EBV-Glykoprotein 125

MATERIALS AND METHODS

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  .

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.