BOTH PU.1 AND NUCLEAR FACTOR-KAPPA-B 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 fa ctors, Phosphorylation of PU.1 by protein kinase CK2 was found to up-r egulate its trans-activation function, but not its DNA binding activit y. previous studies suggested that Ets proteins could bind to NF-kappa B motifs at the tetrameric core sequence TTCC, In macrophages, LPS-in ducible HIV-1 gene expression is mediated in part by binding of NF-kap pa B to identical tandem binding sites located within the long termina l repeat (LTR), Thus, we performed additional studies to determine whe ther PU.1 also played a role in regulating HIV-1 gene expression in ma crophages, Our functional studies revealed that activation of the HIV- 1 LTR in LPS-stimulated cells requires both NF-kappa B and PU.1. Exten sive mutagenesis of the HIV-1 LTR revealed that PU.1-dependent activat ion requires the Ets moth within the, upstream NF-kappa B site, wherea s NF-kappa B itself binds to the downstream site. We also found that i nsertion of five additional nucleotides between the NF-kappa B sites a bolished LPS inducibility, suggesting a direct interaction between fac tors that bind these sites. Lastly, we found that mutation of PU.1 at serine 148, which prevents its phosphorylation by CK2, blocked its abi lity 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-kappa B-dependent promoter, While these data do not demonstrate direct binding of PU.1 to the HIV-1 LTR, they illustrate a novel sole for PU.1 in activation of the HIV-1 LTR by LPS.