Expression of Epstein-Barr virus membrane antigen gp350/220 in E. coli and in insect cells

Nuebling, C. Micha; Buck, Marion; Boos, Harald; Deimling, Andreas von; Mueller‐Lantzsch, Nikolaus

doi:10.1016/0042-6822(92)90207-6

Cited by 10 publications

(4 citation statements)

References 26 publications

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“…Because this protein contains 18 potential N-glycosylation sites, we conclude that glycosylation contributed 20 kDa to the total size of the molecule. This conclusion is consistent with previous studies of the gp350 protein, which have indicated that it is heavily glycosylated (23,49,50).…”

Section: Characterization Of the Recombinant Proteinssupporting

confidence: 82%

Kinetic Analysis of the Interactions of Complement Receptor 2 (CR2, CD21) with Its Ligands C3d, iC3b, and the EBV Glycoprotein gp350/220

Sarrias¹,

Franchini²,

Canziani

et al. 2001

The Journal of Immunology

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The molecular mechanisms involved in the interaction of complement receptor 2 (CR2) with its natural ligands iC3b and C3d are still not well understood. In addition, studies regarding the binding site(s) of the receptor on C3 as well as the affinities of the C3 fragments for CR2 have produced contradictory results. In the present study, we have used surface plasmon resonance technology to study the interaction of CR2 with its ligands C3d, iC3b, and the EBV surface glycoprotein gp350/220. We measured the kinetics of binding of the receptor to its ligands, examined the influence of ionic contacts on these interactions, and assessed whether immobilized and soluble iC3b bound with similar kinetics to CR2. Our results indicate that 1) gp350 binding to CR2 follows a simple 1:1 interaction, whereas that of the C3 fragments is more complex and involves more than one intramolecular component; 2) kinetic differences exist between the binding of C3d and iC3b to CR2, which may be due to an additional binding site found on the C3c region of iC3b; and 3) iC3b binds to CR2 with different kinetics, depending on whether the iC3b is in solution or immobilized on the surface. These findings suggest that binding of CR2 to iC3b and C3d is more complex than previously thought.

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Section: Characterization Of the Recombinant Proteinssupporting

confidence: 82%

Kinetic Analysis of the Interactions of Complement Receptor 2 (CR2, CD21) with Its Ligands C3d, iC3b, and the EBV Glycoprotein gp350/220

Sarrias¹,

Franchini²,

Canziani

et al. 2001

The Journal of Immunology

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“…It has also been reported that gp350-immunized marmosets are protected from EBV infection, developing gp350-reacting antibodies, several of which exhibit virus-neutralizing activity (57). Neutralizing antibody epitopes on gp350/220 are not generally glycosylation-dependent (58); some of them are located on the amino acid backbone (59), suggesting that the amino acid sequence of this protein induces protective antibodies. The gp350/220 B-lymphocyte-binding regions are thus suitable targets for inducing protective immunity against EBV infection.…”

Section: Figure 4 the Effect Of Anti-habp Antibodies On Ebv Binding mentioning

confidence: 99%

Identification of Three gp350/220 Regions Involved in Epstein-Barr Virus Invasion of Host Cells

