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 amplified from virus DNA by PCR. The active enzyme was expressed in Escherichia coli and in insect cells as a non-fusion protein. The protein from E. coli specifically converted dUTP to dUMP and did not react with other dNTPs or NTPs. Preliminary experiments yielded a Km value of about 0.8 microM for dUTP. MAbs against the dUTPase reacted with a protein of approximately 31 kDa in 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-stimulated B cells harbouring either type 1 or type 2 EBV. The protein was found in untreated cells at low levels, whereas induction of the lytic replication cycle by TPA treatment or by providing the immediate early transactivator BZLF1 in trans resulted in increased expression. 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 normal, healthy carriers and from patients with various diseases. While the sera of EBV-negative individuals (0/3) or healthy carriers (0/33) did not contain detectable levels of antibodies, patients with mononucleosis (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 expressed during EBV replication and reactivation. The enzyme might therefore be a potential target for drug therapy under conditions of active DNA replication.
Sommer
P
P
Institut für Mikrobiologie und Hygiene, Abteilung Virologie, Universitätskliniken, Homburg, Germany.
Kremmer
E
E
Bier
S
S
König
S
S
Zalud
P
P
Zeppezauer
M
M
Jones
J F
JF
Mueller-Lantzsch
N
N
Grässer
F A
FA
D000208:Acute Disease; D000818:Animals; D000914:Antibodies, Viral / Q000097:blood*; D001402:B-Lymphocytes / Q000201:enzymology; D002908:Chronic Disease; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000032:analysis; D006566:Herpesviridae Infections / Q000276:immunology*; D004854:Herpesvirus 4, Human / Q000201:enzymology* / Q000276:immunology; D006801:Humans; D016366:Open Reading Frames; D011755:Pyrophosphatases / Q000235:genetics / Q000276:immunology*; D051381:Rats; D015534:Trans-Activators / Q000032:analysis; D014407:Tumor Cells, Cultured; D014412:Tumor Virus Infections / Q000276:immunology*; D014764:Viral Proteins
D000914:Antibodies, Viral; C063676:BZLF1 protein, Herpesvirus 4, Human; D004268:DNA-Binding Proteins; D015534:Trans-Activators; D014764:Viral Proteins; D011755:Pyrophosphatases; C020020:dUTP pyrophosphatase