A single amino acid in EBNA-2 determines superior B lymphoblastoid cell line growth maintenance by Epstein-Barr virus type 1 EBNA-2.
Sequence differences in the EBNA-2 protein mediate the superior ability of type 1 Epstein-Barr virus (EBV) to transform human B cells into lymphoblastoid cell lines compared to that of type 2 EBV. Here we show that changing a single amino acid (S442D) from serine in type 2 EBNA-2 to the aspartate found in type 1 EBNA-2 confers a type 1 growth phenotype in a lymphoblastoid cell line growth maintenance assay. This amino acid lies in the transactivation domain of EBNA-2, and the S442D change increases activity in a transactivation domain assay. The superior growth properties of type 1 EBNA-2 correlate with the greater induction of EBV LMP-1 and about 10 cell genes, including CXCR7. In chromatin immunoprecipitation assays, type 1 EBNA-2 is shown to associate more strongly with EBNA-2 binding sites near the LMP-1 and CXCR7 genes. Unbiased motif searching of the EBNA-2 binding regions of the differentially regulated cell genes identified an ETS-interferon regulatory factor composite element motif that closely corresponds to the sequences known to mediate EBNA-2 regulation of the LMP-1 promoter. It appears that the superior induction by type 1 EBNA-2 of the cell genes contributing to cell growth is due to their being regulated in a manner different from that for most EBNA-2-responsive genes and in a way similar to that for the LMP-1 gene.


OBJECTIVE
The EBNA-2 transcription factor plays a key role in B cell transformation by EBV and defines the two EBV types. Here we identify a single amino acid (Ser in type 1 EBV, Asp in type 2 EBV) of EBNA-2 that determines the superior ability of type 1 EBNA-2 to induce a key group of cell genes and the EBV LMP-1 gene, which mediate the growth advantage of B cells infected with type 1 EBV. The EBNA-2 binding sites in these cell genes have a sequence motif similar to the sequence known to mediate regulation of the EBV LMP-1 promoter. Further detailed analysis of transactivation and promoter binding provides new insight into the physiological regulation of cell genes by EBNA-2.
Section of Virology, Imperial College Department of Medicine, St. Mary's Hospital Campus, London, United Kingdom.
Stelios
S
Tzellos
Section of Virology, Imperial College Department of Medicine, St. Mary's Hospital Campus, London, United Kingdom.
Paulo B
PB
Correia
Section of Virology, Imperial College Department of Medicine, St. Mary's Hospital Campus, London, United Kingdom.
Claudio Elgueta
CE
Karstegl
Section of Virology, Imperial College Department of Medicine, St. Mary's Hospital Campus, London, United Kingdom.
Laila
L
Cancian
School of Life Sciences, University of Sussex, Falmer, Brighton, United Kingdom.
Julian
J
Cano-Flanagan
School of Life Sciences, University of Sussex, Falmer, Brighton, United Kingdom.
Michael J
MJ
McClellan
School of Life Sciences, University of Sussex, Falmer, Brighton, United Kingdom.
Michelle J
MJ
West
Section of Virology, Imperial College Department of Medicine, St. Mary's Hospital Campus, London, United Kingdom p.farrell@imperial.ac.uk.
Paul J
PJ
Farrell
D000596:Amino Acids / Q000235:genetics / Q000378:metabolism*; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism; D001402:B-Lymphocytes / Q000378:metabolism* / Q000821:virology*; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D047369:Chromatin Immunoprecipitation / Q000379:methods; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D019309:Epstein-Barr Virus Nuclear Antigens / Q000235:genetics / Q000378:metabolism*; D005814:Genes, Viral / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D011401:Promoter Regions, Genetic / Q000235:genetics; D054387:Receptors, CXCR / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation / Q000235:genetics; D014763:Viral Matrix Proteins / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
C501896:ACKR3 protein, human; D000596:Amino Acids; D004268:DNA-Binding Proteins; C402641:EBNA-2 protein, Human herpesvirus 4; C037662:EBV-associated membrane antigen, Epstein-Barr virus; D019309:Epstein-Barr Virus Nuclear Antigens; D054387:Receptors, CXCR; D014763:Viral Matrix Proteins; D014764:Viral Proteins; D001224:Aspartic Acid; D012694:Serine