Two major outer envelope glycoproteins of Epstein-Barr virus are encoded by the same gene.

Two major outer envelope glycoproteins of Epstein-Barr virus, gp350 and gp220, are known to be encoded by 3.2- and 2.5-kilobase RNAs which map to the same DNA fragment (M. Hummel, D. Thorley-Lawson, and E. Kieff, J. Virol. 49:413-417). These RNAs have the same 5' and 3' ends. The larger RNA is encoded by a 2,777-base DNA segment which is preceded by TATTAAA, has AATAAA near its 3' end, and contains a 2,721-base open reading frame. The smaller RNA has one internal splice which maintains the same open reading frame. Translation of the 3.2- and 2.5-kilobase RNAs yielded proteins of 135 and 100 kilodaltons (Hummel et al., J. Virol. 49:413-417). The discrepancy between the 907 codons of the open reading frame and the 135-kilodalton size of the gp350 precursor is due to anomalous behavior of the protein in gel electrophoresis, since a protein translated from most of the Epstein-Barr virus open reading frame in Escherichia coli had similar properties. Antisera raised in rabbits to the protein expressed in E. coli specifically immunoprecipitated gp350 and gp220, confirming the mapping and sequencing results and the translational reading frame. The rabbit antisera also reacted with the plasma membranes of cells that were replicating virus and neutralized virus, particularly after the addition of complement. This is the first demonstration that the primary amino acid sequence of gp350 and gp220 has epitopes which can induce neutralizing antibody. We propose a model for the gp350 protein based on the theoretical analysis of its primary sequence.     

Synthesis and processing of the three major envelope glycoproteins of Epstein-Barr virus.

In pulse-chase experiments, the three major Epstein-Barr virus envelope glycoproteins, gp350/300, gp250/200, and gp85, were shown to be synthesized from separate precursors of 190,000, 160,000, and 83,000 daltons, respectively. These three pulse-labeled species were chased into the mature forms of the glycoproteins between 1 and 3 h after transfer to nonradioactive medium. Digestion of precursor forms with endo-beta-N-acetylglucosaminidase H (endo H) yielded polypeptides of 160,000, 120,000, and 75,000 daltons. Comparison of these results with those from experiments with tunicamycin, which specifically blocks N-linked glycosylation, indicated that some other post-translational modification(s), probably O-linked glycosylation, contributes about 100,000 and 60,000 daltons of apparent molecular mass to gp350/300 and gp250/200, respectively. Experiments with endo H showed that mature gp350/300 and gp250/200 contain complex-type (endo H-resistant) N-linked glycosyl chains, whereas gp85 contains both high-mannose (endo H-sensitive)- and complex-type oligosaccharides. In contrast to the results obtained with the three envelope glycoproteins, no precursor forms of the two unglycosylated protein, p160 (the major Epstein-Barr virus capsid antigen) and p140 (an envelope protein), were detected. The partial proteolytic maps of gp350/300 and gp250/200 were quite similar, suggesting that polypeptide sequence homology could account for at least part of the observed serological cross-reactivity of the two proteins. Taken together, these results demonstrate that the polypeptide portions of gp350/300 and gp250/200 are closely related but not derived from a common precursor. Furthermore, the polypeptide portions comprise half or less of the apparent molecular weight of the mature glycoproteins on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Soluble gp350/220 and deletion mutant glycoproteins block Epstein-Barr virus adsorption to lymphocytes.

The Epstein-Barr virus (EBV) major outer envelope glycoprotein complex, gp350/220, was known to be a ligand for CR2, a B-lymphocyte plasma membrane protein. By Scatchard analysis, soluble EBV gp350/220 binds with high affinity (KD, 1.2 x 10(-8) M) to approximately the same number of B-lymphocyte surface sites as do CR2-specific monoclonal antibodies. Soluble gp350, gp220, or an amino-terminal, 576-amino-acid gp220 derivative binds similarly to B-lymphocyte receptors. Soluble gp350/220, gp220, or even a 470-amino-acid, amino-terminal gp220 derivative blocks EBV adsorption or infection. These experiments demonstrate that (i) gp350/220 is the predominant or exclusive EBV ligand for B lymphocytes; (ii) ligand-receptor blockade can prevent lymphocyte infection by EBV; and (iii) the amino-terminal, 470-amino-acid domain of gp350/220 contains the key ligand domain(s). Consistent with the ligand domain(s) being in the amino-terminal half of gp220 are the findings that the gp350/220-specific, EBV-neutralizing monoclonal antibody 72A1 blocks EBV adsorption by recognizing an epitope in the amino-terminal 470 (probably within the amino-terminal 162) amino acids and a deletion of amino-terminal amino acids 28 and 29 from gp350/220 inactivates ligand activity.     

