Type B leukemogenic virus has a T-cell-specific enhancer that binds AML-1

Type B leukemogenic virus (TBLV) induces rapidly appearing T-cell tumors in mice. TBLV is highly related to mouse mammary tumor virus (MMTV) except th at TBLV long terminal repeats (LTRs) have a deletion of negative regulatory elements and a triplication of sequences flanking the deletion. To determi ne if the LTR triplication represents a viral enhancer element, we inserted the triplication upstream and downstream in either orientation relative to the thymidine kinase promoter linked to the luciferase gene. These experim ents showed that upregulation of reporter gene activity by the TBLV triplic ation was relatively orientation independent, consistent with the activity of eukaryotic enhancer elements. TBLV enhancer activity was observed in T-c ell lines but not in fibroblasts, B cells, or mammary cells, suggesting tha t enhancer function is cell type dependent. To analyze the transcription fa ctor binding sites that are important for TBLV enhancer function, we prepar ed substitution mutations in a reconstituted C3H MMTV LTR that recapitulate s the deletion observed in the TBLV LTR, Transient transfections showed tha t a single mutation (556M) decreased TBLV enhancer activity at least 20-fol d in two different T-cell lines. This mutation greatly diminished AML-1 (re cently renamed RUNX1) binding in gel shift assays with a mutant oligonucleo tide, whereas AML-1 binding to a wild-type TBLV oligomer was specific, as j udged by competition and supershift experiments. The 556 mutation also redu ced TBLV enhancer binding of two other protein complexes, called NF-A and N F-B, that did not appear to be related to c-Myb or Ets. AML-1 overexpressio n in a mammary cell line enhanced expression from the TBLV LTR approximatel y 30-fold. These data suggest that binding of AML-1 to the TBLV enhancer, l ikely in combination with other factors, is necessary for optimal enhancer function.