Epstein-Barr virus nuclear antigen-1-dependent and -independent oriP-binding cellular proteins.
OBJECTIVE
Epstein-Barr virus (EBV) nuclear antigen-1 (EBNA-1) and the replication origin, oriP, are essential for the replication and maintenance of latent EBV DNA in cells, but no enzymatic activity has been associated with EBNA-1 protein alone. In this study, we have searched for host cellular proteins that interact with EBNA-1 protein in various B cell lines latently infected with EBV, including a recently EBV growth-transformed cell line.


METHODS
By using gel shift analysis, we investigated the interactions of an oligonucleotide containing a single EBNA-1 recognition site, derived from the family of repeats (FR) element of oriP, with protein from cell extracts.


RESULTS
The FR oligonucleotide bound a (72-kD) cellular protein in the absence of EBNA-1 and without induction of the previously reported 'anti-EBNA-1 proteins'. The FR oligonucleotide formed complexes with additional proteins from EBNA-1-synthesizing cell lines; these complexes were abolished or supershifted by anti-EBNA-1 monoclonal antibodies. SDS-PAGE analyses of 35S-Met-labeled proteins that bound to a biotin- conjugated FR oligonucleotide, fractionated by a glycerol gradient centrifugation and affinity-purified with streptavidin, showed three major bands, a 72-kD protein, the FR binding of which seemed to be independent of EBNA-1, a 64-kD protein in both EBNA-1-transfected and latently EBV-infected cell lines, and a 45-kD protein in EBV-infected cell lines, which was most prominent in a recently EBV growth-transformed cell line.


CONCLUSIONS
The FR element forms complexes with cellular proteins in the absence and presence of EBNA-1. These 72-, 64- and 45-kD cellular proteins might be involved in the function of the oriP and EBNA-1 system.
Kusano
S
S
Department of Virology I, National Institute of Infectious Diseases, Tokyo, Japan.
Tamada
K
K
Senpuku
H
H
Harada
S
S
Ito
S
S
Yanagi
K
K
D001665:Binding Sites; D002051:Burkitt Lymphoma; D002457:Cell Extracts; D002499:Centrifugation, Density Gradient; D016384:Consensus Sequence / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D019309:Epstein-Barr Virus Nuclear Antigens / Q000235:genetics / Q000502:physiology*; D004854:Herpesvirus 4, Human / Q000235:genetics*; D006801:Humans; D009838:Oligodeoxyribonucleotides / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D018741:Replication Origin / Q000235:genetics*; D014162:Transfection; D014407:Tumor Cells, Cultured
D002457:Cell Extracts; D004268:DNA-Binding Proteins; D019309:Epstein-Barr Virus Nuclear Antigens; D009838:Oligodeoxyribonucleotides; C417029:EBV-encoded nuclear antigen 1