Epstein-Barr Virus Recombinant Lacking Expression of Glycoprotein gp150 Infects B Cells Normally but Is Enhanced for Infection of Epithelial Cells

Glycoprotein gp150 is a highly glycosylated protein encoded by the BDLF3 open reading frame of Epstein-Barr virus (EBV). It does not have a homolog in the alpha- and betaherpesviruses, and its function is not known. To determine whether the protein is essential for replication of EBV in vitro, a recombinant virus which lacked its expression was made. The recombinant virus had no defects in assembly, egress, binding, or infectivity for B cells or epithelial cells. Infection of epithelial cells was, however, enhanced. The glycoprotein was sensitive to digestion with a glycoprotease that digests sialomucins, but no adhesion to cells that express selectins that bind to sialomucin ligands could be detected.     

Glycoprotein B Cleavage Is Important for Murid Herpesvirus 4 To Infect Myeloid Cells

Glycoprotein B (gB) is a conserved herpesvirus virion component implicated in membrane fusion. As with many—but not all—herpesviruses, the gB of murid herpesvirus 4 (MuHV-4) is cleaved into disulfide-linked subunits, apparently by furin. Preventing gB cleavage for some herpesviruses causes minor infection deficits in vitro, but what the cleavage contributes to host colonization has been unclear. To address this, we mutated the furin cleavage site (R-R-K-R) of the MuHV-4 gB. Abolishing gB cleavage did not affect its expression levels, glycosylation, or antigenic conformation. In vitro, mutant viruses entered fibroblasts and epithelial cells normally but had a significant entry deficit in myeloid cells such as macrophages and bone marrow-derived dendritic cells. The deficit in myeloid cells was not due to reduced virion binding or endocytosis, suggesting that gB cleavage promotes infection at a postendocytic entry step, presumably viral membrane fusion. In vivo, viruses lacking gB cleavage showed reduced lytic spread in the lungs. Alveolar epithelial cell infection was normal, but alveolar macrophage infection was significantly reduced. Normal long-term latency in lymphoid tissue was established nonetheless.     

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.     

细胞间接触是EB病毒自发感染人类上皮细胞的有效途径

选用产EB病毒的绒猴淋巴细胞B95-8系和补体受体2型(complement receptor 2,CR2)与多聚免疫球蛋白受体(polymeric immunoglobulin receptor,plgR)表达阴性的人水生化上皮细胞Hacat系共培养,进行细胞接触感染实验。一周后去除B95-8细胞,仅留Hacat细胞,并以自行改进的方法鉴定前者是否得以彻底去除。在证实没有．B95-8残留后,PCR和原位杂交分别检验剩余Hacat细胞中EB病毒的感染结果。实验结果表明：改进的方法能够灵敏和简便地判断B95-8细胞的污染与否,并且与．B95-8细胞接触共培养的Hacat细胞能被EB病毒有效地感染,后者暗示了EB病毒对上皮细胞可能存在细胞融合和CR2或plgR介导之外新的感染途径。本研究在一定程度上简化了前人的细胞接触感染方法,也为建立天然的EB病毒自发有效地感染上皮细胞的模型奠定了基础。     

细胞间接触是EB病毒自发感染人类上皮细胞的有效途径

选用产EB病毒的绒猴淋巴细胞B95-8系和补体受体2型(complement receptor 2, CR2)与多聚免疫球蛋白受体(polymeric immunoglobulin receptor, pIgR)表达阴性的人永生化上皮细胞Hacat系共培养,进行细胞接触感染实验.一周后去除B95-8细胞,仅留Hacat细胞,并以自行改进的方法鉴定前者是否得以彻底去除.在证实没有B95-8残留后,PCR和原位杂交分别检验剩余Hacat细胞中EB病毒的感染结果.实验结果表明改进的方法能够灵敏和简便地判断B95-8细胞的污染与否,并且与B95-8细胞接触共培养的Hacat细胞能被EB病毒有效地感染,后者暗示了EB病毒对上皮细胞可能存在细胞融合和CR2或pIgR介导之外新的感染途径.本研究在一定程度上简化了前人的细胞接触感染方法,也为建立天然的EB病毒自发有效地感染上皮细胞的模型奠定了基础.     

