CLONING AND EXPRESSION OF THE EPSTEIN-BARR VIRUS-ENCODED DUTPASE - PATIENTS WITH ACUTE, REACTIVATED OR CHRONIC VIRUS-INFECTION DEVELOP ANTIBODIES AGAINST THE ENZYME

The gene encoding the Epstein-Barr virus (EBV)-specific dUTPase was am plified from virus DNA by PCR. The active enzyme was expressed in Esch erichia coli and in insect cells as a non-fusion protein. The protein from E. coli specifically converted dUTP to dUMP and did not react wit h other dNTPs or NTPs. Preliminary experiments yielded a K-m value of about 0.8 mu M for dUTP. MAbs against the dUTPase reacted with a prote in of approximately 31 kDa in 12-O-tetradecanoyl-phorbol-13-acetate (T PA)-stimulated B cells harbouring either type 1 or type 2 EBV. The pro tein was found in untreated cells at low levels, whereas induction of the lytic replication cycle by TPA treatment or by providing the immed iate early transactivator BZLF1 in trans resulted in increased express ion. We demonstrated that the virus dUTPase isolated from EBV-infected cells is a phosphoprotein. The protein expressed in insect cells was used to test for the presence of specific antibodies in sera from norm al, healthy carriers and from patients with various diseases. While th e sera of EBV-negative individuals (0/3) or healthy carriers (0/33) di d not contain detectable levels of antibodies, patients with mononucle osis (5/18), chronic EBV infection (2/7), EBV reactivation (7/20) and human immunodeficiency virus infection (5/24) showed elevated antibody titres against the enzyme. This indicated that the dUTPase is express ed during EBV replication and reactivation. The enzyme might therefore be a potential target for drug therapy under conditions of active DNA replication.