CBF, Myb, and Ets Binding Sites Are Important for Activity of the Core I Element of the Murine Retrovirus SL3-3 in T Lymphocytes

Abstract

Transcriptional enhancers within the long terminal repeats of murine leukemia viruses are major determinants of the pathogenic properties of these viruses. Mutations were introduced into the adjacent binding sites for three transcription factors within the enhancer of the T-cell-lymphomagenic virus SL3-3. The sites that were tested were, in 5′-to-3′ order, a binding site for core binding factor (CBF) called core II, a binding site for c-Myb, a site that binds members of the Ets family of factors, and a second CBF binding site called core I. Mutation of each site individually reduced transcriptional activity in T lymphocytes. However, mutation of the Myb and core I binding sites had larger effects than mutation of the Ets or core II site. The relative effects on transcription in T cells paralleled the effects of the same mutations on viral lymphomagenicity, consistent with the idea that the role of these sequences in viral lymphomagenicity is indeed to regulate transcription in T cells. Mutations were also introduced simultaneously into multiple sites in the SL3-3 enhancer. The inhibitory effects of these mutations indicated that the transcription factor in T cells that recognizes the core I element of SL3-3, presumably CBF, needed to synergize with one or more factors bound at the upstream sites to function. This was tested further by generating a multimer construct that contained five tandem core I elements linked to a basal long terminal repeat promoter. This construct was inactive in T cells. However, transcriptional activity was detected with a multimer construct in which the transcription factor binding sites upstream of the core were also present. These results are consistent with the hypothesis that CBF requires heterologous transcription factors bound at nearby sites to function in T cells.