Bel1-mediated Transactivation of the Spumaretroviral Internal Promoter Is Repressed by Nuclear Factor I

Gene expression of the internal and long terminal repeat promoters of the spuma retrovirus is specifically activated by the transactivator Bel1, the key regulator of viral gene expression. Bel1 directly binds to and activates DNA target sites of viral promoters and those of distinct cellular genes. To determine the contribution of cellular transcription factors to viral transactivation, the viral internal promoter (IP) was analyzed by transient expression, electrophoretic mobility shift assays), and supershifts. Here we report that Bel1-mediated transactivation of the full-length and shortened versions of the Bel1 response element (BRE) were repressed by nuclear factor I (NFI). Electrophoretic mobility shift assays using nuclear extracts from transfected 293T cells revealed that different DNA-protein complexes consisting of DNA target sites of NFI and Bel1 proteins were formed. The specificity of the repressor and transactivator DNA binding was shown by NFI- and Bel1-specific antibodies that led to supershifts of the different nuclear protein-oligodeoxynucleotide complexes. The specificity of the complexes was confirmed by using unlabeled, shortened, and mutated IP.BRE oligodeoxynucleotides in competition experiments with the authentic IP.BRE. Cotransfection of the infectious spumavirus DNA genome with a human NFI-X1 expression plasmid into cell cultures greatly reduced the expression of viral structural and Bel1 proteins. These data demonstrate the relevance of NFI-mediated repression of Bel1-driven transactivation in vivo.