Both PU.1 and Nuclear factor-κB mediate lipopolysaccharide-induced HIV- 1 long terminal repeat transcription in macrophages

We recently reported that LPS stimulation of monocytic cells leads to the activation of PU.1, a member of the Ets family of transcription factors. Phosphorylation of PU.1 by protein kinase CK2 was found to up-regulate its trans-activation function, but not its DNA binding activity. Previous studies suggested that Ets proteins could bind to NF-κB motifs at the tetrameric core sequence TTCC. In macrophages, LPS-inducible HIV-1 gene expression is mediated in part by binding of NF-κB to identical tandem binding sites located within the long terminal repeat (LTR). Thus, we performed additional studies to determine whether PU.1 also played a role in regulating HIV-1 gene expression in macrophages. Our functional studies revealed that activation of the HIV-1 LTR in LPS-stimulated cells requires both NF-κB and PU.1. Extensive mutagenesis of the HIV-1 LTR revealed that PU.l-dependent activation requires the Ets motif within the upstream NF-κB site, whereas NF-κB itself binds to the downstream site. We also found that insertion of five additional nucleotides between the NF-κB sites abolished LPS inducibility, suggesting a direct interaction between factors that bind these sites. Lastly, we found that mutation of PU.1 at serine 148, which prevents its phosphorylation by CK2, blocked its ability to activate the HIV-1 LTR in response to LPS. These effects were promoter specific because PU.1 did not affect LPS-inducible activation of a distinct NF-κB-dependent promoter. While these data do not demonstrate direct binding of PU.1 to the HIV-1 LTR, they illustrate a novel role for PU.1 in activation of the HIV-1 LTR by LPS.