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Epstein-Barr virus glycoprotein gp85 associates with the BKRF2 gene product and is incompletely processed as a
L R Yaswen1, E B Stephens, L C Davenport
1Department of Pathology and Laboratory Medicine, University of Florida, Gainesville 32610.
Abstract:
The Epstein-Barr virus (EBV) glycoprotein gp85 is the EBV gH homologue and is thought to be involved in penetration of virus through the B-cell membrane. However, although the glycoprotein is functionally important, it is found in very low abundance in infected cells and in the virion. To facilitate analysis of the structure and function of gp85, recombinant vaccinia viruses were constructed to express the glycoprotein. Recombinant gp85 was recognized by polyclonal antibody made to a peptide derived from the gp85 sequence, but not by monoclonal antibodies that reacted with the native molecule. Unlike native gp85, the recombinant protein contained no sugars that were resistant to endoglycosidase H and it was not transported to the cell surface. The native protein was found to be associated with two additional glycoproteins with apparent M(r) of 25,000 and 42,000. Antibody made to a peptide derived from a sequence in the BKRF2 open reading frame immunoprecipitated glycoproteins with the mobilities of gp85 and its associated 25,000-Da molecule. These data suggest that the BKRF2 gene product, like that encoded by its positional homologues gL of herpes simplex virus and the UL115 gene product of human cytomegalovirus, associates with gp85 and may be required for glycoprotein processing.
Insights
Epstein-Barr virus (EBV) glycoprotein gp85 is crucial for B-cell entry but difficult to study. Recombinant expression revealed it associates with BKRF2, essential for its processing and cell surface transport.
Area of Science:
- Virology
- Molecular Biology
- Immunology
Background:
- Epstein-Barr virus (EBV) glycoprotein gp85, the EBV gH homologue, is implicated in viral entry into B-cells.
- gp85 is functionally important but present in very low abundance, hindering structural and functional analysis.
Purpose of the Study:
- To facilitate the structural and functional analysis of EBV gp85.
- To investigate the association of gp85 with other viral proteins and its processing.
Main Methods:
- Construction of recombinant vaccinia viruses to express EBV gp85.
- Use of polyclonal and monoclonal antibodies for gp85 recognition.
- Endoglycosidase H treatment to analyze glycosylation.
- Immunoprecipitation assays using antibodies against BKRF2 peptide.
Main Results:
- Recombinant gp85 was recognized by anti-peptide antibodies but not by antibodies against native gp85.
- Recombinant gp85 lacked endoglycosidase H-resistant sugars and was not cell surface-transported.
- Native gp85 associated with two other glycoproteins (25 kDa and 42 kDa).
- Antibody to BKRF2 peptide immunoprecipitated gp85 and the 25 kDa glycoprotein.
Conclusions:
- The BKRF2 gene product associates with EBV gp85.
- BKRF2 may be required for proper processing and cell surface transport of gp85.
- This association is analogous to gL in HSV and UL115 in human cytomegalovirus.