Sendai virus envelopes can mediate Epstein-Barr virus binding to and penetration into Epstein-Barr virus receptor-negative cells

Epstein-Barr virus (EBV) receptor-negative cells were treated with UV-inactivated Sendai virus (SV) or with reconstituted SV envelopes having a low hemolytic activity and then assayed for EBV binding or for susceptibility to EBV infection. EBV binding was assessed by using both unlabeled and fluoresceinated EBV preparations. It was found that SV or SV envelope treatment renders these cells able to bind EBV. Various experiments were performed to clarify the mechanism of this SV-induced binding. The EBV receptor-negative 1301 cells were treated with SV either at 0°C or at both 0 and 37°C successively and then examined for EBV binding at 0°C. It was thus found that when SV treatment was performed exclusively at 0°C, the target cells showed higher fluorescence intensity after their incubation with fluoresceinated EBV. In addition, Clostridium perfringens neuraminidase treatment of 1301 cells did not induce any EBV binding to these cells. These data indicate that EBV binding is not due to the disturbance of the cell membrane by SV envelope fusion or to the uncovering of EBV binding sites on the cells after the enzymatic action of SV neuraminidase. Moreover, bound EBV was partly eluted from SV-treated 1301 cells at 37°C, and the treatment of EBV with C. perfringens neuraminidase inhibited its SV-mediated binding. These data indicate that EBV binds to the hemagglutinin-neuraminidase of SV on the target cell surface and that a fraction of the bound EBV becomes irreversibly associated with the SV-treated cell membrane. Our data also show that EBV can penetrate into 1301 cells which have incorporated SV envelopes into their membrane, as demonstrated by the induction of the EBV-determined nuclear antigen by B95-8 EBV in SV envelope-treated 1301 cells.     

Electron microscopic studies of tumor viruses. II. Entry and uncoating of Epstein-Barr virus.

Entry of Epstein-Barr virus into human lymphoblastoid cells (Daudi cells) was studied by electron microscopy. At the site of viral attachment, two distinct interactions conducive to penetration of the virus occurred between the viral envelope and cell membrane, namely, (i) simultaneous dissolution of both the envelope and cell membrane, presumably resulting in passage of viral capsids into the cytoplasm and (ii) dissolution confined to the cell membrane with resulting penetration of enveloped virus. In the latter case envelope dissolution appears to occur subsequently in the cytoplasm with release of capsids. Fusion of the viral envelope with the cell membrane was not observed. The capsids exhibited two distinct structural forms--one dense, the other translucent or light in appearance. The former disrupted near the cell membrane with release of viral cores into the cytoplasm whereas the light capsids containing dense cores migrated toward the nucleus and accumulated in the perinuclear region. Apparently the process of releasing deoxyribonucleic acid (DNA) from the light capsid is slowed down or prevented in Daudi cells. A hypothesis is presented concerning the manner in which these two types of capsids initiate infection.     

Use of fluoresceinated Epstein-Barr virus to study Epstein-Barr virus-lymphoid cell interactions.

As a direct approach to visualize Epstein-Barr virus (EBV) binding to its cellular receptors and to learn more on the nature of this binding, virus preparations were conjugated to fluorescein isothiocyanate and used to detect EBV receptors on lymphoid cells. Different enzymatic and chemical treatments were also applied either to the virus or to target cells or to both, and the effect of these treatments on virus binding was then examined. The results obtained show that: (i) EBV can be fluoresceinated without affecting its infectivity or cell binding ability, and the fluoresceinated virus represents an important tool to investigate the biology and nature of EBV interactions with its cellular receptors; (ii) the two virus strains (P3HR-1 and B95-8) share common receptors on Raji cells; (iii) protease treatment of EBV or target cells abrogates virus binding; (iv) EBV receptors regenerate after removal of the protease, and this regeneration is inhibited by cycloheximide or sucrose; (v) EBV particles bear concanavalin A receptors, and this lectin hinders the interaction of the virus with its cellular receptors; (vi) neuraminidase treatment, various monosaccharides, ovalbumin, and glycopeptides derived from EBV or cell surface do not inhibit virus binding. Taken together, the above data also demonstrate that some cellular and viral surface (glyco-) proteins are required for EBV binding to its targets.     