Urquiza

López

Patino

et al. 2005

Journal of Biological Chemistry

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Epstein-Barr virus (EBV) invasion of B-lymphocytes involves Epstein-Barr virus (EBV)2 invades B-lymphocytes through molecular interactions involving the EBV gp350/220 protein. It is known that gp350 binds to B-lymphocyte CR2 (known as C3d receptor) with a 3.2 nM affinity constant (12 nM affinity has been determined for B-lymphocyte surface) (1, 2). This simple 1:1 interaction is involved in adsorption, capping, and EBV endocytosis and is considered to be a primary determinant of EBV tissue tropism (3-5). The CR2 region binding to gp350 overlaps the CR2 region binding to C3dg. The gp350 N-terminal region contains part of the B-lymphocyte binding domain (5).The gp350 21 EDPGFFNVE 29 peptide, having significant homology with C3d-amino acid sequences (6), binds to purified CR2 and to CR2(ϩ) but not to CR2(Ϫ) B-and T-lymphoblastoid cell lines; this peptide coupled to BSA inhibits CR2 binding to EBV and gp350 (7). This peptide inhibits C3dg binding to B-cells (as well as EBV) and also inhibits C3-induced Raji cell proliferative response (7,8).However, the 470-mer N-terminal recombinant protein and gp220, containing this peptide sequence, present single-component binding, whereas gp350/220 presents a higher affinity two-component binding to CR2 (5, 9) and inhibits only 50% of EBV binding and EBV infection of Raji cells (5) (5). On the other hand, the C3dg region (homologous to this gp350 peptide) is not in contact with CR2 in the reported complex structure (10). It is thus very likely that other gp350/220 regions are involved in EBV binding to B-lymphocytes.The gp350/220 region containing the epitope recognized by neutralizing monoclonal antibody 72A1 is one of the most important regions involved in gp350/220 binding to B-lymphocyte CR2, because the mAb 72A1 Fab fragment inhibits EBV binding and invasion of host cells (5, 11). Moreover, mAb 72A1 inhibits EBV invasion of monocytes (12), neutrophils (13), and T-cells (14). Anti-gp350/220 mAb 72A1 also inhibits interleukin-6 (IL-6) protein synthesis induced in PBLs by gp350/220 binding to CR2 (15); it also inhibits EBV invasion of B-lymphocytes (5). It is known that the gp350/220 region involved in EBV invasion of host cells is recognized by this monoclonal antibody; however, its precise localization remains unknown. The purpose of this work was thus to identify B-lymphocyte binding sequences that are involved in EBV virus infection of B-lymphocytes. MATERIALS AND METHODSPeptide Synthesis-Forty-six peptides, covering the total length of EBV gp350-reported sequence (16), were synthesized by the solid phase method (17) with N-terminal t-Boc-protected amino acids. Peptides were cleaved by a low-high hydrogen fluoride protocol (18). These peptides were analyzed by mass spectrometry and reverse-phase high performance liquid chromatography. Synthesized peptide sequences are shown in Fig. 1. Tyrosine (Y) was C-terminally added, allowing those peptides not containing this amino acid to be radiolabeled.Lymphoblastoid Cell Line Cultures-The cloned Raji (19), Ramos (20), and P3HR-1 ...

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“…4A ). Gp350 is a membrane protein ( 61 ) and therefore would localize to the outer cell membrane. This localized expression was seen predominantly at the cell membrane by 7 and 9 dpi ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Gammaherpesvirus Infection of Human Neuronal Cells

Jha

Mehta

et al. 2015

mBio

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Gammaherpesviruses human herpesvirus 4 (HHV4) and HHV8 are two prominent members of the herpesvirus family associated with a number of human cancers. HHV4, also known as Epstein-Barr virus (EBV), a ubiquitous gammaherpesvirus prevalent in 90 to 95% of the human population, is clinically associated with various neurological diseases such as primary central nervous system lymphoma, multiple sclerosis, Alzheimer’s disease, cerebellar ataxia, and encephalitis. However, the possibility that EBV and Kaposi’s sarcoma-associated herpesvirus (KSHV) can directly infect neurons has been largely overlooked. This study has, for the first time, characterized EBV infection in neural cell backgrounds by using the Sh-Sy5y neuroblastoma cell line, teratocarcinoma Ntera2 neurons, and primary human fetal neurons. Furthermore, we also demonstrated KSHV infection of neural Sh-Sy5y cells. These neuronal cells were infected with green fluorescent protein-expressing recombinant EBV or KSHV. Microscopy, genetic analysis, immunofluorescence, and Western blot analyses for specific viral antigens supported and validated the infection of these cells by EBV and KSHV and showed that the infection was efficient and productive. Progeny virus produced from infected neuronal cells efficiently infected fresh neuronal cells, as well as peripheral blood mononuclear cells. Furthermore, acyclovir was effective at inhibiting the production of virus from neuronal cells similar to lymphoblastoid cell lines; this suggests active lytic replication in infected neurons in vitro. These studies represent a potentially new in vitro model of EBV- and KSHV-associated neuronal disease development and pathogenesis.

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Expression of Epstein-Barr virus membrane antigen gp350/220 in E. coli and in insect cells

Cited by 10 publications

References 26 publications

Kinetic Analysis of the Interactions of Complement Receptor 2 (CR2, CD21) with Its Ligands C3d, iC3b, and the EBV Glycoprotein gp350/220

Kinetic Analysis of the Interactions of Complement Receptor 2 (CR2, CD21) with Its Ligands C3d, iC3b, and the EBV Glycoprotein gp350/220

Identification of Three gp350/220 Regions Involved in Epstein-Barr Virus Invasion of Host Cells

Gammaherpesvirus Infection of Human Neuronal Cells

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