Basal transcription of the HIV-1 genome is controlled by a variety of ubiquitous and inducible regulatory factors, some with the ability to associate with the viral DNA sequences within the promoter spanning the long terminal repeat (LTR). In this report we demonstrate that activation of the HIV-1 promoter through the inducible DNA binding NF-kB transcription factors can be affected by cdk9 in human astrocytic cells. Our results show that ectopic expression of cdk9, but not its mutant variant which lacks the domain responsible for its kinase activity, augments transcription of the LTR. Moreover, we demonstrate that induction of the NF-kB pathway by PMA, or overexpression of its subunits including p50/p65 have a negative effect on the ability of cdk9 to stimulate viral gene transcription in these cells. Results from bandshift experiments demonstrated significant suppression of p50/p65 association to its DNA target motif by cdk9. Further, data from GST pull-down and combined immunoprecipitation/Western blot analysis of the protein extracts from cells expressing cdk9, p50 and p65 have revealed the interaction of cdk9 with both p50 and p65 in the absence of DNA containing the kB motif. All of these observations led us to conclude that the interaction of cdk9 with the NF-kB factors can determine the ability of NF-kB to modulate HIV-1 gene transcription.

The single-stranded DNA and RNA binding protein, Puralpha, has recently received special attention as this protein, by associating with the specific nucleotide sequence (GGN repeats) and/or several important cellular and viral proteins regulates crucial biological events such as transcription, replication, and cell proliferation. In this study, we focused on the promoter activity of the Puralpha upstream DNA sequence and demonstrated that the sequence spanning 6,000 nucleotides upstream of the Puralpha transcription start site has promoter activity in various cell types. Results from promoter deletion studies revealed that this region encompasses various regulatory motifs which differentially participate in the promoter activity of Puralpha in various cells. The transcription start site of Puralpha is surrounded by the GA/GC-rich sequence which exhibits the ability to interact with Puralpha, suggesting a role for autoregulation of Puralpha transcription. Results from co-transfection studies revealed that ectopic expression of Puralpha reduced transcriptional activity of the Puralpha promoter and the region located between amino acid residues, 1-85 of Puralpha is important for the observed autoregulatory event. The regulatory protein of the human neurotropic virus, JCV, T-antigen, which interacts with Puralpha, decreased transcriptional activity of the Puralpha promoter. Co-expression of JCV T-antigen and Puralpha had no significant effect on the suppression of Puralpha gene transcription by either protein. The importance of this finding in light of earlier results showing down regulation of Puralpha during JCV infection of glial cells is discussed.

The single-stranded DNA and RNA binding protein, Puralpha, has recently received special attention as this protein, by associating with the specific nucleotide sequence (GGN repeats) and/or several important cellular and viral proteins regulates crucial biological events such as transcription, replication, and cell proliferation. In this study, we focused on the promoter activity of the Puralpha upstream DNA sequence and demonstrated that the sequence spanning 6,000 nucleotides upstream of the Puralpha transcription start site has promoter activity in various cell types. Results from promoter deletion studies revealed that this region encompasses various regulatory motifs which differentially participate in the promoter activity of Puralpha in various cells. The transcription start site of Puralpha is surrounded by the GA/GC-rich sequence which exhibits the ability to interact with Puralpha, suggesting a role for autoregulation of Puralpha transcription. Results from co-transfection studies revealed that ectopic expression of Puralpha reduced transcriptional activity of the Puralpha promoter and the region located between amino acid residues, 1-85 of Puralpha is important for the observed autoregulatory event. The regulatory protein of the human neurotropic virus, JCV, T-antigen, which interacts with Puralpha, decreased transcriptional activity of the Puralpha promoter. Co-expression of JCV T-antigen and Puralpha had no significant effect on the suppression of Puralpha gene transcription by either protein. The importance of this finding in light of earlier results showing down regulation of Puralpha during JCV infection of glial cells is discussed.

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