The Epstein-Barr virus (EBV) gN homolog BLRF1 encodes a 15-kilodalton glycoprotein that cannot be authentically processed unless it is coexpressed with the EBV gM homolog BBRF3

Abstract

The Epstein-Barr virus (EBV) homolog of the conserved herpesvirus glycoprotein gN is predicted to be encoded by the BLRF1 open reading frame (ORF). Antipeptide antibody to a sequence corresponding to residues in the predicted BLRF1 ORF immunoprecipitated a doublet of approximately 8 kDa from cells expressing the BLRF1 ORF as a recombinant protein. In addition, four glycosylated proteins of 113, 84, 48, and 15 kDa could be immunoprecipitated from virus-producing cells by the same antibody. The 15-kDa species was the mature form of gN, which carried alpha2,6-sialic acid residues. The remaining glycoproteins which associated with gN were products of the BBRF3 ORF of EBV, which encodes the EBV gM homolog. The 8-kDa doublet seen in cells expressing recombinant gN comprised precursors of the mature 15-kDa gN. Coexpression of EBV gM with EBV gN was required for authentic processing of the 8-kDa forms to the 15-kDa form.

FIG. 1

Electrophoretic analysis (in 18% polyacrylamide gels) of proteins immunoprecipitated with anti-gN from CV-1 cells transfected with pTM1 vector or pTM1-gN, infected with vvT7 and labeled with [³H] leucine or [³H] glucosamine. Sizes are indicated on the left in kilodaltons.

FIG. 2

Electrophoretic analysis in 18% polyacrylamide of proteins immunoprecipitated from Akata cells that had been induced with anti-immunoglobulin and labeled with [³H] glucosamine or [³H] leucine. Proteins were immunoprecipitated with anti-gN or with preimmune antibody from the same rabbit. Sizes are indicated on the left in kilodaltons.

FIG. 3

Electrophoretic analysis in 9 to 18% polyacrylamide of proteins immunoprecipitated from Akata cells that had been induced with anti-immunoglobulin and labeled with [³H] glucosamine. Proteins were immunoprecipitated with a rabbit anti-peptide antibody to the EBV gL (gp25), which immunoprecipitates the EBV gH-gL complex of gp85, gp42/38, and gp25, with rabbit anti-peptide antibody anti-gN, or with preimmune rabbit antibody. Immunoprecipitated proteins were eluted from protein A-agarose beads by heating at 37°C. Sizes are indicated on the left in kilodaltons.

FIG. 4

Electrophoretic analysis in 9 to 18% polyacrylamide of proteins immunoprecipitated from Akata cells that had been induced with anti-immunoglobulin and labeled with [³H] glucosamine. Proteins were immunoprecipitated with anti-peptide antibody anti-gN, anti-peptide antibody anti-gM, anti-peptide antibody to EBV gL, or pre-immune antibody. Immunoprecipitated proteins were eluted from protein A-agarose beads by heating at 37°C. Sizes are indicated on the right in kilodaltons.

FIG. 5

Electrophoresis in 18% polyacrylamide of proteins immunoprecipitated with anti-gM or preimmune rabbit antibody from CV-1 cells labeled with [³H] glucosamine, infected with vvT7, and transfected with pTM1-gM or cotransfected with pTM1-gM and pTM1-gN. Immunoprecipitated proteins were eluted from protein A-agarose beads by heating at 37°C. Arrows indicate three glycoproteins specifically immunoprecipitated by anti-gM antibody.

FIG. 6

Electrophoretic analysis in 18% polyacrylamide of proteins immunoprecipitated with anti-gN from induced Akata cells and labeled with [³H] leucine. After immunoprecipitation and before electrophoresis, the proteins were incubated overnight in buffer at pH 7.2 or 5.0 or buffer at either pH containing neuraminidase from Arthrobacter (na/Arth), neuraminidase and O-glycanase (na/Arth/O-g), or neuraminidase from Newcastle disease virus (na/NDV). Sizes are indicated on the right in kilodaltons.

FIG. 7

(A) Electrophoretic analysis in 18% polyacrylamide of proteins immunoprecipitated with anti-gN or anti-gM from CV-1 cells labeled with [³H] leucine, infected with vvT7, and transfected with pTM1 vector alone, pTM1-gN (gN), or pTM1-gN and pTM1-gM (gN/gM). (B) All proteins were immunoprecipitated with anti-gN and were incubated overnight in buffer or buffer containing neuraminidase from Arthrobacter (na/Arth) before being subjected to electrophoresis. Sizes are indicated on the right in kilodaltons.

FIG. 8

Electrophoretic analysis in 9 to 18% polyacrylamide under nonreducing conditions of proteins immunoprecipitated by preimmune antibody, anti-peptide antibody to EBV gL, anti-gN, or anti-gM from Akata cells that had been induced with anti-immunoglobulin and labeled with [³H] glucosamine. Immunoprecipitated proteins were eluted from protein A-agarose beads by heating at 37°C. Positions of molecular size markers (in kilodaltons) electrophoresed under reducing conditions several lanes away from the samples are indicated on the right.