Nucleic Acids Research The characterization of the EBV alkaline deoxyribonuclease doned and expressed in E.coli

Abstract

Studies of nucleic acid homology suggest the BGLF5 open reading frame of Epstein-Barr virus (EBV) encodes an alkaline deoxyribonuclease (DNase) sharing some homology with that of herpes simplex virus. We report here the expression of the BGLF5 open reading frame in £. coli and the expression of high levels of a novel alkaline DNase activity in induced cells. This alkaline DNase has been purified to apparent homogeneity as a single protein species. This is the first report of the expression of a herpesvirus coded DNase in a prokaryotic system and of the purification of the EBV DNase to demonstrable purity. It has the biochemical characteristics of a typical herpesvirus alkaline exonuclease showing a high pH optimum, an absolute requirement for Mg for activity and sensitivity to high salt concentrations and polyamines. The enzyme activity was neutralized by sera from patients with nasopharyngeal carcinoma and was reactive with these sera in Western blot analysis. Thus the prokaryotic expression system described here provides an economical and efficient source of the EBV DNase for biochemical and seroepidemiological analysis. INTRODUCTION Epstein-Barr virus (EBV) (1) is a human gammaherpesvirus (2) which has long been recognised as the aetiologic agent of infectious mononucleosis (TM) (3) and has been associated with the development of a number of tumours: endemic Burkitt's lymphoma (BL) (1), undifferentiated nasopharyngeal carcinoma (NPC) (4), and B cell lymphomas in immunodeficient individuals (5). Chemical treatment of Epstein-Barr virus producer cell lines with agents such as 12-Otetradecanoylphorbol-13-acetate (TPA) results in the successive production of early antigens (EA), virus capsid antigens (VCA) and the synthesis of infectious virus (6). In the case of the non-producer cell line Raji only early antigens are synthesized, superinfection of such cells (with virus obtained from producer cell lines) leads to viral antigen expression and the production of infectious virus (7). A number of novel virus-specified enzymatic activities have been identified in chemically induced and superinfected cells, including a DNA polymerase (8,9,10), and a thymidine kinase (11). These enzymes together with other non-structural proteins are believed to make up the early antigen complex. However the lack of an effective lytic system for EBV has limited the quantity of proteins available for analysis. A viral DNase has been detected in EBV positive lymphoid cell lines expressing EA proteins (12,13,14,15). By analogy with the herpes simplex virus (HSV) enzyme we would expect that the EBV enzyme would be essential for DNA synthesis, repair and recombination. Biochemical studies of the HSV DNase show that the enzyme has endonuclease activity and both 5' and 3' exonuclease activities. Its precise role in viral replication is unclear. In the case of the EBV coded enzyme its biochemical properties

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