Intracellular trafficking of two major Epstein-Barr virus glycoproteins, gp350/220 and gp110.

The processing and intracellular localization of the two predominant Epstein-Barr virus glycoproteins expressed in late lytic infection were investigated. Immune light or electron microscopy of frozen fixed sections revealed that gp110 colocalized to the endoplasmic reticulum and to the nuclear membrane with the endoplasmic reticulum-resident protein, heavy-chain-binding protein (BiP), while gp350/220 accumulated in low abundance in the endoplasmic reticulum and was present in higher abundance in cytoplasmic structures presumed to be Golgi and in plasma membranes. Consistent with endoplasmic reticulum and nuclear membrane localization, the bulk of gp110 was sensitive to endoglycosidase H, indicating high-mannose, pre-Golgi, N-linked glycosylation; while consistent with Golgi and plasma membrane localization, gp350/220 was mostly resistant to endoglycosidase H because of complex N- and O-linked glycosylation. gp350/220 was as abundant in extracellular enveloped virus as in the plasma membrane but was much less abundant or undetected in internal cytoplasmic or nuclear membranes. In contrast, gp110-specific antibodies did not label extracellular or intracellular virus. These data indicate that the major antigenic components of gp110 are not incorporated into or are occluded in virions and that gp350/220 is added to virus in cytoplasmic transit through a process of de-envelopment and re-envelopment at the plasma membrane or at post-Golgi vesicles. Consistent with cytoplasmic de-envelopment and re-envelopment at the plasma membrane was the finding of some free nucleocapsids in the cytoplasm of cells with intact nuclear membranes and nucleocapsids which appeared to bud through the plasma membrane.     

Antibodies to gp350/220 enhance the ability of Epstein-Barr virus to infect epithelial cells

Epstein-Barr virus (EBV) is a persistent, orally transmitted herpesvirus that replicates in B cells and epithelial cells and is associated with lymphoid and epithelial malignancies. The virus binds to CD21 on B cells via glycoprotein gp350/220 and infects efficiently. Infection of cultured epithelial cells has not typically been efficient but can occur in the absence of gp350/220 and CD21 and in vivo is thought to be important to the development of nasopharyngeal carcinoma. We report here that antibodies to gp350/220, which inhibit EBV infection of B cells, enhance infection of epithelial cells. The effect is not mediated by Fc receptor binding but is further enhanced by antibody cross-linking, which may patch gp350/220 in the virus envelope. Saliva from EBV-seropositive individuals has similar effects that can be reversed by depletion of antibody. The results are consistent with a model in which gp350/220 interferes with the access of other important players to the epithelial cell surface. The results may have implications for the development of nasopharyngeal carcinoma in high-risk populations in which elevated titers of antibody to EBV lytic cycle proteins are prognostic.     

Epitope mapping of gp350/220 conserved domain of epstein barr virus to develop nasopharyngeal carcinoma (npc) vaccine

Nasopharyngeal carcinoma (NPC) is a malignant tumor in the nasopharyngeal epithelial cells that caused by many factors, one of which is the viral infection of EBV (Epstein Barr Virus). The standard treatments to cure NPC still have not been encouraging. The prevention through vaccination is an effective way to stop the disease. However, EBV vaccine being able to cover all variants of virus is still not available yet. Therefore, we identified the conserved region of glycoprotein 350/220 of EBV which has immunogenic and antigenic properties. The glycoprotein 350/220 is viral surface protein responsible to bind CR2 receptor, mediated EBV to enter the host cell. The conserved domain is crucial for EBV in infecting host cells. Further, by blocking CR2 binding domain of gp350/220 using antibody will inhibit EBV's spreading, and provoke an immune system to eliminate the virus in a patient. Glycoprotein 350/220 from all variants of Epstein-Barr virus was retrieved from NCBI. The conserved domain of gp350/220 was identified by aligning the protein sequences and structures. The polymorphic structure was used as a template for docking analysis to identify the resemblance of amino acid from polymorphic variants of gp350/220 that binds CR2. The epitope mapping of gp350/220 was done by Discotope BepiPred method. The result revealed that the conserved region of gp350/220 was predicted to have an epitope, QNPVYLIPETVPYIKWDNC residue, and it does not have any similarities to the human's cell surface protein. Therefore, it can be used as a reference to develop vaccine to prevent NPC.     