Chemically induced infection of CD4-negative HeLa cells with HIV-1

Infection with human immunodeficiency virus type-1 (HIV-1) requires the presence of a CD4 molecule and chemokine receptors such as CXCR4 or CCR5 on the surface of target cells. However, it is still not clear how the virus enters the cells. Although CD4 was initially identified as the primary receptor for HIV-1, the expression of CD4 or one of the chemokine receptors alone is not sufficient to render susceptibility to infection with the virus. To ascertain whether or not adsorption of the virus needs charge-to-charge interaction between viral envelope and host cell membrane protein(s) and if binding alone promotes penetration of the virus into the cells, we have developed a chemically induced infection system targeting a CD4-negative and CXCR4-positive HeLa cell clone (N7 HeLa) which is usually not susceptible to infection with the LAI strain of HIV-1. Use of a poly-L-lysine (PLL)-coated culture plate to enhance the attachment of the virus to the cells made N7 HeLa cells infectable with HIV-1 at very low efficiency. PLL alone cannot fully substitute for the function of the CD4 molecule. However, trypsin-treated viruses, which have largely lost infectivity to CD4-positive MT-4 cells that are highly susceptible to HIV-1 infection, enhanced infectivity against N7 HeLa cells when the PLL-coated plate was used. These results provide evidence that infection with HIV-1 requires both high binding affinity between viruses and cells, and then needs a modification of the viral envelope such as cleavage of gp120/160 to enhance the infection, probably resulting in exposure of the hydrophobic fusion domain of gp41. HIV-1 infection of N7 HeLa cells was also enhanced by treatment with low pH, 12-O-tetradecanoylphorbol-13-acetate (TPA) and some factor(s) from the MT-4 cell culture supernatant. Not only tight viral adsorption with cleavage of the viral envelope but also some activated status of the cells may be required for sufficient HIV-1 infection in this artificial condition.     

Depletion of glycoprotein gp85 from virosomes made with Epstein-Barr virus proteins abolishes their ability to fuse with virus receptor-bearing cells.

Entry of an enveloped virus such as Epstein-Barr virus (EBV) into host cells involves fusion of the virion envelope with host cell membranes either at the surface of the cell or within endocytic vesicles. Previous work has indirectly implicated the EBV glycoprotein gp85 in this fusion process. A neutralizing monoclonal antibody to gp85, F-2-1, failed to inhibit binding of EBV to its receptor but interfered with virus fusion as measured with the self-quenching fluorophore octadecyl rhodamine B chloride (R18) (N. Miller and L. M. Hutt-Fletcher, J. Virol. 62:2366-2372, 1988). To test further the hypothesis that gp85 functions as a fusion protein, EBV virion proteins including or depleted of gp85 were incorporated into lipid vesicles to form virosomes. Virosomes were labeled with R18, and those that were made with undepleted protein were shown to behave in a manner similar to that of R18-labeled virus. They bound to receptor-positive but not to receptor-negative cells and fused with Raji cells but not with receptor-positive, fusion-incompetent Molt 4 cells; monoclonal antibodies that inhibited binding or fusion of virus inhibited binding and fusion of virosomes, and virus competed with virosomes for attachment to cells. In contrast, virosomes made from virus proteins depleted of gp85 by immunoaffinity chromatography remained capable of binding to receptor-positive cells but failed to fuse. These results are compatible with the hypothesis that gp85 is actively involved in the fusion of EBV with lymphoblatoid cell lines and suggest that the ability of antibody F-2-1 to neutralize infectivity of EBV represents a direct effect on the function of gp85 as a fusion protein.     

Neutralizing antibodies specific for glycoprotein H of herpes simplex virus permit viral attachment to cells but prevent penetration.

Monoclonal antibodies specific for gH of herpes simplex virus were shown previously to neutralize viral infectivity. Results presented here demonstrate that these antibodies (at least three of them) block viral penetration without inhibiting adsorption of virus to cells. Penetration of herpes simplex virus is by fusion of the virion envelope with the plasma membrane of a susceptible cell. Electron microscopy of thin sections of cells exposed to virus revealed that neutralized virus bound to the cell surface but did not fuse with the plasma membrane. Quantitation of virus adsorption by measuring the binding of purified radiolabeled virus to cells revealed that the anti-gH antibodies had little or no effect on adsorption. Monitoring cell and viral protein synthesis after exposure of cells to infectious and neutralized virus gave results consistent with the electron microscopic finding that the anti-gH antibodies blocked viral penetration. On the basis of the results presented here and other information published elsewhere, it is suggested that gH is one of three glycoproteins essential for penetration of herpes simplex virus into cells.     

