EBV-Glykoprotein 125

The Epstein-Barr virus (EBV) homologue of the conserved herpesvirus glycoprotein gN is predicted to be encoded by the BLRF1 open reading frame (ORF). Antipeptide antibodies against a sequence corresponding to residues in the predicted BLRF1 ORF immunoprecipitated an approximately 8 kDa doublet from cells expressing the BLRF1 ORF as a recombinant protein. In addition, four glycosylated proteins of 113, 84, 48, and 15 kDa from virus-producing cells could be immunoprecipitated by the same antibody. The 15 kDa species was the mature form of gN bearing α2,6 sialic acid residues. The remaining glycoproteins associated with gN were products of the EBV BBRF3 ORF, which encodes the EBV gM homolog. The 8 kDa doublet seen in cells expressing recombinant gN

The envelopes of herpesviruses comprise several glycoproteins that are known or suspected to play diverse roles in viral attachment, entry, exit, and spread.

Some of these glycoproteins appear to be unique to individual members or subfamilies of herpesviruses, presumably reflecting adaptations to specialized biological niches; others are more apparently conserved either in sequence or in genomic organization, reflecting a common evolutionary origin, if not actual commonality of function. By general consensus, five glycoproteins, gB, gH, gL, gN, and gM, fall under the heading of conserved molecules, although not all have been well characterized, even in some of the most extensively studied herpesviruses.

  • The five Epstein-Barr virus (EBV) open reading frames (ORFs) predicted to encode members of the conserved set of glycoproteins are BXLF2 (encodes gH), BKRF2 (encodes gL), BALF4 (encodes gB) , BLRF1 (encoding gN) and BBRF3 (encoding gM).
  • The glycoprotein gp85, EBV gH , the glycoprotein gp25, EBV gL , and the glycoprotein gp110, EBV gB have all been characterized biochemically and functionally, and a mixed picture emerges their behavior is compared to that of their counterparts in other herpesviruses.
  •  The EBV gB is not a major virion component as in many herpesviruses, but is mainly localized in the nuclear membrane of virion-producing cells , and although gB is critical for viral entry in many herpesviruses, in EBV it plays a major role in viral assembly.
  • In contrast, the EBV-gH-gL complex behaves generally like that of other herpesviruses, although it  contains a third, poorly conserved but functionally important glycoprotein, gp42 (18). Glycoprotein gH is retained in the endoplasmic reticulum in the absence of its partner gL , and the virion-associated complex plays a crucial role in virus entry.
  • Neither the gN nor the gM homologues of EBV have been described to date. All gN homologues are predicted to be small type 1 membrane proteins. The EBV BLRF1 ORF is predicted to encode a type 1 membrane protein of 102 amino acids with a  r  of 10,944 without post-translational modification.
  •  The putative sequence contains no potential N-linked glycosylation sites, but 12 of the 102 amino acids are serine or threonine residues that are predicted to lie outside the signal sequence or the carboxyl-terminal transmembrane domain and could be targets for O-linked glycosylation. However, descriptions of gN homologues in the alphaherpesviruses indicate a variety of processing differences. The gN homologue of pseudorabies virus (PRV) is a 14 kDa O-glycosylated protein associated with the virion.
  • The homolog of herpes simplex virus type 1 (HSV-1) is a 12 kDa protein that does not appear to bear any post-translational modifications. The bovine herpesvirus 1 homologue is a 9-kDa non-glycosylated membrane protein found on the surface of the virion. It is reported to be disulfide-bonded to a second virion component and more tightly associated with the tegument than the envelope glycoprotein gD. The varicella-zoster virus homologue is a 7 kDa protein that, after cleavage of the signal peptide and without further post-translational modifications, is very close to the predicted size of the molecule. Recently, the PRV gN homologue was shown to exist in a disulfide-linked complex with glycoprotein gM and is dependent on gM for localization in the virion.

To study the structure and processing of the EBV gN homologue, we expressed the protein as a recombinant molecule and compared its biosynthesis to that of the native protein in the virus. We report here that the EBV BLRF1 ORF encodes a 15 kDa glycoprotein bearing O-linked sugars whose processing differs for the recombinant and the native proteins. Like all glycoprotein gH homologues, EBV gN associates with a second glycoprotein and requires the presence of a second glycoprotein for authentic processing. As in PRV, the EBV glycoprotein gN associates with the gM homologue, the product of the BBRF3 ORF.

MATERIALS AND METHODS

Akata, a Burkitt lymphoma-derived cell line that carries EBV  and can be induced to produce it (a gift from John Sixbey, St. Jude Children’s Research Hospital, Memphis, Tennessee), was identified in RPMI 1640 (Sigma Chemical Co ., St. Louis, Mo.) supplemented with 10% heat-inactivated fetal bovine serum (Gibco/BRL Life Technologies, Grand Island, NY). CV-1 cells were grown in Dulbecco’s modified Eagle’s medium containing 10% heat-inactivated fetal bovine serum.

virus production.