Expression of the Epstein-Barr virus gp350/220 gene in rodent and primate cells.

The gene encoding the Epstein-Barr virus envelope glycoproteins gp350 and gp220 was inserted downstream of the cytomegalovirus immediate-early, Moloney murine leukemia virus, mouse mammary tumor virus, or varicella-zoster virus gpI promoters in vectors containing selectable markers. Host cell and recombinant vector systems were defined which enabled the isolation of rodent or primate cell clones which expressed gp350/220 in substantial quantities. Continued expression of gp350/220 required maintenance of cells under positive selection for linked markers and periodic cloning. gp350/220 expressed in various host cells varied slightly in electrophoretic mobility, probably reflecting differences in glycosylation. Insertion of a stop codon into the gp350/220 open reading frame, upstream of the putative membrane anchor sequence, resulted in efficient secretion of truncated gp350 and gp220 from rat pituitary (GH3) cells. gp350/220 expressed in mammalian cells is highly immunogenic and elicits virus-neutralizing antibodies when administered to mice.     

Induction of interleukin-6 after stimulation of human B-cell CD21 by Epstein-Barr virus glycoproteins gp350 and gp220.

The cellular receptor for Epstein-Barr virus (EBV) is the type 2 complement receptor, CD21. At initial infection, EBV virion glycoproteins gp350 and gp220 bind to CD21. We report here that the cross-linking of CD21 by gp350/220 results in increased amounts of interleukin 6 (IL-6) RNA and IL-6 protein. This effect could be blocked with anti-gp350/220 and anti-CD21 monoclonal antibodies. Induction of IL-6 in B cells by EBV could be mimicked by treatment with the protein kinase C (PKC) activator phorbol 12,13-dibutyrate but not with the calcium ionophore ionomycin. IL-6 induction by EBV was inhibited with the PKC-specific inhibitor bisindolylmaleimide or the protein tyrosine kinase inhibitors methyl 2,5-dihydroxycinnamate and herbimycin A, indicating that the induction of IL-6 following CD21 cross-linking is mediated through PKC- and protein tyrosine kinase-dependent pathways.     

Identification and procaryotic expression of the gene coding for the highly immunogenic 28-kilodalton structural phosphoprotein (pp28) of human cytomegalovirus.

Human cytomegalovirus contains a structural polypeptide that is 28 kilodaltons in apparent molecular size and is reactive in Western blot (immunoblot) analysis with the majority of human sera. The gene coding for this polypeptide was mapped on the genome of human cytomegalovirus strain AD169. A monoclonal antibody specific for the 28-kilodalton polypeptide was used to screen a cDNA library constructed from poly(A)+ RNA of human cytomegalovirus-infected cells in the procaryotic expression vector lambda gt11. Hybridization of cDNA with cosmid and plasmid clones mapped the gene to the HindIII R fragment. The gene was transcribed into a late 1.3-kilobase RNA. The nucleotide sequence of the coding region was determined. Parts of the 28-kilodalton polypeptide were expressed in Escherichia coli as hybrid proteins fused to beta-galactosidase. In Western blots these proteins were recognized by human sera. Antibodies raised against the hybrid proteins reacted specifically with the viral antigen in immunoprecipitations and Western blots. In vitro phosphorylation of HCMV virions and immunoprecipitation showed that the 28-kilodalton polypeptide was phosphorylated.     

Identification of three gp350/220 regions involved in Epstein-Barr virus invasion of host cells

Epstein-Barr virus (EBV) invasion of B-lymphocytes involves EBV gp350/220 binding to B-lymphocyte CR2. The anti-gp350 monoclonal antibody (mAb)-72A1 Fab inhibits this binding and therefore blocks EBV invasion of target cells. However, gp350/220 regions interacting with mAb 72A1 and involved in EBV invasion of target cells have not yet been identified. This work reports three gp350/220 regions, defined by peptide 11382, 11389, and 11416 sequences, that are involved in EBV binding to B-lymphocytes. Peptides 11382, 11389, and 11416 bound to CR2(+) but not to CR2(-) cells, inhibited EBV invasion of cord blood lymphocytes (CBLs), were recognized by mAb 72A1, and inhibited mAb 72A1 binding to EBV. Peptides 11382 and 11416 binding to peripheral blood lymphocytes (PBLs) induced interleukin-6 protein synthesis in these cells, this phenomenon being inhibited by mAb 72A1. The same behavior has been reported for gp350/220 binding to PBLs. Anti-peptide 11382, 11389, and 11416 antibodies inhibited EBV binding and EBV invasion of PBLs and CBLs. Peptide 11382, 11389, and 11416 sequences presented homology with the C3dg regions coming into contact with CR2 (C3dg and gp350 bound to similar CR2 regions). These peptides could be used in designing strategies against EBV infection.     

