Co-expression of the Epstein--Barr virus BXLF2 and BKRF2 genes with a recombinant baculovirus produces gp85 on the cell surface with antigenic similarity to the native protein

Pulford, David; Lowrey, Pauline; Morgan, Andrew

doi:10.1099/0022-1317-76-12-3145

Cited by 19 publications

(16 citation statements)

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“…Infection of cells with recombinant baculovirus for full-length gH alone did not allow proper transport of the protein to the cell surface. However, the coexpression of full-length EBV gH and gL proteins in insect cells did result in expression at the cell surface, and deletion of 27 C-terminal gH residues allowed secretion of gH (25). Following these results, we designed secreted, soluble EBV gH constructs that removed the C-terminal transmembrane domain and cytoplasmic tail residues.…”

Section: Resultsmentioning

confidence: 99%

“…Insect cells are well suited to the task of expressing soluble EBV glycoproteins in liters of cells in shaker suspension (20,25,26), and the pBACgus4x-1 vector allowed simultaneous expression of both glycoproteins from a single recombinant baculovirus. The soluble gp42 and gH/gL proteins were optimally expressed 48 to 72 h after infection, based on test infections following protein expression by Western blotting of supernatants (data not shown).…”

Section: Resultsmentioning

confidence: 99%

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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

Kirschner

Omerović

Попов

et al. 2006

J Virol

110

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Epstein-Barr virus (EBV) is a herpesvirus that infects cells by fusing its lipid envelope with the target cellmembrane. 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.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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

Kirschner

Omerović

Попов

et al. 2006

J Virol

110

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“…The soluble form of gHgL was expressed by baculovirus (27). Sf9 cells were infected at a multiplicity of infection of 3, and 5 days later, the culture medium was clarified by low-speed centrifugation to remove cells and centrifuged at 16,000 ϫ g for 90 min to remove virus.…”

Section: Methodsmentioning

confidence: 99%

Use of gHgL for Attachment of Epstein-Barr Virus to Epithelial Cells Compromises Infection

Borza

Morgan

Turk

et al. 2004

J Virol

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Epstein-Barr virus (EBV) is a lymphotropic herpesvirus. However, access to B lymphocytes during primary infection may be facilitated by replication in mucosal epithelial cells. Attachment and penetration of EBV into these two cell types are fundamentally different. Both the distribution of receptors and the cellular origin of the virus impact the efficiency of infection. Epithelial cells potentially offer a wide range of receptors with which virus can interact. We report here on analyses of epithelial cells expressing different combinations of receptors. We find that the stoichiometry of the virus glycoprotein complex that includes gHgL and gp42 affects the use of gHgL not just for entry into epithelial cells but also for attachment. Penetration can be mediated efficiently with either a coreceptor for gp42 or gHgL, but the use of gHgL for attachment as well as penetration greatly compromises its ability to mediate entry.Epstein-Barr virus (EBV) is predominantly a lymphotropic herpesvirus. It is the etiologic agent of most cases of infectious mononucleosis and has been implicated in development of immunoblastic lymphoma, endemic Burkitt's lymphoma, and certain types of Hodgkin's disease. However, the virus also has tropism for epithelial cells. It causes oral hairy leukoplakia, a wart-like lesion of the oral cavity, and is associated with development of nasopharyngeal and gastric carcinomas (28). Initiation of infection of these two key targets, B lymphocytes and epithelial cells, is substantially different. It probably involves different routes (18) and certainly involves different envelope glycoproteins and cell receptors.B-cell infection is initiated by attachment of glycoprotein gp350/220 to the complement receptor type 2 (CR2) (6,21,22,34). Entry requires fusion of virus with the B-cell membrane, which is mediated by glycoprotein gB (8), and a noncovalently linked complex of three glycoproteins, gH, gL, and gp42 (9,20,37). Glycoprotein gL serves as a chaperone for gH (40), and a recombinant virus with gH deleted also lacks gL (20). Thus, with few exceptions, the functions of gH and gL cannot be mapped to either one of the two components. However, the third protein, gp42, plays no known role in gHgL maturation and is unique among human herpesviruses. It interacts with HLA class II (32), which functions as an essential coreceptor for B-cell infection (7,14). A monoclonal antibody (MAb) to gp42 that blocks the interaction with HLA class II inhibits virus cell fusion (15,19), and a MAb to HLA class II that blocks gp42 binding neutralizes virus infection. In further support of a critical role for gp42 in B-cell infection, a virus that lacks gp42 fails to infect B cells unless cells and bound virus are fused with polyethylene glycol (37) or a soluble form of gp42 which lacks a transmembrane domain but retains the ability to bind to gH and gL is added in trans (38).In contrast, not only is gp42 completely dispensable for entry into epithelial cells that do not constitutively express HLA class II, its presence is al...

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“…It also forms a complex with a smaller glycoprotein, glycoprotein L (gL), which is required for normal processing and transport of gH to the cell membrane and thus acts as a molecular chaperone (Hutchinson et al, 1992 ;Kaye et al, 1992 ;Klupp et al, 1994 ;Pulford et al, 1995 ;Duus et al, 1995 ;Duus & Grose, 1996 ;Khattar et al, 1996 ;Mukai et al, 1997 ;Stokes et al, 1996). In the case of Epstein-Barr virus (EBV), a third viral glycoprotein, which is encoded by the BZLF2 open reading frame, associates with the gH-gL complex (Li et al, 1995).…”

Section: Introductionmentioning

confidence: 99%

Identification of a variant B-specific neutralizing epitope on glycoprotein H of human herpesvirus-6.

Takeda

Haque²,

Sunagawa³

et al. 1997

Journal of General Virology

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We have identified the human herpesvirus-6 variant B (HHV-6B)-specific neutralizing epitope on glycoprotein H (gH) which is recognized by monoclonal antibody (MAb) OHV3, with complement-independent neutralizing activity. HHV-6 gHs from HHV-6A (strain U1102) and HHV-6B (strain HST) were expressed in a T7-vaccinia virus transient expression system. OHV3 reacted with HST gH, but not with U1102 gH, in an immunoprecipitation assay and an indirect immunofluorescence assay. In addition,

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Co-expression of the Epstein--Barr virus BXLF2 and BKRF2 genes with a recombinant baculovirus produces gp85 on the cell surface with antigenic similarity to the native protein

Cited by 19 publications

References 19 publications

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

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

Use of gHgL for Attachment of Epstein-Barr Virus to Epithelial Cells Compromises Infection

Identification of a variant B-specific neutralizing epitope on glycoprotein H of human herpesvirus-6.

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