Demarchi F, d'Adda di Fagagna F, Gutierrez MI, Falaschi A, Giacca M; International Conference on AIDS.
Int Conf AIDS. 1992 Jul 19-24; 8: A82 (abstract no. PoA 2474).
International Centre for Genetic Engineering and Biotechnology, Trieste, Italy.
OBJECTIVES: Gene expression of HIV-1 is finely modulated by its 5' Long Terminal Repeat, which consists in an overlapping array of binding sites for nuclear proteins with positive and negative function. We have studied DNA-protein interactions of three sites of the Negative Regulatory Element (NRE), a region upstream of nucleotide-167 whose overall effect is to downregulate transcription promoted by the downstream domains. METHODS: Protein-DNA interaction assays in vitro (gel retardation, DNase I footprinting, methylation interference. South-Western blotting, UV-crosslinking) were utilized to describe the interaction of purified proteins from HeLa and H9 cells with the LTR. In vivo function of the same sites were tested with mutants after transfection in Jurkat and HeLa cells. RESULTS: We have previously described a binding site for transcription factor USF/MLTF at position -152-174, which behaves as a downregulator of transcription in HeLa cells (Giacca et al. 1992, Virology 186, 133-147). This site is the target of at least three human proteins, of 44 KDa (corresponding to USF/MLTF). 70 KDa and 110 KDa. The 44 and 70 KDa proteins have been purified to homogeneity. Transfection of an expression vector for the USF/MLTF cDNA results in downregulation of promoter efficiency in HeLa cells. At position from -203 to -242 a binding domain was located, partially overlapping to the previously described binding site for NFAT-1. On the contrary of NFAT-1, however, this binding activity is not restricted to activated T cells, but it is constitutive in several T cell lines and present also in many non-T cells. The protein binding to this sequence was purified by a combination of ion-exchange and specific affinity chromatography. Finally, the interactions of a previously unrecognized binding site, from nucleotides -260 to -275 of the NRE, initially identified by in vivo footprinting (Demarchi et al., J. Virology, in press), with nuclear proteins from HeLa and Jurkat cells were studied. Mutation of this site in the context of a functionally active LTR results in downregulation of transcription. CONCLUSIONS: Several human proteins interact with the Long Terminal Repeat of HIV-1, and the final rate of gene expression is dependent on the combined effect of factors having negative and positive function. Two sites with negative function in the context of the NRE have been described in this work. The study of these interactions is essential to describe the modulation of viral expression by cellular factors, and ultimately can contribute to unravelling the mechanism of viral latency and reactivation.
Publication Types:
Keywords:
- Base Sequence
- Binding Sites
- Cell Line
- DNA-Binding Proteins
- HIV Long Terminal Repeat
- HIV-1
- Hela Cells
- Humans
- In Vitro
- Nuclear Proteins
- Promoter Regions (Genetics)
- Transcription Factors
- Transcription, Genetic
- genetics
Other ID:
UI: 102198512
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