Bel1-mediated Transactivation of the Spumaretroviral
Internal Promoter Is Repressed by Nuclear Factor I*
Kenji
Kido
,
Helmut
Bannert,
Richard
M.
Gronostajski§, and
Rolf M.
Flügel¶
From the Division of Retroviral Gene Expression,
Research Program Applied Tumor Virology German Cancer Research
Center, Im Neuenheimer Feld 242, 69009 Heidelberg, Germany and
§ The State University of New York at Buffalo,
Department of Biochemistry, Buffalo, New York 14214
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.
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
We dedicate this article to Harald zur Hausen on the occasion of his
retirement as head of the German Cancer Research Center (DKFZ) with
gratitude and appreciation.
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