Diverse IgG serum response to novel glycopeptide epitopes detected within immunodominant stretches of Epstein-Barr virus glycoprotein 350/220: diagnostic potential of O-glycopeptide microarrays

The Epstein-Barr virus (EBV) envelope glycoprotein 350/220 (gp350/220) is the most abundant molecule on the viral surface and it is responsible for the initial viral attachment to cell surface of the host. As many other viral envelope proteins, it is highly glycosylated, not least with O-linked glycans, most of which essential for EBV life cycle. EBV gp350/220 is also a primary target for neutralizing antibodies in the human hosts and a promising candidate for an EBV vaccine. Here we showed that recombinant GalNAc transferases can glycosylate scan peptides of the EBV gp350/220 envelope protein immobilized on microarray glass slides. We also identified serum IgG antibodies to a selection of peptides and O-glycopeptides, whereas sera from EBV-IgG negative individuals remained negative. We here describe novel glycopeptide epitopes present within immunodominant stretches of EBV gp350/220 and demonstrate a remarkable variability between individual samples with respect to their reactivity patterns to peptides and glycopeptides. The study provides additional insights into the complex B-cell response towards the EBV gp350/220 envelope protein, which may have implications for diagnostic and vaccine developments.     

Identification and isolation of the main component (gp350-gp220) of Epstein-Barr virus responsible for generating neutralizing antibodies in vivo.

The majority of hybridomas we have characterized against Epstein-Barr virions react with the major glycoproteins gp350 and gp220 (gp350/220). One of these antibodies, ID4C-1, neutralizes virus infection in vitro. The presence of gp350/220 on the viral envelope could be confirmed directly by immunoelectron microscopy. We used lectin affinity (ricin) and immunoaffinity (ID4C-1) to purify gp350/220 and show that this material is able to induce potent virus-neutralizing antibodies. Absorption of four human and one rabbit anti-Epstein-Barr virus sera with purified gp350/220 suggests that this is the primary component responsible for generating neutralizing antibodies in vivo.     

Immunochemical characterization of gp115, a yeast glycoprotein modulated by the cell cycle

A cell cycle-modulated glycoprotein (gp115, 115 kDa, isoelectric point 4.8-5) of Saccharomyces cerevisiae has been purified by Concanavalin A-affinity chromatography, followed by preparative two-dimensional gel electrophoresis, from yeast membrane proteins solubilized in Triton X-100. Antisera have been generated against the electrophoretically purified protein. Their specificity has been established by immunoblot analysis and by comparison of the partial proteolytic map obtained for the immunoprecipitated 35S-labeled 115 kDa polypeptide with that of the in vivo [35S]methionine-labeled gp115 isolated from two-dimensional gels. In tunicamycin-treated cells the immunoblot analysis identifies an unglycosylated precursor (86-88 kDa) and in sec18 mutant cells at the restrictive temperature an intermediary precursor of about 100 kDa. Six to seven carbohydrate chains have been estimated to be present on the gp115 protein, accounting for an electrophoretic shift corresponding to about 27 to 29 kDa of its relative molecular mass. Affinity-purified antibodies against the unglycosylated precursor (86-88 kDa) of gp115 were prepared and used to localize gp115 by indirect immunofluorescence microscopy. The similarity between the pattern of fluorescence obtained with these antibodies and that obtained using anti-plasma membrane H+-ATPase antibodies suggests an association of gp115 with the plasma membrane.     

表达EB病毒主要膜蛋白gp350／220的非复制型重组痘苗病毒的构建

利用非复制痘苗病毒质粒载体pNEOCK11β75及pNEOCK,改造了表达EB病毒主要膜蛋白gp350/22的复制型重组痘苗病毒VMA,构建了非复制型重组痘苗病毒VMA△CK。该病毒能在鸡胚原代成纤维细胞（CEF）中正常繁殖,而在人源细胞中不能正常繁殖。在CEF中连续传代至第25代,经PCR证明,该病毒符合非复制型重组痘苗病毒的特征。经免疫荧光及免疫酶斑法证实,VMA△CK可稳定表达gp350/220,且表达水平与VAM无明显差异。VMA△CK经腹腔免疫Balb/C小鼠,4周后能诱生一定水平的抗gp350/220特异性抗体,加强免疫2周后该抗体水平明显升高。这一结果类似于VMA免疫Balb/C小鼠的结果,初免后,VMA△CK且抗痘苗抗体水平明显低于VMA免疫组;加强免疫2周后,两组小鼠的抗痘苗抗体水平趋于一致。上述结果证明,所构建的非复制痘苗病毒不影响目的抗原的表达,也不影响该抗原的免疫原性,但导致病毒毒力下降,而且用该病毒免疫小鼠后小鼠抗痘苗病毒载体的免疫反应明显下降。     

Identification and characterization of a human cytomegalovirus gene coding for a membrane protein that is conserved among human herpesviruses.