The Ebola Virus Glycoprotein Contributes to but Is Not Sufficient for Virulence In Vivo

Among the Ebola viruses most species cause severe hemorrhagic fever in humans; however, Reston ebolavirus (REBOV) has not been associated with human disease despite numerous documented infections. While the molecular basis for this difference remains unclear, in vitro evidence has suggested a role for the glycoprotein (GP) as a major filovirus pathogenicity factor, but direct evidence for such a role in the context of virus infection has been notably lacking. In order to assess the role of GP in EBOV virulence, we have developed a novel reverse genetics system for REBOV, which we report here. Together with a previously published full-length clone for Zaire ebolavirus (ZEBOV), this provides a unique possibility to directly investigate the role of an entire filovirus protein in pathogenesis. To this end we have generated recombinant ZEBOV (rZEBOV) and REBOV (rREBOV), as well as chimeric viruses in which the glycoproteins from these two virus species have been exchanged (rZEBOV-RGP and rREBOV-ZGP). All of these viruses could be rescued and the chimeras replicated with kinetics similar to their parent virus in tissue culture, indicating that the exchange of GP in these chimeric viruses is well tolerated. However, in a mouse model of infection rZEBOV-RGP demonstrated markedly decreased lethality and prolonged time to death when compared to rZEBOV, confirming that GP does indeed contribute to the full expression of virulence by ZEBOV. In contrast, rREBOV-ZGP did not show any signs of virulence, and was in fact slightly attenuated compared to rREBOV, demonstrating that GP alone is not sufficient to confer a lethal phenotype or exacerbate disease in this model. Thus, while these findings provide direct evidence that GP contributes to filovirus virulence in vivo, they also clearly indicate that other factors are needed for the acquisition of full virulence.     

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

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

Pseudorabies Virus Glycoprotein gK Is a Virion Structural Component Involved in Virus Release but Is Not Required for Entry

