable gH-gL complex that is transported to the cell surface. It also has no significant effect on the egress of virus, and its absence does not alter the specificity of virus binding to CR2-positive cells. Defects in virus that lacks gp42 appear to be limited to postbinding events.

The virus that fails to express gp42 may not be a true null mutant. The insertion of the Neo^r gene 182 bp from the initiation codon of the BZLF2 ORF that encodes gp42 might permit synthesis of a truncated 61-amino-acid protein. Unfortunately, we currently have no reagents that might identify such a species. Such a protein, if made, would include the putative signal sequence of gp42, and it is possible that the 61 amino acids still include the domain responsible for the interaction between gp42 and gH and gL. A truncated protein would, however, retain none of the four potential N-linked glycosylation sites and none of the 11 cysteine residues of the native molecule. As demonstrated experimentally (see above), it would also no longer express the epitope recognized by MAb F-2-1, which is identical with, or very close to, the site of interaction with MHC class II. It would therefore not be expected to be functional.

Most of the studies done to date suggest that gp42 is involved in the fusion of virus and cell membranes, and the ability of polyethylene glycol to restore infectivity of virus that fails to express the protein is consistent with this hypothesis. Polyethylene glycol has been used both to enhance virus binding and to facilitate fusion of membranes (7, 12, 14, 16). There is, however, no reason to suppose that the recombinant virus made here is in any way deficient in binding. Unlike the primary interactions of the alphaherpesviruses, like herpes simplex virus, with cell surfaces, which are of low affinity, the gp350-CR2 interaction is a high-affinity reaction. Although gp42 binds to MHC class II on the cell surface, neither antibodies to gp42 nor antibodies to MHC class II that block this binding have any effect on the ability of virus to bind to cells via CR2; rather, in the case of antibody F-2-1, which blocks the interaction of gp42 and MHC class II, virus-cell fusion is inhibited (29).

Glycoprotein gp42 is a type II membrane protein with an uncleaved signal peptide (25, 26, 39). Its amino acid sequence includes no other domain that is predicted to be hydrophobic, and it seems unlikely that the molecule is directly involved in the fusion process. Of the three proteins that make up the EBV gH-gL complex, gH, as the most hydrophobic of the three molecules, is perhaps the most likely to play that role and has in fact been implicated as doing so in other herpesviruses (4, 5, 8-11). It is clear, however, that even if gH is a prime mover in EBV fusion with the B lymphocyte surface, the protein is not functional in the absence of gp42. We have speculated (25) that the interaction of gp42 with MHC class II might be required simply to bring virus closer to the cell membrane than does the initial interaction between gp350 and CR2 or that perhaps signaling via MHC class II is important in some way for the induction of changes in cell surface behavior. The association of gp42 with gH and gL as an integral part of the complex rather than as a separate entity in the membrane would suggest that, whatever its role is, it is one that is closely linked, either spatially or temporally, to those of its partners. The relatively low number of receptors for EBV, estimated to be approximately 30,000 per cell (44) even on the high-level-expressing Raji line, does not lend itself well to definitive electron microscopic analysis of the sequential events in EBV entry. However, the blocking of virus lacking gp42 at the cell surface opens up new possibilities in this regard. Complementation of recombinant virus with gp42 which contains defined mutations should also provide new insight into the complexity of the role that the molecule plays in B-cell infection.