CCAAT Enhancer Binding Protein and Nuclear Factor of Activated T Cells Regulate HIV-1 LTR via a Novel Conserved Downstream Site in Cells of the Monocyte-Macrophage Lineage

Satinder Dahiya,Yujie Liu,Will Dampier,Michael R Nonnemacher,Brian Wigdahl,Shilpa J Buch

doi:10.1371/journal.pone.0088116

Satinder Dahiya, Yujie Liu + Show 4 more

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https://doi.org/10.1371/journal.pone.0088116

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Journal: PLoS ONE	Publication Date: Feb 14, 2014
Citations: 22	License type: CC BY 4.0

Affiliation: Drexel University, Institute for Molecular Medicine

Abstract

Transcriptional control of the human immunodeficiency virus type 1 (HIV-1) promoter, the long terminal repeat (LTR), is achieved by interactions with cis-acting elements present both upstream and downstream of the start site. In silico transcription factor binding analysis of the HIV-1 subtype B LTR sequences revealed a potential downstream CCAAT enhancer binding protein (C/EBP) binding site. This binding site (+158 to+172), designated DS3, was found to be conserved in 67% of 3,858 unique subtype B LTR sequences analyzed in terms of nucleotide sequence as well as physical location in the LTR. DS3 was found to be well represented in other subtypes as well. Interestingly, DS3 overlaps with a previously identified region that bind members of the nuclear factor of activated T cells (NFAT) family of proteins. NFATc2 exhibited a higher relative affinity for DS3 as compared with members of the C/EBP family (C/EBP α and β). DS3 was able to compete efficiently with the low-affinity upstream C/EBP binding site I with respect to C/EBP binding, suggesting utilization of both NFAT and C/EBP. Moreover, cyclosporine A treatment, which has been shown to prevent dephosphorylation and nuclear translocation of NFAT isoforms, resulted in enhanced C/EBPα binding. The interactions at DS3 were also validated in an integrated HIV-1 LTR in chronically infected U1 cells. A binding knockout of DS3 demonstrated reduced HIV-1 LTR-directed transcription under both basal and interleukin-6-stimulated conditions only in cells of the monocyte-macrophage lineage cells and not in cells of T-cell origin. Thus, the events at DS3 positively regulate the HIV-1 promoter in cells of the monocyte-macrophage lineage.

Highlights

Human immunodeficiency virus type 1 (HIV-1) is transcriptionally regulated by cellular and viral proteins interacting with the cis-regulating elements in the viral promoter, the long terminal repeat (LTR)
We characterized a downstream element in the LTR (+158 to +172, subtype B LTR as reference) that is bound by nuclear factor of activated T cells (NFAT) and CCAAT enhancer binding protein (C/EBP) family members
We propose an experimental model (Figure 7) that involves the preferential occupation of DS3 by NFAT under basal conditions based on its higher relative affinity for DS3 compared with C/EBP family members

Summary

Introduction

Human immunodeficiency virus type 1 (HIV-1) is transcriptionally regulated by cellular and viral proteins interacting with the cis-regulating elements in the viral promoter, the long terminal repeat (LTR). With the constant evolution in our understanding of the nucleosome packaging within the HIV-1 LTR [4,5], the contributions of transcription factor (TF) binding sites (TFBSs) located downstream of the start site have been examined previously [6,7,8,9]. In this regard, the downstream binding sites for AP-1, AP3-like, DBF1 (dehydrationresponsive element [DRE]-binding factor), and Sp1 (specificity protein 1) have been previously demonstrated to regulate basal transcription within the context of the integrated LTR [7]. The relative importance of these sites depends on the cellular phenotype of the infected cell with respect to cell type and activation and differentiation state, as the subset of TFs available at a given point in time is regulated by both the differentiation status of the cell and the activation signals received by a given cell population

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