The pseudorabies virus (PrV) gene homologous to herpes simplex virus type 1 (HSV-1) UL53, which encodes HSV-1 glycoprotein K (gK), has recently been sequenced (J. Baumeister, B. G. Klupp, and T. C. Mettenleiter, J. Virol. 69:5560–5567, 1995). To identify the corresponding protein, a rabbit antiserum was raised against a 40-kDa glutathione S-transferase–gK fusion protein expressed in Escherichia coli. In Western blot analysis, this serum detected a 32-kDa polypeptide in PrV-infected cell lysates as well as a 36-kDa protein in purified virion preparations, demonstrating that PrV gK is a structural component of virions. After treatment of purified virions with endoglycosidase H, a 34-kDa protein was detected, while after incubation with N-glycosidase F, a 32-kDa protein was specifically recognized. This finding indicates that virion gK is modified by N-linked glycans of complex as well as high-mannose type. For functional analysis, the UL53 open reading frame was interrupted after codon 164 by insertion of a gG-lacZ expression cassette into the wild-type PrV genome (PrV-gKβ) or by insertion of the bovine herpesvirus 1 gB gene into a PrV gB⁻ genome (PrV-gK^gB). Infectious mutant virus progeny was obtained only on complementing gK-expressing cells, suggesting that gK has an important function in the replication cycle. After infection of Vero cells with either gK mutant, only single infected cells or small foci of infected cells were visible. In addition, virus yield was reduced approximately 30-fold, and penetration kinetics showed a delay in entry which could be compensated for by phenotypic gK complementation. Interestingly, the plating efficiency of PrV-gKβ was similar to that of wild-type PrV on complementing and noncomplementing cells, pointing to an essential function of gK in virus egress but not entry. Ultrastructurally, virus assembly and morphogenesis of PrV gK mutants in noncomplementing cells were similar to wild-type virus. However, late in infection, numerous nucleocapsids were found directly underneath the plasma membrane in stages typical for the entry process, a phenomenon not observed after wild-type virus infection and also not visible after infection of gK-complementing cells. Thus, we postulate that presence of gK is important to inhibit immediate reinfection.Herpesvirions are complex structures consisting of a nucleoprotein core, capsid, tegument, and envelope. They comprise at least 30 structural proteins (35). Pseudorabies virus (PrV), a member of the Alphaherpesvirinae, is an economically important animal pathogen, causing Aujeszky’s disease in swine. It is also highly pathogenic for most other mammals except higher primates, including humans (28, 45), and a wide range of cultured cells from different species support productive virus replication, reflecting the wide in vivo host range. Envelope glycoproteins play major roles in the early and late interactions between virion and host cell. They are required for virus entry and participate in release of free virions and viral spread by direct cell-to-cell transmission (27, 37). For PrV, 10 glycoproteins, designated gB, gC, gD, gE, gG, gH, gI, gL, gM, and gN, have been characterized (20, 27); these glycoproteins are involved in the attachment of virion to host cell (gC and gD), fusion of viral envelope and cellular cytoplasmic membrane (gB, gD, gH, and gL), spread from infected to noninfected cells (gB, gE, gH, gI, gL, and gM), and egress (gC, gE, and gI) (27, 37). Homologs of these glycoproteins are also present in other alphaherpesviruses (37). The gene coding for a potential 11th PrV glycoprotein, gK, has been described recently (3), but the protein and its function have not been identified.The product of the homologous UL53 open reading frame (ORF) of herpes simplex virus type 1 (HSV-1) is gK (13, 32). gK was detected in nuclear membranes and in membranes of the endoplasmic reticulum but was not observed in the plasma membrane (14). Also, it did not appear to be present in purified virion preparations (15). The latter result was surprising since earlier studies identified several mutations in HSV-1 gK resulting in syncytium-inducing phenotypes (7, 14), which indicates participation of gK in membrane fusion events during HSV-1 infection. Moreover, HSV-1 mutants in gK exhibited a delayed entry into noncomplementing cells, which is difficult to reconcile with absence of gK from virions (31). Mutants deficient for gK expression have been isolated and investigated by different groups (16, 17). Mutant F-gKβ carries a lacZ gene insertion in the HSV-1 strain F gK gene, which interrupts the ORF after codon 112 (16). In mutant ΔgK, derived from HSV-1 KOS, almost all of the UL53 gene was deleted (17). Both mutants formed small plaques on Vero cells, and virus yield was reduced to an extent which varied with the different confluencies of the infected cells, cell types, and mutants used for infection. However, both HSV-1 gK mutants showed a defect in efficient translocation of virions from the cytoplasm to the extracellular space, and only a few enveloped virions were present in the extracellular space after infection of Vero cells (16, 17). The authors therefore suggested that HSV-1 gK plays a role in virion transport during egress.Different routes of final envelopment and egress of alphaherpesvirions are discussed. It has been suggested that HSV-1 nucleocapsids acquire their envelope at the inner nuclear membrane and are transported as enveloped particles through the endoplasmic reticulum to the Golgi stacks, where glycoproteins are modified in situ during transport (5, 6, 19, 39), although other potential egress pathways cannot be excluded (4). In contrast, maturation of varicella-zoster virus and PrV involves primary envelopment at the nuclear membrane, followed by release of nucleocapsids into the cytoplasm and secondary envelopment in the trans-Golgi area (10, 12, 43). Final egress of virions appears to occur via transport vesicles containing one or more virus particles by fusion of vesicle and cell membrane. The possibility of different routes of virion egress is supported by studies of other proteins involved in egress, e.g., the UL20 proteins of HSV-1 and PrV and the PrV UL3.5 protein, which lacks a homolog in the HSV-1 genome (1, 8, 9). In UL20-negative HSV-1, virions accumulated in the perinuclear cisterna of Vero cells (1), while PrV UL20⁻ virions accumulated and were retained in cytoplasmic vesicles (9). PrV UL3.5 is important for budding of nucleocapsids into Golgi-derived vesicles during secondary envelopment (8). Thus, there appear to be profound differences in the egress pathways. Since HSV-1 gK was also implicated in egress, we were interested in identifying the PrV homolog and analyzing its function.     

