Identification and characterization of a PU.1/Spi-B binding site in the bovine leukemia virus long terminal repeat.
Bovine leukemia virus (BLV) is a B-lymphotropic oncogenic retrovirus whose transcriptional promoter is located in the viral 5' long terminal repeat (LTR). To date, no B-lymphocyte-specific cis-regulatory element has been identified in this region. Since ETS proteins are known to regulate transcription of numerous retroviruses, we searched for the presence in the BLV promoter region of binding sites for PU.1/Spi-1, a B-cell- and macrophage-specific ETS family member. In this report, nucleotide sequence analysis of the viral LTR identified a PUbox located at -95/-84 bp. We demonstrated by gel shift and supershift assays that PU.1 and the related Ets transcription factor Spi-B interacted specifically with this PUbox. A 2-bp mutation (GGAA-->CCAA) within this motif abrogated PU.1/Spi-B binding. This mutation caused a marked decrease in LTR-driven basal gene expression in transient transfection assays of B-lymphoid cell lines, but did not impair the responsiveness of the BLV promoter to the virus-encoded transactivator Tax(BLV). Moreover, ectopically expressed PU.1 and Spi-B proteins transactivated the BLV promoter in a PUbox-dependent manner. Taken together, our results provide the first demonstration of regulation of the BLV promoter by two B-cell-specific Ets transcription factors, PU.1 and Spi-B. The PU.1/Spi-B binding site identified here could play an important role in BLV replication and B-lymphoid tropism.
Dekoninck
Ann
A
Laboratoire de Virologie Moléculaire, Service de Chimie Biologique, Institut de Biologie et de Médecine Moléculaires (IBMM), Université Libre de Bruxelles, Rue des Profs Jeener et Brachet 12, 6041 Gosselies, Belgium.
Calomme
Claire
C
Nizet
Séverine
S
de Launoit
Yvan
Y
Burny
Arsène
A
Ghysdael
Jacques
J
Van Lint
Carine
C
D000818:Animals; D001402:B-Lymphocytes; D001665:Binding Sites; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D001909:Leukemia Virus, Bovine / Q000235:genetics*; D016328:NF-kappa B / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D012756:Sheep; D020079:Terminal Repeat Sequences; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation / Q000502:physiology; D000099232:Proto-Oncogene Protein Spi-1
D004268:DNA-Binding Proteins; D016328:NF-kappa B; D011518:Proto-Oncogene Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D000099232:Proto-Oncogene Protein Spi-1; C076877:SPIB protein, human