Adaptability in herpesviruses: glycoprotein D-independent infectivity of pseudorabies virus.

Initial contact between herpesviruses and host cells is mediated by virion envelope glycoproteins which bind to cellular receptors. In several alphaherpesviruses, the nonessential glycoprotein gC has been found to interact with cell surface proteoglycans, whereas the essential glycoprotein gD is involved in stable secondary attachment. In addition, gD is necessary for penetration, which involves fusion between virion envelope and cellular cytoplasmic membrane. As opposed to other alphaherpesvirus gD homologs, pseudorabies virus (PrV) gD is not required for direct viral cell-to-cell spread. Therefore, gD- PrV can be passaged in noncomplementing cells by cocultivating infected and noninfected cells. Whereas infectivity was found to be strictly cell associated in early passages, repeated passaging resulted in the appearance of infectivity in the supernatant, finally reaching titers as high as 10(7) PFU/ml (PrV gD- Pass). Filtration experiments indicated that this infectivity was not due to the presence of infected cells, and the absence of gD was verified by Southern and Western blotting and by virus neutralization. Infection of bovine kidney cells constitutively expressing PrV gD interfered with the infectivity of wild-type PrV but did not inhibit that of PrV gD- Pass. Similar results were obtained after passaging of a second PrV mutant, PrV-376, which in addition to gD also lacks gG, gI, and gE. Penetration assays demonstrated that PrV gD- Pass entered cells much more slowly than wild-type PrV. In summary, our data demonstrate the existence of a gD-independent mode of initiation of infection in PrV and indicate that the essential function(s) that gD performs in wild-type PrV infection can be compensated for after passaging. Therefore, regarding the requirement for gD, PrV seems to be intermediate between herpes simplex virus type 1, in which gD is necessary for penetration and cell-to-cell spread, and varicella-zoster virus (VZV), which lacks a gD gene. Our data show that the relevance of an essential protein can change under selective pressure and thus demonstrate a way in which VZV could have evolved from a PrV-like ancestor.     

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.     

HIV infection does not require endocytosis of its receptor, CD4

The T cell surface molecule CD4 interacts with class II MHC molecules on the surface of target cells as well as with the envelope glycoprotein of human immunodeficiency virus (HIV). Internalization of CD4 molecules is observed after exposure of CD4+ T cells to either phorbol esters or appropriate antigen-bearing target cells. To determine whether HIV entry proceeds via receptor-mediated endocytosis or direct viral fusion with the cell membrane, we have constructed two mutants in the cytoplasmic domain of the CD4 protein that severely impair the ability of CD4 molecules to undergo endocytosis. Quantitative infectivity studies reveal that HeLa cell lines expressing wild-type or mutant CD4 molecules are equally susceptible to HIV infection. In addition, HIV binding does not lead to CD4 endocytosis. These studies indicate that although the CD4 molecule can be internalized, HIV entry proceeds via direct fusion of the viral envelope with the cell membrane.     

Activation of the Epstein-Barr virus replicative cycle by human herpesvirus 6.

One common attribute of herpesviruses is the ability to establish latent, life-long infections. The role of virus-virus interaction in viral reactivation between or among herpesviruses has not been studied. Preliminary experiments in our laboratory had indicated that infection of Epstein-Barr virus (EBV) genome-positive human lymphoid cell lines with human herpesvirus 6 (HHV-6) results in EBV reactivation in these cells. To further our knowledge of this complex phenomenon, we investigated the effect of HHV-6 infection on expression of the viral lytic cycle proteins of EBV. Our results indicate that HHV-6 upregulates, by up to 10-fold, expression of the immediate-early Zebra antigen and the diffuse and restricted (85 kDa) early antigens (EA-D and EA-R, respectively) in both EBV producer and nonproducer cell lines (i.e., P3HR1, Akata, and Raji). Maximal EA-D induction was observed at 72 h post-HHV-6 infection. Furthermore, expression of late EBV gene products, namely, the viral capsid antigen (125 kDa) and viral membrane glycoprotein gp350, was also increased in EBV producer cells (P3HR1 and Akata) following infection by HHV-6. By using dual-color membrane immunofluorescence, it was found that most of the cells expressing viral membrane glycoprotein gp350 were also positive for HHV-6 antigens, suggesting a direct effect of HHV-6 replication on induction of the EBV replicative cycle. No expression of late EBV antigens was observed in Raji cells following infection by HHV-6, implying a lack of functional complementation between the deleted form of EBV found in Raji cells and the superinfecting HHV-6. The susceptibility of the cell lines to infection by HHV-6 correlated with increased expression of various EBV proteins in that B95-8 cells, which are not susceptible to HHV-6 infection, did not show an increase in expression of EBV antigens following treatment with HHV-6. Moreover, UV light-irradiated or heat-inactivated HHV-6 had no upregulating effect on the Zebra antigen or EA-D in Raji cells, indicating that infectious virus is required for the observed effects of HHV-6 on these EBV products. These results show that HHV-6, another lymphotropic human herpesvirus, can activate EBV replication and may thus contribute to the pathogenesis of EBV-associated diseases.     