Mapping the N-Terminal Residues of Epstein-Barr Virus gp42 That Bind gH/gL by Using Fluorescence Polarization and Cell-Based Fusion Assays

Epstein-Barr virus (EBV) requires at a minimum membrane-associated glycoproteins gB, gH, and gL for entry into host cells. B-cell entry additionally requires gp42, which binds to gH/gL and triggers viral entry into B cells. The presence of soluble gp42 inhibits membrane fusion with epithelial cells by forming a stable heterotrimer of gH/gL/gp42. The interaction of gp42 with gH/gL has been previously mapped to residues 36 to 81 at the N-terminal region of gp42. In this study, we further mapped this region to identify essential features for binding to gH/gL by use of synthetic peptides. Data from fluorescence polarization, cell-cell fusion, and viral infection assays demonstrated that 33 residues corresponding to 44 to 61 and 67 to 81 of gp42 were indispensable for maintaining low-nanomolar-concentration gH/gL binding affinity and inhibiting B-cell fusion and epithelial cell fusion as well as viral infection. Overall, specific, large hydrophobic side chain residues of gp42 appeared to provide critical interactions, determining the binding strength. Mutations of these residues also diminished the inhibition of B-cell and epithelial cell fusions as well as EBV infection. A linker region (residues 62 to 66) between two gH/gL binding regions served as an important spacer, but individual amino acids were not critical for gH/gL binding. Probing the binding site of gH/gL and gp42 with gp42 peptides is critical for a better understanding of the interaction of gH/gL with gp42 as well as for the design of novel entry inhibitors of EBV and related human herpesviruses.Epstein-Barr virus (EBV) is a large DNA virus belonging to the family of gammaherpesviruses. The virus is transmitted through saliva, and it can infect epithelial cells, as well as B cells, which provide the host latency reservoir (1, 22). Reactivation of the virus can occur intermittently, allowing virus infection of new hosts (1). Viral reactivation from latency is quickly controlled by the immune system. Primary infection with EBV can lead to the development of infectious mononucleosis. In addition, EBV infection is associated with a variety of human cancers, such as nasopharyngeal carcinoma, Hodgkin''s lymphoma, and Burkitt''s lymphoma (4, 8, 9, 27, 30). EBV is an enveloped virus which contains a number of membrane glycoproteins required for membrane fusion and viral entry into the host cell. EBV-mediated entry into epithelial cells requires the three viral glycoproteins gB, gH, and gL, which are conserved among herpesviruses, and entry into B cells additionally requires the viral glycoprotein gp42 (7, 16, 17). EBV lacking gp42 can attach to B cells but cannot enter them (29). However, EBV lacking gp42 can still efficiently infect epithelial cells. In fact, gp42 acts as an inhibitor of epithelial cell infection, and recent studies suggest that the level of gp42 in the virion regulates whether EBV preferentially infects epithelial cells or B cells (2). EBV gp42 has been shown to play an essential role in membrane fusion with B cells (7, 16, 17). It binds to human leukocyte antigen class II (HLA class II) proteins expressed on B cells to trigger virus-cell membrane fusion (6, 7, 10, 16, 25).Interestingly, EBV gp42 occurs in two forms in infected cells, a full-length membrane-bound form and a soluble form generated by proteolytic cleavage that is secreted from infected cells due to loss of the N-terminal transmembrane domain (21). Both the full-length form and the secreted gp42 form bind to gH/gL and HLA class II, and the functional significance of gp42 cleavage is not completely clear. In a virus-free cell-cell fusion assay, enhanced secretion of gp42 promotes fusion with B lymphocytes. Cleavage and secretion of gp42 are necessary for membrane fusion with B lymphocytes (24). However, membrane fusion with epithelial cells is inhibited by the presence of gp42 for both virus infection and cell-cell fusion (14, 29). This is likely due to the formation of a heterotrimeric gH/gL/gp42 complex that is unable to mediate membrane fusion with epithelial cells, possibly due to steric hindrance of gH/gL receptor binding (3, 11).The interaction of gH/gL and gp42 plays a key role in membrane fusion, but it has not yet been fully understood. The crystal structures of a gH/gL/gp42 complex and gH/gL alone have not been available. Although the crystal structures of gp42 alone and gp42/HLA class II complex have been solved (15, 19), the N-terminal region of gp42 (bound to gH/gL) is not visible in the structures, most likely due to its flexibility. Previous studies have shown that the N-terminal region of gp42 contains multiple functional regions, including a cleavage site that results in the secretion of gp42, a potential homodimerization region, and two segments (15 residues each) required for gH/gL binding (Fig. ​(Fig.11 A) (13, 14). Extensive gp42 N-terminal deletion analysis demonstrated that residues 37 to 56 and 72 to 96 include functional regions of the N terminus of gp42 required to trigger fusion and suggested that some of the residues within residues 67 to 71 are also important. Additional experiments showed that amino acids within segments from residues 47 to 61 and 67 to 81 are critical for binding gH/gL (13). Those two segments are spaced by five residues, which appear to act as a linker. Previous studies also showed that a 46-mer peptide spanning residues 36 to 81, mimicking the full-length gp42, binds to gH/gL and inhibits the formation of gH/gL/gp42 complex, thus blocking membrane fusion with epithelial cells and fusion with B cells (13, 14).Open in a separate windowFIG. 1.Schematic representation of EBV gp42. (A) Representation of wild-type gp42 showing the relative locations of known functional domains. The transmembrane domain is predicted to span residues 9 to 22 and is shown as a gray box. The site of gp42 cleavage is between residues 40 and 42 and is indicated by a black bar. The two gH/gL binding regions, spanning residues 47 to 61 and 67 to 87, are indicted with hatched boxes, flanking the five-residue linker. The C-terminal C-type lectin domain, including the hydrophobic pocket and HLA class II-binding region, is indicated by cross-hatched boxes. The putative dimerization region is indicated by a dotted box. (B) Amino acid sequence of gp42 peptide spanning residues 36 to 81 of the gp42 protein.In order to obtain a better understanding of the interaction of gp42 with gH/gL, and the role of this interaction in membrane fusion, we probed the gH/gL binding site by using 27 synthetic peptide analogs spanning residues 36 to 81 of gp42. Peptides were tested for binding affinity to soluble gH/gL by using a fluorescence polarization (FP) assay, probed for inhibition of B-cell and epithelial cell fusion in cell-based assays, and finally investigated for their ability to block epithelial cell infection. The data from the FP assay agreed very well with cell-cell fusion data and infection data, providing correlative data for peptide binding affinity and inhibition of cell-cell fusion and infection in an apparent competitive manner. We have defined the minimal length requirements for high-affinity binding to gH/gL and obtained a more detailed map of the key amino acids of the gp42 N terminus that are necessary for optimal gH/gL binding and inhibition of epithelial cell and B-cell membrane fusion.     

