Summary auto-generated
This study examines how the cellular protein YB-1 regulates HIV-1 gene transcription in three cell types: astrocytic (U-87MG), neuronal (SK-N-MC), and T-cell (Jurkat) lines. The researchers used transfection assays with HIV-1 long terminal repeat (LTR) reporter constructs to show that YB-1 enhances viral promoter activity. Through deletion analysis, they identified the GC-rich motif (nucleotides -80 to -43) as a critical YB-1 response element in CNS-derived cells, whereas Jurkat cells require upstream sequences. Using electrophoretic mobility shift assays with purified proteins, the authors determined that SP1 and YB-1 bind the same GC-rich region but in different DNA configurations: SP1 binds double-stranded DNA while YB-1 preferentially binds single-stranded DNA. Importantly, when both proteins are present together, each inhibits the other's DNA binding and transcriptional activity, without forming a direct protein-protein complex. The study also demonstrates that YB-1 and Tat cooperate additively to enhance LTR transcription. These findings suggest that YB-1 acts as a cell-type-dependent regulator of HIV-1 expression and that protein-DNA interactions at the GC-rich motif are dynamically regulated by competing transcription factors.
Key findings
- YB-1 enhances HIV-1 LTR transcription in astrocytic and neuronal cells primarily through the GC-rich motif (nucleotides -80 to -43), while in T cells it requires upstream regulatory sequences
- SP1 and YB-1 bind the same GC-rich DNA region but in different configurations: SP1 binds double-stranded DNA while YB-1 preferentially binds single-stranded DNA
- Co-expression of SP1 and YB-1 results in mutual inhibition of their DNA binding and transcriptional activities without direct protein-protein interaction
- YB-1 and Tat cooperate additively rather than synergistically to enhance LTR transcription in CNS cells
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Abstract
Transcription of human immunodeficiency virus type 1 (HIV-1) is regulated by a series of host DNA-binding proteins which interact with the upstream viral promoter sequences, and the viral regulatory protein Tat which associates with TAR RNA sequences located in the leader of the viral transcript. Here we have examined the ability of the cellular protein YB-1 to modulate transcription of the HIV-1 promoter in a human astrocytic cell line (U-87MG), a neuronal cell line (SK-N-MC) and lymphoid cells (Jurkat) by transfection assay. Ectopic expression of YB- 1 in U-87MG and SK-N-MC augments basal transcriptional activity of the viral sequence located between -80 and -43, which encompasses the GC- rich motif. In accord with the previous report, in Jurkat cells YB-1- mediated activation of the HIV-1 promoter required sequences which are located further upstream from the GC-rich motif. Combined overexpression of YB-1 and the GC-rich binding protein, SP1, in the transfected cells decreased the level of activation of the viral promoter, suggesting that YB-1 and SP1 may exert negative effects on each other's function. Results from band shift assay with purified YB-1 and SP1 indicated that SP1 and YB-1 bind to the GC-rich DNA sequence in the double-stranded and single-stranded configurations, respectively. However, efficient binding of SP1 to the double-stranded GC-rich motif corresponding to the HIV-1 long terminal repeat (LTR) is diminished in the presence of YB-1. Similarly, in the presence of SP1, YB-1 loses its ability to become associated with its target single-stranded DNA probe. No evidence for direct association of YB-1 and SP1 either in the presence or in the absence of DNA was observed. These data suggest that while YB-1 stimulates expression of the LTR in central nervous system cells, the level of activity of other cellular proteins, such as SP1, may dictate binding of YB-1 to its target sequence, and therefore affect the regulatory function of this protein.