Anti-idiotype antibodies that mimic gp86 of human cytomegalovirus inhibit viral fusion but not attachment.

Human cytomegalovirus (CMV) infects cells by sequential processes involving attachment, fusion with the cell membrane, and penetration of the capsid. We used two monoclonal anti-idiotype that mimic one of the CMV envelope glycoproteins, gp86, to study its role in the early phases of CMV infection. Neither of two such antibodies inhibited virus binding to human embryonic lung (HEL) fibroblasts; however, both antibodies inhibited the fusion of CMV with HEL cells, as measured by an assay in which viral envelope is labeled with a fluorescent amphiphile (octadecyl rhodamine B chloride, or R18), resulting in increased fluorescence during fusion of virus with the cell membrane. Because these anti-idiotype antibodies were shown previously to bind to specific receptors on HEL cell membranes, these findings suggest that both gp86 and its cell membrane receptor may function in the fusion of human CMV with HEL cells.     

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.     

Epstein-Barr virus transforming protein LMP1 plays a critical role in virus production

The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1), which is critical for EBV-induced B-cell transformation, is also abundantly expressed during the lytic cycle of viral replication. However, the biological significance of this strong LMP1 induction remains unknown. We engineered a bacterial artificial chromosome clone containing the entire genome of Akata strain EBV to specifically disrupt the LMP1 gene. Akata cell clones harboring the episomes of LMP1-deleted EBV were established, and the effect of LMP1 loss on virus production was investigated. We found that the degree of viral DNA amplification and the expression levels of viral late gene products were unaffected by LMP1 loss, demonstrating that the LMP1-deleted EBV entered the lytic replication cycle as efficiently as the wild-type counterpart. This was confirmed by our electron microscopic observation that nucleocapsid formation inside nuclei occurred even in the absence of LMP1. By contrast, loss of LMP1 severely impaired virus release into culture supernatants, resulting in poor infection efficiency. The expression of truncated LMP1 in Akata cells harboring LMP1-deleted EBV rescued the virus release into the culture supernatant and the infectivity, and full-length LMP1 partially rescued the infectivity. These results indicate that inducible expression of LMP1 during the viral lytic cycle plays a critical role in virus production.     

Inhibition of human immunodeficiency virus (HIV) infection in vitro by anticarbohydrate monoclonal antibodies: peripheral glycosylation of HIV envelope glycoprotein gp120 may be a target for virus neutralization.

Carbohydrate structures are often involved in the initial adhesion of pathogens to target cells. In the present study, a panel of anticarbohydrate monoclonal antibodies (MAbs) was tested for their ability to inhibit in vitro human immunodeficiency virus infectivity. MAbs against three different N- and O-linked carbohydrate epitopes (LeY, A1, and sialyl-Tn) were able to block infection by cell-free virus as well as inhibit syncytium formation. Inhibition of virus infectivity was independent of virus strain (HTLVIIIB or patient isolate SSI-002), the cell line used for virus propagation (H9 or MT4), and the cell type used as the infection target (MT4, PMC, or selected T4 lymphocytes). Inhibition was observed when viruses were preincubated with MAbs but not when cells were preincubated with MAbs before inoculation, and the MAbs were shown to precipitate 125I-labeled gp120. The MAbs therefore define carbohydrate structures expressed by the viral envelope glycoprotein gp120, indicating that glycans of the viral envelope are possible targets for immunotherapy or vaccine development or both.     

Infectious rotavirus enters cells by direct cell membrane penetration, not by endocytosis.

