High-Affinity Interaction between HIV-1 Vpr and Specific Sequences That Span the C/EBP and Adjacent NF-κB Sites within the HIV-1 LTR Correlate with HIV-1-Associated Dementia

Abstract

Numerous host and viral factors likely participate in the onset and progression of HIV-1-associated dementia (HIVD). Previous studies have suggested that viral gene expression in resident central nervous system (CNS) cells of monocyte/macrophage lineage play a central role in the production of neurotoxic viral proteins and infectious virus, deregulation of cellular gene expression, and/or dysfunction of glial and neuronal cell populations. HIV-1 replication is regulated, in part, by interactions between cellular transcription factors and the viral trans-activators, Tat and viral protein R (Vpr), with cis-acting promoter elements within the LTR. We have previously demonstrated that Vpr binds with high affinity to selected sequence configurations within CCAAT/enhancer binding protein (C/EBP) site I and downstream sequences immediately adjacent to this site. Studies reported herein establish a correlation between the diagnosis of HIVD and the increased prevalence of HIV-1 LTRs containing a C/EBP binding site I that exhibits high affinity for Vpr. To this end, the interaction of Vpr with C/EBP site I variants in 47 LTRs from three nondemented patients and 96 LTRs from seven demented patients was examined. Competition electrophoretic mobility shift (EMS) analyses were utilized to examine Vpr binding to oligonucleotide probes containing C/EBP site I variants. We demonstrated that 89% of LTRs derived from patients exhibiting clinical dementia contained C/EBP site I configurations that displayed a high relative affinity for Vpr, while only 11% of LTRs contained C/EBP site I configurations that exhibited a low relative affinity Vpr binding phenotype. In contrast, examination of LTRs derived from patients lacking clinically evident dementia revealed that only 53% of brain-derived LTRs contained C/EBP site I configurations that displayed a high relative affinity for Vpr, while 47% of LTRs contained C/EBP site I configurations that exhibited a low relative affinity Vpr binding phenotype. We propose that sequence-specific interactions between cis-acting elements in the LTR, members of the C/EBP family of transcription factors, and the virion-associated trans-activator protein Vpr play important roles in the pathogenesis of HIVD.

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

“…The 5 T SNP results in dramatically reduced binding of Sp1, Sp3, and the truncated form of Sp3 [23]. Approximately 5.1% of the patients within this cohort have a SNP within the LTR at C/EBP site I (AGCTTTCTACAA), where there is a C-to-T change at position 3 (3 T) that also results in a knockout binding phenotype [23,25-27,37,38]. Finally, a small number of the patients (1.0%) in the DrexelMed HIV/AIDS Genetic Analysis Cohort have been found to have the 3T5T double variant.…”

“…This differential regulation may ultimately impact viral replication in both a cell type- and a tissue-specific manner [13,21,34,35] that may impact the course of HIV-1 disease [36]. Due to sequence variation within the LTR, cis -acting transcription factor binding sites in the LTR may be functionally altered during the evolution of quasispecies, resulting in altered promoter activity [20-22] and altered transcription factor binding [23,25-27]. Studies in the pre-HAART era have demonstrated that specific HIV-1 LTR CCAAT/enhancer binding protein (C/EBP) and Sp protein binding site configurations that arise as a consequence of quasispecies evolution, such as a C-to-T SNP at nucleotide position 3 in C/EBP site I within the context of a subtype B consensus sequence binding site (designated 3 T) and the C-to-T change at nucleotide position 5 in Sp binding site III (designated 5 T), were preferentially encountered in patients with more severe disease [23,37] and the brains of patients with HIVD [37].…”

“…Studies in the pre-HAART era have demonstrated that specific HIV-1 LTR CCAAT/enhancer binding protein (C/EBP) and Sp protein binding site configurations that arise as a consequence of quasispecies evolution, such as a C-to-T SNP at nucleotide position 3 in C/EBP site I within the context of a subtype B consensus sequence binding site (designated 3 T) and the C-to-T change at nucleotide position 5 in Sp binding site III (designated 5 T), were preferentially encountered in patients with more severe disease [23,37] and the brains of patients with HIVD [37]. Specific nucleotide changes within the LTR, such as the 3 T and 5 T, abrogate binding of cognate transcription factors to their corresponding binding site [23,25-27,37,38]. Variations in the Sp GC box array of the viral promoter result in altered rates of viral gene expression and viral replication [39-44].…”

HIV-1 Vpr Potently Induces Programmed Cell Death in the CNSin Vivo

“…However, R77Q, a mutant of Vpr, was unable to bind ANT and impaired the induction of apoptosis, indicating the sequence-specific interaction of Vpr with ANT. A recent study demonstrated that Vpr directly binds with high affinity to cellular transcriptional factors during the progression of AIDS (Burdo et al, 2004). This study was also able to correlate the presence of increased numbers of C/EBP sites within HIV-1 long terminal repeats (LTRs), in clinically defined HIV-associated dementia (HAD) patients, compared to non-HAD patients.…”

Role of β-Catenin and TCF/LEF Family Members in Transcriptional Activity of HIV in Astrocytes

“…The association between ␤-catenin signaling and C/EBP activity is particularly interesting, given recent evidence from Burdo et al showing that different regions of the brain contain LTRs with distinct C/EBP binding sites (5). Furthermore, C/EBP sequence variants that bind Vpr with high affinity occur at a higher frequency in brain tissue from patients with HAD (6). In addition to enhancing HIV transcription, excessive C/EBP␤ and C/EBP␦ activity can increase neuroinflammation that contributes to HAND by enhancing production of proinflammatory cytokines such as interleukin-6 (IL-6), IL-1␤, and tumor necrosis factor alpha (TNF-␣) by astrocytes (31).…”