EBV was obtained from Akata cells resuspended at 2 x 10  6  per ml and induced with 100 µg anti-human immunoglobulin G per ml for 5 days. Stocks of vaccinia virus expressing T7 RNA polymerase (vvT7)  were grown in CV-1 cells infected at a multiplicity of infection of 0.01 and harvested by freeze-thawing and sonication of the cells.

Expression in the pTM1 vector.

The BLRF1  ORF was amplified from the EcoRI G fragment of Akata virus DNA by a PCR method. The 5′ primer (GGC G  CC ATG G  GG AAG GTC CT) comprised the first ATG of the ORF and an  NcoI  site. The 3′ primer (GGC G  C↓T CGA G  CA TCT AAT CCG) included the BLRF1 stop codon and an  XhoI  site. The DNA amplified with these primers was cut with  NcoI  and  XhoI  and inserted into pTM1 previously cut with the same enzymes to prepare plasmid pTM1-gN. The BBRF3 ORF was amplified from Akata virus DNA. The 5′ primer (GGC G  T↓ C ATG A AG TCC AAG A) contained the first ATG of the ORF and an  example HI website. The 3′ primer (GGC G  GA GCT↓ C TT AGG GGA AGA T) included the  BBRF3  stop codon and a SacI site.

The DNA amplified with these primers was cut with Bsp HI and Sac I  and inserted  into  pTM1  that had been cut with  Sac  I and  Nco  I (leaving ends compatible with those produced by  Bsp HI) to create plasmid pTM1 -gM to produce. Plasmids were transfected into CV-1 cells 30 min after cells were infected with vvT7. For single plasmid transfections, 5 µg DNA was mixed with 50 µl Lipofectin(Gibco/BRL), made up to a total volume of 800 µl with serum-free medium. For transfections of two plasmids, the total amount of DNA used was 5 µg. Each mixture was incubated at room temperature for 45 min before being added to the cells.

Antibody.

Three antipeptide antibodies were raised as previously described against synthetic peptides encoding residues 125 to 137 of the BKRF2 ORF (anti-gL), residues 44 to 55 and 55 to 69 of the predicted BLRF1 -ORF (anti-gN) and to residues 346 to 364 of the BBRF3 ORF (anti-gM). A cysteine ​​residue, not present in the viral sequence, was added to the amino terminus of the BBRF3 and BKRF2 peptides to facilitate coupling to keyhole limpet hemocyanin. All antibodies were purified by chromatography on Protein A (Sigma) coupled to Affi-Gel-15 (Bio-Rad, Richmond, CA).

Radiolabeling and immunoprecipitation.

EBV was extrinsically labeled with  125  I (Amersham Corp., Arlington Heights, Illinois) after concentration by centrifugation from 4 liters of spent culture medium as previously described . EBV proteins were biosynthesized with [  3  H]-leucine or [  3  H]-glucosamine (20 Ci/mmol; Amersham) for 20 h 6 h after induction with anti-human immunoglobulin G as previously described  marked. CV-1 cells infected with vvT7 and transfected with pTM1-DNA, pTM1-gN-DNA, pTM1-gM-DNA or a mixture of plasmids were treated with [  3  H]-leucine or [  3  H]- Glucosamine labeled as previously described . Labeled cells were suspended in radioimmunoprecipitation buffer (50mM Tris-HCl [pH 7.2], 0.15M NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 0.1mM phenylmethylsulfonyl fluoride, 100 U aprotinin per ml) and immunoprecipitated with antibody and protein A-Sepharose CL4B (Sigma). Immunoprecipitated proteins were washed, dissociated either by boiling for 2 min or by heating at 37°C for 30 min in sample buffer with or without 2-mercaptoethanol and analyzed by SDS-polyacrylamide gel electrophoresis in 18% acrylamide cross-linked with 0.09% , analyzed. Bisacrylamide or in 9 to 18% polyacrylamide crosslinked with 0.28%  N  ,  N‘-diallyltartardiamide followed by fluorography  .

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Oligosaccharid-Verdauung.

To remove sugars, immunoprecipitated proteins were washed in radioimmunoprecipitation buffer and suspended in buffer (0.1% SDS, 50mM EDTA, 1% 2-mercaptoethanol, 100mM sodium phosphate, 0.5%  N  -octylglucoside [pH 7.2 or pH 5.0]) resuspended. Boiled for 2 min, cooled and incubated for 20 h at 37° C. with the addition of either 5 mU neuraminidase from Newcastle disease virus or 5.0 mU neuraminidase from  Arthrobacter  or neuraminidase and 2.5 mU  O  -glycosidase. All enzymes were from Boehringer Mannheim. Digested samples were analyzed by SDS-polyacrylamide gel electrophoresis and fluorography.

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