| Transcriptional control of the human immunodeficiency virus type 1 (HIV-1) promoter, the long terminal repeat (LTR), is achieved by interactions with cis-acting binding elements present both upstream and downstream of the start site. The functional impact of two distinct regions of the LTR was studied with specific focus on cells of the monocyte-macrophage lineage. First, a novel downstream transcription factor binding site (+158 to + 172; designated DS3) was identified and found to be conserved in 67% of 3,858 unique subtype B LTR sequences analyzed. DS3 was represented well in other subtypes also and exhibited affinity for nuclear factor of activated T cells (NFAT) family of proteins isoform c2 and members of the CCAAT enhancer binding protein family isoforms a and beta. The interactions at DS3 were also validated in an integrated HIV-1 LTR in chronically infected U1/HIV-1 cells. A substitution mutation in DS3 demonstrated reduced HIV-1 LTR-directed transcription under both basal and interleukin-6-stimulated conditions in cells of the monocyte-macrophage lineage cells but not in cells of the T-cell origin. The second region analyzed was the GC box array in the LTR that binds the Sp family of transcription factors. The effect of monocytic differentiation on binding of Sp1, Sp3 and truncated Sp3 to a high affinity HIV-1 Sp element was examined and Sp1 binding was shown to be increased relative to the sum of full-length and truncated Sp3 binding following PMA-induced monocytic differentiation in cell line models as well as primary monocyte-derived macrophages (MDMs). The Sp binding phenotype was shown to be associated with changes in the transcriptional activation mediated by the HIV-1 G/C box array. Analysis of post-translational modifications on Sp1 and Sp3 by mass spectroscopy showed a loss of phosphorylation on certain serine and threonine residues with chemically induced differentiation in promonocytic cells. Additionally, in silico analyses using JASPAR, predicted differential Sp isoform binding at Sp sites between Web-PSSM predicted CXCR4/CCR5 utilizing HIV-1 LTRs. Using these analyses and combining them with a high-throughput NanostringRTM technologies, an attempt is being made to develop a HIV-1-specific Sp binding weight matrix taking into account all variations at these binding sites. |