Rotaviruses are icosahedral viruses with a segmented, double-stranded RNA genome. They are the major cause of severe infantile infectious diarrhea. Rotavirus growth in tissue culture is markedly enhanced by pretreatment of virus with trypsin. Trypsin activation is associated with cleavage of the viral hemagglutinin (viral protein 3 [VP3]; 88 kilodaltons) into two fragments (60 and 28 kilodaltons). The mechanism by which proteolytic cleavage leads to enhanced growth is unknown. Cleavage of VP3 does not alter viral binding to cell monolayers. In previous electron microscopic studies of infected cell cultures, it has been demonstrated that rotavirus particles enter cells by both endocytosis and direct cell membrane penetration. To determine whether trypsin treatment affected rotavirus internalization, we studied the kinetics of entry of infectious rhesus rotavirus (RRV) into MA104 cells. Trypsin-activated RRV was internalized with a half-time of 3 to 5 min, while nonactivated virus disappeared from the cell surface with a half-time of 30 to 50 min. In contrast to trypsin-activated RRV, loss of nonactivated RRV from the cell surface did not result in the appearance of infection, as measured by plaque formation. Endocytosis inhibitors (sodium azide, dinitrophenol) and lysosomotropic agents (ammonium chloride, chloroquine) had a limited effect on the entry of infectious virus into cells. Purified trypsin-activated RRV added to cell monolayers at pH 7.4 medicated 51Cr, [14C]choline, and [3H]inositol released from prelabeled MA104 cells. This release could be specifically blocked by neutralizing antibodies to VP3. These results suggest that MA104 cell infection follows the rapid entry of trypsin-activated RRV by direct cell membrane penetration. Cell membrane penetration of infectious RRV is initiated by trypsin cleavage of VP3. Neutralizing antibodies can inhibit this direct membrane penetration.     

A broad antiviral neutral glycolipid, fattiviracin FV-8, is a membrane fluidity modulator

To screen for an effective antiviral compound which acts as a membrane fluidity modulator, dichotomous effects on human immunodeficiency virus type 1 (HIV-1) infection due to different treatments of several glycolipids and lipids were examined. Continuous treatment of infected cells with 40 microg ml(-1) fattiviracin FV-8, a neutral glycolipid isolated from Streptomycetes, inhibited HIV-1 infection by 96%, whereas pretreatment with 400 microg ml(-1) enhanced infectivity 4.7-fold. The glycolipid showed similar effects as glycyrrhizin; it inhibited infection by broad enveloped viruses, blocked cell-cell fusion, reduced the infectivity of treated virions and enhanced susceptibility to viral infection and cell-cell fusion of cells pretreated with high doses of the compound. Suppression and enhancement was correlated with decreased and increased fluidity of plasma membrane of the fattiviracin FV-8-treated cells. Restricted movement of membrane molecules might impede the formation of a wide fusion pore, and therefore be critical to the entry of viruses. Thus, this can be applied as a new strategy to inhibit viral infections.     

Binding to sialic acids is not an essential step for the entry of animal rotaviruses to epithelial cells in culture.

The infection of target cells by animal rotaviruses requires the presence of sialic acids on the cell surface. Treatment of the cells with neuraminidases or incubation of the viruses with some sialoglycoproteins, such as glycophorin A, greatly reduces virus binding, with the consequent reduction of viral infectivity. In this work, we report the isolation of animal rotavirus variants whose infectivity is no longer dependent on the presence of sialic acids on the cell surface. In addition, although these variants bind to glycophorin A as efficiently as the wild-type virus, this interaction no longer inhibit viral infectivity. These observations indicate that the initial interaction of the mutants with the cell occurs at a site different from the sialic acid-binding site located on VP8, the smaller trypsin cleavage product of VP4. Reassortant analysis showed that the mutant phenotype segregates with the VP4 gene. Neutralizing monoclonal antibodies directed to VP4 and VP7 were tested for their ability to neutralize the variants. Antibodies to VP7 and VP5, the larger trypsin cleavage product of VP4, neutralized the mutants as efficiently as the wild-type virus. In contrast, although antibodies to VP8 were able to bind to the mutants, they showed little or no neutralizing activity. The implications of these findings in rotavirus attachment to and penetration of epithelial cells in culture are discussed.     

