Mechanism of Transcriptional Downregulation of HIV Gene Expression by U/A Base Pairs in Proviral DNA

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PROJECT SUMMARY/ABSTRACT Monocyte-derived macrophages (MDM) differentiated from primary human monocytes express high levels of the dNTP triphosphohydrolase SAMHD1 and undetectable dUTPase, leading to elevated ratios of dUTP/TTP in these non-dividing cells. This imbalanced dNTP pool has a profound effect on HIV infection by a mechanism involving incorporation of dUMP into viral DNA by reverse transcriptase, producing abundant U/A base pairs ("uracilation"). This is a unique aspect of HIV infection in this target cell type. Due to the low expression level of uracil DNA glycosylase in MDM, integrated uracilated proviruses persist for at least one month in vitro and can also be detected in monocytes and alveolar macrophages isolated from HIV infected people on long-term anti- retroviral therapy (ART). One emerging aspect of persistent proviral U/A pairs is their ability to decrease viral gene expression and induce transcriptional mutagenesis suggesting they may serve as a previously unrecognized mechanism of viral latency. In three aims, the broad goal of this proposal is to understand the effects of U/A base pairs on transcription factor (TF) occupancy, chromatin structure and RNA pol II activity with respect to HIV proviral DNA in MDM. In the first aim we will use a novel uracil sequencing method with single- nucleotide resolution to map U/A pairs within the 5'-long terminal repeat (LTR) promoter of HIV proviral DNA in MDM. This methodology is enabling because all other polymerase-based sequencing methods read uracil as thymidine. High-resolution mapping of the U/A pairs will establish which TF binding sites are modified during HIV infection of MDM and whether the RNA Pol II transcription initiation site also contains destabilizing U/A pairs. In a second aim, we will quantify the effects of U/A pairs on TF binding and RNA Pol II initiation and elongation in vitro and in human cells. In vitro TF DNA binding measurements will employ cognate binding sequences with site-specific T/A→U/A substitutions to quantify the effect of single- and multiple-site substitutions. Human cell studies will involve transfection of an eGFP reporter DNA containing site-specific U/A base pairs in the LTR region into an engineered cell line that is deficient in uracil excision and also expresses HIV transactivator protein. The relative levels of transcription from uracilated and reference (all-T) reporter constructs will be quantified by fluorescence and RT-PCR measurements. We will also perform ChIP experiments using TF specific antibodies to determine if the occupancy of TFs is affected by specific U/A pairs. The third aim will elucidate the effect of U/A pairs on nucleosome and TF occupancy and chromatin structure in infected MDM target cells. High-resolution nucleosome mapping by micrococcal nuclease (MNase) sensitivity will be used to determine differences in chromatin structure arising from U/A pairs in proviral DNA. These experiments will extend our understanding of the transcriptional effects of uracilation to the most relevant cell-type and explore the effects on chromatin structure that are relevant to infection of cells.