Monoclonal Antibodies That Bind to Domain III of Dengue Virus E Glycoprotein Are the Most Efficient Blockers of Virus Adsorption to Vero Cells

The specific mechanisms by which antibodies neutralize flavivirus infectivity are not completely understood. To study these mechanisms in more detail, we analyzed the ability of a well-defined set of anti-dengue (DEN) virus E-glycoprotein-specific monoclonal antibodies (MAbs) to block virus adsorption to Vero cells. In contrast to previous studies, the binding sites of these MAbs were localized to one of three structural domains (I, II, and III) in the E glycoprotein. The results indicate that most MAbs that neutralize virus infectivity do so, at least in part, by the blocking of virus adsorption. However, MAbs specific for domain III were the strongest blockers of virus adsorption. These results extend our understanding of the structure-function relationships in the E glycoprotein of DEN virus and provide the first direct evidence that domain III encodes the primary flavivirus receptor-binding motif.     

Herpes Simplex Virus Glycoprotein D Can Bind to Poliovirus Receptor-Related Protein 1 or Herpesvirus Entry Mediator, Two Structurally Unrelated Mediators of Virus Entry

Several cell membrane proteins have been identified as herpes simplex virus (HSV) entry mediators (Hve). HveA (formerly HVEM) is a member of the tumor necrosis factor receptor family, whereas the poliovirus receptor-related proteins 1 and 2 (PRR1 and PRR2, renamed HveC and HveB) belong to the immunoglobulin superfamily. Here we show that a truncated form of HveC directly binds to HSV glycoprotein D (gD) in solution and at the surface of virions. This interaction is dependent on the native conformation of gD but independent of its N-linked glycosylation. Complex formation between soluble gD and HveC appears to involve one or two gD molecules for one HveC protein. Since HveA also mediates HSV entry by interacting with gD, we compared both structurally unrelated receptors for their binding to gD. Analyses of several gD variants indicated that structure and accessibility of the N-terminal domain of gD, essential for HveA binding, was not necessary for HveC interaction. Mutations in functional regions II, III, and IV of gD had similar effects on binding to either HveC or HveA. Competition assays with neutralizing anti-gD monoclonal antibodies (MAbs) showed that MAbs from group Ib prevented HveC and HveA binding to virions. However, group Ia MAbs blocked HveC but not HveA binding, and conversely, group VII MAbs blocked HveA but not HveC binding. Thus, we propose that HSV entry can be mediated by two structurally unrelated gD receptors through related but not identical binding with gD.     

Attachment but Not Penetration of Bovine Herpesvirus 1 Is Necessary To Induce Apoptosis in Target Cells

Bovine herpesvirus 1 (BHV-1) induces apoptotic cell death in bovine peripheral blood mononuclear cells and B-lymphoma cells. Using a BHV-1 glycoprotein H null mutant, we have demonstrated that although penetration of BHV-1 is not required, attachment of BHV-1 viral particles is essential for the induction of apoptosis.     

Apolipoprotein E but Not B Is Required for the Formation of Infectious Hepatitis C Virus Particles

Our previous studies have found that hepatitis C virus (HCV) particles are enriched in apolipoprotein E (apoE) and that apoE is required for HCV infectivity and production. Studies by others, however, suggested that both microsomal transfer protein (MTP) and apoB are important for HCV production. To define the roles of apoB and apoE in the HCV life cycle, we developed a single-cycle HCV growth assay to determine the correlation of HCV assembly with apoB and apoE expression, as well as the influence of MTP inhibitors on the formation of HCV particles. The small interfering RNA (siRNA)-mediated knockdown of apoE expression remarkably suppressed the formation of HCV particles. However, apoE expressed ectopically could restore the defect of HCV production posed by the siRNA-mediated knockdown of endogenous apoE expression. In contrast, apoB-specific antibodies and siRNAs had no significant effect on HCV infectivity and production, respectively, suggesting that apoB does not play a significant role in the HCV life cycle. Additionally, two MTP inhibitors, CP-346086 and BMS-2101038, efficiently blocked secretion of apoB-containing lipoproteins but did not affect HCV production unless apoE expression and secretion were inhibited. At higher concentrations, however, MTP inhibitors blocked apoE expression and secretion