Epstein-Barr virus that lacks glycoprotein gN is impaired in assembly and infection

The Epstein-Barr virus (EBV) glycoproteins N and M (gN and gM) are encoded by the BLRF1 and BBRF3 genes. To examine the function of the EBV gN-gM complex, recombinant virus was constructed in which the BLRF1 gene was interrupted with a neomycin resistance cassette. Recombinant virus lacked not only gN but also detectable gM. A significant proportion of the recombinant virus capsids remained associated with condensed chromatin in the nucleus of virus-producing cells, and cytoplasmic vesicles containing enveloped virus were scarce. Virus egress was impaired, and sedimentation analysis revealed that the majority of the virus that was released lacked a complete envelope. The small amount of virus that could bind to cells was also impaired in infectivity at a step following fusion. These data are consistent with the hypothesis that the predicted 78-amino-acid cytoplasmic tail of gM, which is highly charged and rich in prolines, interacts with the virion tegument. It is proposed that this interaction is important both for association of capsids with cell membrane to assemble and release enveloped particles and for dissociation of the capsid from the membrane of the newly infected cell on its way to the cell nucleus. The phenotype of EBV lacking the gN-gM complex is more striking than that of most alphaherpesviruses lacking the same complex but resembles in many respects the phenotype of pseudorabies virus lacking glycoproteins gM, gE, and gI. Since EBV does not encode homologs for gE and gI, this suggests that functions that may have some redundancy in alphaherpesviruses have been concentrated in fewer proteins in EBV.     

Soluble Epstein-Barr virus glycoproteins gH, gL, and gp42 form a 1:1:1 stable complex that acts like soluble gp42 in B-cell fusion but not in epithelial cell fusion

Epstein-Barr virus (EBV) is a herpesvirus that infects cells by fusing its lipid envelope with the target cell membrane. The fusion process requires the actions of viral glycoproteins gH, gL, and gB for entry into epithelial cells and additionally requires gp42 for entry into B cells. To further study the roles of these membrane-associated glycoproteins, purified soluble forms of gp42, gH, and gL were expressed that lack the membrane-spanning regions. The soluble gH/gL protein complex binds to soluble gp42 with high affinity, forming a stable heterotrimer with 1:1:1 stoichiometry, and this complex is not formed by an N-terminally truncated variant of gp42. The effects of adding soluble gp42, gH/gL, and gH/gL/gp42 were examined with a virus-free cell-cell fusion assay. The results demonstrate that, in contrast to gp42, membrane fusion does not proceed with secreted gH/gL. The addition of soluble gH/gL does not inhibit or enhance B-cell or epithelial cell fusion when membrane-bound gH/gL, gB, and gp42 are present. However, the soluble gH/gL/gp42 complex does activate membrane fusion with B cells, similarly to soluble gp42, but it does not inhibit fusion with epithelial cells, as observed for gp42 alone. A gp42 peptide, derived from an N-terminal segment involved in gH/gL interactions, binds to soluble gH/gL and inhibits EBV-mediated epithelial cell fusion, mimicking gp42. These observations reveal distinct functional requirements for gH/gL and gp42 complexes in EBV-mediated membrane fusion.     

Identification and characterization of fusion and processing domains of the human immunodeficiency virus type 2 envelope glycoprotein.

The envelope glycoprotein of the human immunodeficiency virus type 2 (HIV-2) is synthesized as a polyprotein precursor which is proteolytically processed to produce the mature surface and transmembrane envelope glycoproteins. The processed envelope glycoprotein species are responsible for the fusion between the viral envelope and the host cell membrane during the infection process. The envelope glycoprotein also induces syncytium formation between envelope-expressing cells and receptor-bearing cells. To characterize domains of the HIV-2 envelope glycoprotein involved in membrane fusion and in proteolytic processing, we introduced single amino acid mutations into the region of the HIV-2 surface glycoprotein corresponding to the principal neutralizing determinant (the V3 loop) of HIV-1, the putative HIV-2 envelope precursor-processing sequence, and the hydrophobic amino terminus of the HIV-2 transmembrane envelope glycoprotein. The effects of these mutations on syncytium formation, virus infectivity, envelope expression, envelope processing, and CD4 binding were analyzed. Our results suggest that the V3-like region of the HIV-2 surface glycoprotein and the hydrophobic amino terminus of the transmembrane glycoprotein are HIV-2 fusion domains and characterize the effects of mutations in the HIV-2 envelope glycoprotein precursor-processing sequence.