OVEREXPRESSION, PURIFICATION AND HELIX-DESTABILIZING PROPERTIES OF EPSTEIN-BARR-VIRUS SSDNA-BINDING PROTEIN

The Epstein-Barr virus (EBV) ssDNA-binding protein (SSB) encoded by th e BALF2 gene is one of the essential replication proteins in the lytic phase of EBV DNA replication. In order to obtain the amount of EBV SS B required for characterization, a recombinant baculovirus containing the complete sequence of the BALF2 open reading frame under the contro l of the baculovirus polyhedrin promoter was constructed. Insect cells infected with the recombinant virus produced a protein of 130 kDa, re cognized by anti-BALF2 protein-specific polyclonal antibody. The overe xpressed EBV SSB was purified homogeneously from the cytosolic fractio n of the recombinant virus-infected cells. The purified protein displa ced short DNA strands from their complementary sequences in the single -stranded form of M13. The helix-destabilizing activity was neutralize d by the anti-BALF2 protein-specific antibody. Maximum unwinding occur red at EBV SSB concentrations exceeding saturation level of the DNA su bstrate. The DNA unwinding reaction mediated by the EBV SSB was highly cooperative and extremely rapid. The reaction displayed no directiona lity and required neither ATP nor MgCl2, two essential cofactors for D NA helicase activity. The helix-destabilizing property of the EBV SSB may function to melt out secondary structures on the ssDNA template, t hereby facilitating the movement of the EBV DNA polymerase.