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.
Highlights
HIV-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
Parental and 3T5T LTR results in cell type–specific phenotypes within different promoter backbones The DREXELMED HIV/AIDS Genetic Analysis Cohort is currently comprised of approximately 504 patients, 78.26% of whom are on continuous highly active antiretroviral therapy (HAART)
As one approach to understand the effects of the 3 of the C/EBP site I (3 T) and 5 of the Sp site III (5 T) variations together on LTR-driven gene expression within several backbones, the WT and 3T5T variations were introduced into the LAI, YU-2, and 89.6 HIV-1 LTR backbones and ligated into the pEGFP-N1 vector where the LTR can drive the expression of the green fluorescent protein (GFP)
Summary
HIV-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. HIV-1-infected monocytic cell populations produce only limited quantities of virus due to several host-cell replication blocks including barriers that limit the reverse transcription process [8,9] and nuclear import [10]. These barriers result in a more chronic infection because this cell type is more resistant to the cytopathic effects of HIV-1 gene products [11,12,13] and has a longer lifespan in vivo. The bone marrow may serve as a source of HIV-1-infected macrophages and may play a critical role in neuroinvasion and progression of CNS disease
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