A rabbit antiserum was raised against envelope material from purified human cytomegalovirus strain AD169. The serum recognized polypeptides 200, 170, 160, 75, 58, and 45 kilodaltons in size. It was used to screen a cDNA library constructed from poly(A)+ RNA from human cytomegalovirus-infected cells in the expression vector lambda gt11. A recombinant bacteriophage expressing cytomegalovirus-specific sequences was identified, and the corresponding gene was mapped to the HindIII R fragment. The gene is transcribed into a late 1.5-kilobase RNA. The nucleotide sequence of the coding region was determined. Computer analysis of the gene product revealed a polypeptide containing multiple potential membrane-spanning domains, representing a type of protein not identified in the envelope of herpesviruses before. The protein shows homology on the amino acid level to hypothetical proteins from reading frames BBRF3 of Epstein-Barr virus, UL10 of herpes simplex virus type 1, and ORF50 of varicella-zoster virus. By using an antiserum raised against procaryote-expressed parts of the cytomegalovirus membrane protein, a 45-kilodalton structural component of the virus was identified as the gene product.     

Isolation and deduced amino acid sequence of the gene encoding gp115, a yeast glycophospholipid-anchored protein containing a serine-rich region

gp115 is a N- and O-glycosylated protein of Saccharomyces cerevisiae. It is also modified by addition of glycosylphosphatidylinositol, which anchors the protein to the plasma membrane. The gene encoding gp115 (GGP1) has been cloned by a two-step procedure. By an immunoscreening of a yeast genomic DNA library in the expression vector lambda gt11, a 3'-terminal 0.9-kilobase portion of the gene has been isolated and then used as a molecular probe to screen a yeast genomic DNA library in YEp24. In this way, the whole GGP1 gene has been cloned. Its identity with the gp115 gene has been confirmed by gene disruption, which has also indicated that the function of gp115 is not essential for cell viability. The features of the sequence are also entirely consistent with it corresponding to the gp115 gene. The nucleotide sequence of GGP1 predicts a 60-kDa polypeptide, in agreement with the molecular mass of the gp115 precursor detected in sec53 mutant cells at restrictive temperature. Two hydrophobic sequences, one NH2- and the other COOH-terminal were found. The former has the features of the cleavable signal sequence, which allows the entry of proteins in the secretory pathway. The latter could be the signal sequence that has to be removed during the addition of glycosylphosphatidylinositol. The predicted amino acid sequence of gp115 shows 10 sequons for N-glycosylation and a high proportion of serine-threonine residues (22%) that could provide several sites for O-glycosylation. The unusual concentration of 27 serines in the COOH-terminal portion of the protein shares homology with a similar polyserine repeat of the serine repeat antigen (SERA protein) of Plasmodium falciparum. A two-dimensional analysis of the "in vitro" translational product of the GGP1 mRNA has been carried out, allowing the identification of the "in vivo" gp115 precursor in a two-dimensional gel.     

A protein kinase from wheat germ that phosphorylates the largest subunit of RNA polymerase II.

A protein kinase from wheat germ that phosphorylates the largest subunit of RNA polymerase IIA has been partially purified and characterized. The kinase has a native molecular weight of about 200 kilodaltons. This kinase utilizes Mg2+ and ATP and transfers about 20 phosphates to the heptapeptide repeats Pro-Thr-Ser-Pro-Ser-Tyr-Ser in the carboxyl-terminal domain of the 220-kilodalton subunit of soybean RNA polymerase II. This phosphorylation results in a mobility shift of the 220-kilodalton subunits of a variety of eukaryotic RNA polymerases to polypeptides ranging in size from greater than 220 kilodaltons to 240 kilodaltons on sodium dodecyl sulfate-polyacrylamide gels. The phosphorylation is highly specific to the heptapeptide repeats since a degraded subunit polypeptide of 180 kilodaltons that lacks the heptapeptide repeats is poorly phosphorylated. Synthetic heptapeptide repeat multimers inhibit the phosphorylation of the 220-kilodalton subunit.