EXPRESSION OF THE EPSTEIN-BARR-VIRUS ENVELOPE FUSION GLYCOPROTEIN GP85 GENE BY A RECOMBINANT BACULOVIRUS

The gp85 envelope glycoprotein of Epstein-Barr virus (EBV) has a role in the molecular mechanism of infection, enabling fusion between the v iral and host cell envelopes, a role in common with the homologous gH glycoproteins in other herpesviruses. A glutathione S-transferase bact erial fusion protein (GST85N-S) was generated, containing 178 amino ac ids from the C terminus of gp85 and including a known gp85 linear epit ope. A panel of EBV-positive human antisera contained no antibodies to linear epitopes presented on the purified GST85N-S protein, indicatin g that primary protein structure in this region of gp85 is not a B cel l target. This bacterial fusion protein was used to raise a rabbit mon ospecific polyclonal antiserum capable of detecting gp85 in a Western blot. The majority of recombinant baculovirus-expressed gp85 obtained from cell extracts prepared with SDS appeared on Western blots as hete rogeneous high M(r) protein aggregates and consistently included 84K, 81K and 70K bands. Recombinant gp85 aggregation was increased by boili ng the sample prior to gel electrophoresis. The 84K and 81K proteins w ere completely sensitive to endoglycosidase H treatment, indicating th at these glycosylated species did not undergo further post-translation al processing. Immunofluorescence studies revealed that recombinant gp 85 was not transported to the insect cell surface. It reacted only wit h antibodies recognizing denatured gp85 and not with antibody to nativ e gp85. Therefore expression of the gene encoding gp85, BXLF2, alone i n the baculovirus expression system is insufficient for the synthesis of a correctly transported, processed, folded and antigenically native form of recombinant gp85.