and consequently suppressed the formation of HCV particles. Furthermore, apoE was found to be sensitive to trypsin digestion and to interact with NS5A in purified HCV particles and HCV-infected cells, as demonstrated by coimmunoprecipitation. Collectively, these findings demonstrate that apoE but not apoB is required for HCV assembly, probably via a specific interaction with NS5A.Hepatitis C virus (HCV) is the leading cause of chronic viral hepatitis, affecting approximately 170 million people worldwide (8, 40). HCV coinfection with human immunodeficiency virus (HIV) is also common, occurring overall in 25 to 30% of HIV-positive persons (1). Individuals with chronic HCV infection are at high risk for the development of cirrhosis and hepatocellular carcinoma. A pegylated interferon and ribavirin combination is the standard therapy to treat hepatitis C but suffers from limited efficacy (<50% antiviral response among patients infected with the dominant genotype 1 HCV) and severe side effects (18, 27). More efficacious and safer antiviral drugs for effective treatment of hepatitis C are urgently needed. A thorough understanding of the HCV life cycle will likely provide novel targets for antiviral drug discovery and development to control HCV infection.HCV is an enveloped RNA virus containing a single-stranded, positive-sense RNA genome and is classified as a Hepacivirus in the Flaviviridae family (11, 33). The viral RNA genome carries a single open reading frame flanked by untranslated regions (UTRs) at both the 5′ and 3′ ends. The 5′ and 3′ UTRs contain cis-acting RNA elements important for the initiation of HCV polyprotein translation and viral RNA replication (24). Upon translation, the HCV polyprotein precursor is proteolytically processed by cellular peptidases and viral proteases into at least 10 different viral proteins (C, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B). Studies with subgenomic HCV RNAs demonstrated that the NS3 to NS5B proteins, in association with intracellular membranes and cellular proteins, are essential and sufficient for HCV RNA replication in the cell (5, 14, 25). The newly synthesized HCV proteins and RNA genome are assembled to form progeny HCV particles by undetermined mechanisms.Our earlier work found that infectious HCV particles are highly enriched in apolipoprotein E (apoE), which is a major determinant of HCV infectivity and production in cell culture (10). ApoE-specific monoclonal antibodies (MAbs) effectively neutralized HCV infectivity, in a dose-dependent manner. The knockdown of apoE expression by specific small interfering RNA (siRNA) remarkably suppressed HCV production, suggesting that apoE is also important for the formation of infectious particles and/or egression (10). However, studies by others suggested that HCV assembly and production are dependent on microsomal transfer protein (MTP) and apolipoprotein B (apoB), both of which are essential components required for the assembly and secretion of very-low-density lipoproteins (VLDLs) (19, 21). In those studies, both apoB-specific siRNAs and MTP inhibitors were found to suppress HCV production (19, 21). It was speculated that HCV shares the same assembly and secretion pathway with VLDLs.To define the roles of apoB and apoE in the formation of HCV particles and egression, we developed a single-cycle HCV growth assay. Using this assay system, we have demonstrated that apoE but not apoB is required for the infectivity and formation of infectious HCV particles. First of all, apoB-specific MAb and polyclonal antibodies did not affect HCV infection. Additionally, apoE-specific siRNA potently inhibited the formation of infectious HCV particles, whereas HCV production was unaffected by the siRNA-mediated knockdown of apoB expression. Furthermore, two MTP inhibitors, CP-346086 and BMS-2101038, efficiently blocked apoB secretion but did not significantly affect HCV production prior to the blockage of apoE expression/secretion. At higher concentrations, however, both MTP inhibitors blocked apoE secretion and consequently suppressed the formation of infectious HCV particles. To further understand the role of apoE in HCV assembly, we carried out coimmunoprecipitation (co-IP) experiments and found that apoE-specific MAb pulled down NS5A but not other HCV proteins from lysed HCV particles, suggesting a specific interaction between apoE and NS5A during the formation of infectious HCV particles. Collectively, our findings demonstrate that apoE but not apoB is required for HCV assembly, probably via a specific interaction with NS5A.     

