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Interaction between cell cycle regulator, E2F-1, and NF-kappaB mediates repression of HIV-1 gene transcription
M Kundu1, M Guermah, R G Roeder
1Center for NeuroVirology and NeuroOncology, and Department of Neurology, Allegheny University of the Health Sciences, Philadelphia, Pennsylvania 19102, USA.
Abstract:
The NF-kappaB/Rel family of transcription factors is one of the main targets of cytokines and other agents that induce HIV-1 gene expression. Some of these extracellular stimuli arrest cells in the G1 phase of the mitotic division cycle and modulate the activity of the tumor suppressor protein Rb and its partner E2F-1. Earlier studies indicated that E2F-1, a transcription factor that stimulates expression of S-phase-specific genes, is able to repress transcription directed by the human immunodeficiency virus (HIV-1) type-1 promoter in a variety of cells, including those of glial and lymphocytic origin. Here, we demonstrate that E2F-1 may regulate the activity of the HIV-1 long terminal repeat through its ability to bind sequences in the NF-kappaB enhancer region and to interact with the NF-kappaB subunit, p50. Gel retardation and methylation interference assays show that E2F-1 is able to bind specifically to a site embedded within the two NF-kappaB elements. Gel retardation/immunoblot analysis using purified E2F-1 and p50 homodimers reveals the presence of complexes containing both proteins. Affinity chromatography and co-immunoprecipitation assays provide evidence for direct interaction of E2F-1 and p50 in the absence of their DNA target sequences. In vitro transcription assay demonstrates that E2F-1 represses NF-kappaB mediated transcription in a cell-free system. Functional studies in Jurkat T lymphocytic cells point to the importance of both the E2F and NF-kappaB binding sites in E2F-1 mediated repression of HIV-1 promoter, in vivo. The results of this study suggest that NF-kappaB activity may be regulated by its interaction with the cell cycle regulatory protein, E2F-1.
Insights
The cell cycle protein E2F-1 directly interacts with NF-kappaB subunit p50, repressing HIV-1 gene expression by binding to the NF-kappaB enhancer region. This interaction highlights a novel regulatory mechanism for NF-kappaB activity.
Area of Science:
- Molecular Biology
- Virology
- Cell Biology
Background:
- Nuclear factor kappa B (NF-kappaB) transcription factors regulate HIV-1 gene expression.
- E2F-1, a cell cycle regulator, has been shown to repress HIV-1 transcription.
- Extracellular stimuli influencing HIV-1 can also affect cell cycle regulators like Rb and E2F-1.
Purpose of the Study:
- To investigate the mechanism by which E2F-1 regulates HIV-1 long terminal repeat (LTR) activity.
- To determine if E2F-1 interacts with components of the NF-kappaB pathway.
- To elucidate the role of E2F-1 in modulating NF-kappaB-mediated transcription of HIV-1.
Main Methods:
- Gel retardation and methylation interference assays to assess DNA binding.
- Gel retardation/immunoblot analysis with purified proteins.
- Affinity chromatography and co-immunoprecipitation to study protein interactions.
- In vitro transcription assays.
- Functional studies in Jurkat T lymphocytic cells.
Main Results:
- E2F-1 specifically binds to a site within the NF-kappaB enhancer region of the HIV-1 LTR.
- Direct physical interaction between E2F-1 and the NF-kappaB subunit p50 was demonstrated.
- E2F-1 represses NF-kappaB-mediated transcription in both cell-free systems and in Jurkat T cells.
- Both E2F and NF-kappaB binding sites are crucial for E2F-1 mediated repression of the HIV-1 promoter in vivo.
Conclusions:
- E2F-1 directly regulates HIV-1 LTR activity through interaction with NF-kappaB.
- The cell cycle protein E2F-1 acts as a repressor of NF-kappaB-mediated HIV-1 transcription.
- This study reveals a novel regulatory pathway where E2F-1 modulates NF-kappaB activity, impacting viral gene expression.