Functional properties of the HIV-1 long terminal repeat containing single-nucleotide polymorphisms in Sp site III and CCAAT/enhancer binding protein site I

Abstract

BackgroundHIV-1 gene expression is driven by the long terminal repeat (LTR), which contains many binding sites shown to interact with an array of host and viral factors. Selective pressures within the host as well as the low fidelity of reverse transcriptase lead to changes in the relative prevalence of genetic variants within the HIV-1 genome, including the LTR, resulting in viral quasispecies that can be differentially regulated and can potentially establish niches within specific cell types and tissues.MethodsUtilizing flow cytometry and electromobility shift assays, specific single-nucleotide sequence polymorphisms (SNPs) were shown to alter both the phenotype of LTR-driven transcription and reactivation. Additional studies also demonstrated differential loading of transcription factors to probes derived from the double-variant LTR as compared to probes from the wild type.ResultsThis study has identified specific SNPs within CCAAT/enhancer binding protein (C/EBP) site I and Sp site III (3 T, C-to-T change at position 3, and 5 T, C-to-T change at position 5 of the binding site, respectively) that alter LTR-driven gene transcription and may alter the course of viral latency and reactivation. The HIV-1 LAI LTRs containing the SNPs of interest were coupled to a plasmid encoding green fluorescent protein (GFP), and polyclonal HIV-1 LTR-GFP stable cell lines utilizing bone marrow progenitor, T, and monocytic cell lines were constructed and utilized to explore the LTR phenotype associated with these genotypic changes.ConclusionsAlthough the 3 T and 5 T SNPs have been shown to be low-affinity binding sites, the fact that they can still result in effective HIV-1 LTR-driven gene expression, particularly within the TF-1 cell line, has suggested that the low binding site affinities associated with the 3 T C/EBP site I and 5 T Sp site III are potentially compensated for by the interaction of nuclear factor-κB with its corresponding binding sites under selected physiological and cellular conditions. Additionally, tumor necrosis factor-α and Tat can enhance basal transcription of each SNP-specific HIV-1 LTR; however, differential regulation of the LTR is both SNP- and cell type-specific.

Specific amino acids in HIV-1 Vpr are significantly associated with differences in patient neurocognitive status

“…Neuropsychological assessments of HIV-1-infected patients currently enrolled in the CARES Cohort at the Drexel University College of Medicine (Aiamkitsumrit et al , 2014; Dampier et al , 2016; Li et al , 2011; Nonnemacher et al , 2016; Parikh et al , 2014; Shah et al , 2014) revealed a range of neurocognitive function, as indicated by GDS. Most Cohort patients for which Vpr sequences were available had GDS ≤2 (Fig.…”

Impact of Viral Activators and Epigenetic Regulators on HIV-1 LTRs Containing Naturally Occurring Single Nucleotide Polymorphisms

“…The HIV-1 LAI LTR sequence was amplified by polymerase chain reaction (PCR) from previously published LAI-LTR-Luc reporter constructs [ 41 , 42 ]. The LAI LTR wild type, 3T C/EBP site I, 5T Sp site III, and 3T5T double variant were all constructed into the pEGFP-N1 vector (Clontech, Mountain View, CA) as previously described [ 40 ].…”

“…Basal levels of LTR-driven GFP expression within each cell population and clonal population were measured using flow cytometry. The construction and grouping of these clones are further described in [ 40 ]. Because a different number of clones were generated for each LTR (wt, 3T, 5T, and 3T5T) in each of the different cell lines (TF-1, U937, and Jurkat), three representative clones, which typify all of the clones, were selected for further analysis as shown in the figures.…”

“…Previous studies have shown that populations of cells that were stably transfected with the parental LAI LTR and its sequence variants 3T within C/EBP site I and 5T within Sp site III exhibited altered phenotypic properties of the binding site and LTR when placed within the context of different LTR backbones and under a number of different stimulatory conditions within the context of stably integrated gene expression assays [ 40 ]. Consequently, in order to better understand the mechanisms of the cellular phenotypes observed in the sequence variants with respect to LTR activity and viral replication within the context of a homogeneous chromatin-based microenvironment, clonal cell populations were developed from the previous populations of stably transfected TF-1, U-937, and Jurkat cells.…”

Impact of Naturally Occurring Genetic Variation in the HIV-1 LTR TAR Region and Sp Binding Sites on Tat-Mediated Transcription

“…Given these observations and a growing interest in HIV-1 LTR genetic variation and its impact on HIV-1 transcription, we have examined naturally occurring sequence variation in the LTR to determine what variants may lead to a defective LTR in proviruses present within the peripheral blood and so determined the nature of the defective LTRs. In recent studies with HIV-1-infected patients in the cARTera, the previously identified low Sp affinity 5T variation has been shown to remain prevalent (24.9%), and continued investigations have explored the relative fitness of patientderived peripheral blood HIV-1 LTRs containing the 5T Sp site III sequence configurations [24,25]. These studies demonstrated that transient and stable expression assays of patient-derived LTRs containing the 5T Sp site III configuration as well as many other configurations of this binding site identified within the PBMC compartment performed in a number of T-cell and monocyte cell lines exhibited a wide range of functional activity, but that they were functional.…”