The HIV Envelope but Not VSV Glycoprotein Is Capable of Mediating HIV Latent Infection of Resting CD4 T Cells

HIV fusion and entry into CD4 T cells are mediated by two receptors, CD4 and CXCR4. This receptor requirement can be abrogated by pseudotyping the virion with the vesicular stomatitis virus glycoprotein (VSV-G) that mediates viral entry through endocytosis. The VSV-G-pseudotyped HIV is highly infectious for transformed cells, although the virus circumvents the viral receptors and the actin cortex. In HIV infection, gp120 binding to the receptors also transduces signals. Recently, we demonstrated a unique requirement for CXCR4 signaling in HIV latent infection of blood resting CD4 T cells. Thus, we performed parallel studies in which the VSV-G-pseudotyped HIV was used to infect both transformed and resting T cells in the absence of coreceptor signaling. Our results indicate that in transformed T cells, the VSV-G-pseudotyping results in lower viral DNA synthesis but a higher rate of nuclear migration. However, in resting CD4 T cells, only the HIV envelope-mediated entry, but not the VSV-G-mediated endocytosis, can lead to viral DNA synthesis and nuclear migration. The viral particles entering through the endocytotic pathway were destroyed within 1–2 days. These results indicate that the VSV-G-mediated endocytotic pathway, although active in transformed cells, is defective and is not a pathway that can establish HIV latent infection of primary resting T cells. Our results highlight the importance of the genuine HIV envelope and its signaling capacity in the latent infection of blood resting T cells. These results also call for caution on the endocytotic entry model of HIV-1, and on data interpretation where the VSV-G-pseudotyped HIV was used for identifying HIV restriction factors in resting T cells.