Cooperation of EBV DNA polymerase and EA-D(BMRF1) in vitro and colocalization in nuclei of infected cells.
Expression of the Epstein-Barr virus (EBV) DNA polymerase (EBVpol) open reading frame (BALF5) by in vitro transcription-translation yielded a 116-kDa primary translation product. Enzymatic DNA polymerase activity of the in vitro translated polypeptide required the presence of the 47-kDa BMRF1 (EA-D) gene product. Antiserum raised to the BALF5 gene product expressed in Escherichia coli specifically precipitated a 116-kDa polypeptide in extracts of latently infected lymphoblastoid cells induced for EBV replication. Immunofluorescence microscopy revealed colocalization of the EBVpol and EA-D(BMRF1) to discrete foci within the nuclei of induced cells; however, the blockade of viral DNA synthesis resulted in diffuse nuclear staining patterns for both antigens. Bromodeoxyuridine staining of these discrete foci colocalizing with EBVpol suggests that they are sites of early viral DNA synthesis. These observations suggest that EA-D(BMRF1) may be an accessory protein of the EBV DNA polymerase which colocalizes in vivo with EBVpol to sites of viral DNA replication and cooperates in vitro to form an active EBVpol holoenzyme.
Kiehl
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Division of Infectious Diseases, University of Connecticut Health Center, Farmington 06032.
Dorsky
D I
DI
D000956:Antigens, Viral / Q000032:analysis / Q000378:metabolism*; D002051:Burkitt Lymphoma; D002460:Cell Line; D002467:Cell Nucleus / Q000502:physiology*; D004279:DNA, Viral / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005455:Fluorescent Antibody Technique; D004854:Herpesvirus 4, Human / Q000201:enzymology / Q000235:genetics* / Q000502:physiology; D006801:Humans; D016366:Open Reading Frames; D010750:Phosphoproteins / Q000032:analysis / Q000378:metabolism*; D010957:Plasmids; D014176:Protein Biosynthesis; D012689:Sequence Homology, Nucleic Acid; D018139:Simplexvirus / Q000235:genetics*; D014158:Transcription, Genetic; D014779:Virus Replication
D000956:Antigens, Viral; D004279:DNA, Viral; D010750:Phosphoproteins; D004259:DNA-Directed DNA Polymerase