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

Abstract

Following human immunodeficiency virus type 1 (HIV-1) integration into host cell DNA, the viral promoter can become transcriptionally silent in the absence of appropriate signals and factors. HIV-1 gene expression is dependent on regulatory elements contained within the long terminal repeat (LTR) that drive the synthesis of viral RNAs and proteins through interaction with multiple host and viral factors. Previous studies identified single nucleotide polymorphisms (SNPs) within CCAAT/enhancer binding protein (C/EBP) site I and Sp site III (3T, C-to-T change at position 3, and 5T, C-to-T change at position 5 of the binding site, respectively, when compared to the consensus B sequence) that are low affinity binding sites and correlate with more advanced stages of HIV-1 disease. Stably transfected cell lines containing the wild type, 3T, 5T, and 3T5T LTRs were developed utilizing bone marrow progenitor, T, and monocytic cell lines to explore the LTR phenotypes associated with these genotypic changes from an integrated chromatin-based microenvironment. Results suggest that in nonexpressing cell clones LTR-driven gene expression occurs in a SNP-specific manner in response to LTR activation or treatment with trichostatin A treatment, indicating a possible cell type and SNP-specific mechanism behind the epigenetic control of LTR activation.

Diverse fates of uracilated HIV-1 DNA during infection of myeloid lineage cells

“…These findings include (i) our previous report that multiple uracils silenced transcription from both HIV-1 LTR and CMV promoters in a cell line ( Weil et al, 2013 ), (ii) uracils within the origin of replication in HSV-1 perturb the binding of HSV-1 origin binding protein ( Focher et al, 1992 ), (iii) U/A pairs disrupt AP-1 transcription factor DNA binding ( Rogstad et al, 2002 ), (iv) singly uracilated DNA disrupts RNase H splicing specificity during reverse transcription Klarmann, 2003 , (v) U/A pairs perturb maintenance of telomere length in B cells by disruption of sheltrin binding ( Vallabhaneni et al, 2015 ), and (vi) one or two U/A base pairs within the specific cleavage site of some restriction enzymes prevents DNA strand cleavage ( Roberts et al, 2015 ). In addition, the abasic site product of uracil excision is known to exert a large negative effect on transcription ( Luhnsdorf et al, 2014 ) as would any mutations in transcription factor recognition sequences arising from error prone repair of excised uracils ( Shah et al, 2015 ; Emiliani, 1998 ). Combined, these potential effects of U/A pairs could profoundly silence HIV gene expression.…”

HIV-1 Promoter Single Nucleotide Polymorphisms Are Associated with Clinical Disease Severity

“…LTR-conB108A and 108G sequences were synthesized and cloned into pGL3 by VectorBuilder (Cyagen Biosciences). LTR-LAI plasmids were cloned into the pGL3 luciferase expression vector (promega) as previously described [ 41 , 53 , 54 ]. LTR-LAI108 was mutagenized using GeneArt Site-Directed Mutagenesis System (Thermofisher) and the mutagenized product was transformed into DH5α cells and grown for 24 hours with appropriate antibodies.…”

Computational Analysis Concerning the Impact of DNA Accessibility on CRISPR-Cas9 Cleavage Efficiency

“…[47][48][49][50][51][52] Studies have suggested that the transcription from the integrated proviral HIV-1 genome is highly regulated by the nucleosomes nuc-0 or nuc-1 on the long terminal repeat (LTR) and histone modulators interacting with transcription factors during latent infection. [53][54][55][56][57] The provirus-associated nucleosomes that were maintained in highly heterochromatic status have been thought to be one of the mechanisms to keep viral transcription at a low level. 58 It is therefore important to know what level of DNA accessibility the CRISPR system may be required to facilitate HIV-1 provirus disruption/excision at the HIV-1 integration loci by using gRNAs that target the HIV-1 LTR regions.…”