Bryan Irish

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

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.

AP-1-directed human T cell leukemia virus type 1 viral gene expression during monocytic differentiation

Human T cell leukemia virus type 1 (HTLV‐1) has previously been shown to infect antigen‐presenting cells and their precursors in vivo. However, the role these important cell populations play in the pathogenesis of HTLV‐1‐associated myelopathy/tropical spastic paraparesis or adult T cell leukemia remains unresolved. To better understand how HTLV‐1 infection of these important cell populations may potentially impact disease progression, the regulation of HTLV‐1 viral gene expression in established monocytic cell lines was examined. U‐937 promonocytic cells transiently transfected with a HTLV‐1 long‐terminal repeat (LTR) luciferase construct were treated with phorbol 12‐myristate 13‐acetate (PMA) to induce cellular differentiation. PMA‐induced cellular differentiation resulted in activation of basal and Tax‐mediated transactivation of the HTLV‐1 LTR. In addition, electrophoretic mobility shift analyses demonstrated that PMA‐induced cellular differentiation induced DNA‐binding activity of cellular transcription factors to Tax‐responsive element 1 (TRE‐1) repeat II. Supershift analyses revealed that factors belonging to the activator protein 1 (AP‐1) family of basic region/leucine zipper proteins (Fra‐1, Fra‐2, JunB, and JunD) were induced to bind to TRE‐1 repeat II during cellular differentiation. Inhibition of AP‐1 DNA‐binding activity by overexpression of a dominant‐negative c‐Fos mutant (A‐Fos) in transient expression analyses resulted in severely decreased levels of HTLV‐1 LTR activation in PMA‐induced U‐937 cells. These results have suggested that following infection of peripheral blood monocytes, HTLV‐1 viral gene expression may become up‐regulated by AP‐1 during differentiation into macrophages or dendritic cells.

Genetic variation and HIV-associated neurologic disease.

HIV-associated neurologic disease continues to be a significant complication in the era of highly active antiretroviral therapy. A substantial subset of the HIV-infected population shows impaired neuropsychological performance as a result of HIV-mediated neuroinflammation and eventual central nervous system (CNS) injury. CNS compartmentalization of HIV, coupled with the evolution of genetically isolated populations in the CNS, is responsible for poor prognosis in patients with AIDS, warranting further investigation and possible additions to the current therapeutic strategy. This chapter reviews key advances in the field of neuropathogenesis and studies that have highlighted how molecular diversity within the HIV genome may impact HIV-associated neurologic disease. We also discuss the possible functional implications of genetic variation within the viral promoter and possibly other regions of the viral genome, especially in the cells of monocyte-macrophage lineage, which are arguably key cellular players in HIV-associated CNS disease.

HIV-1 LTR Activity is Altered by Recruitment of Sp Transcription Factors During Monocytic Differentiation

Viral replication, in part, is mediated by interactions between the HIV-1 long terminal repeat (LTR) and a variety of host cell and viral proteins. Basal and activated LTR activity is dependent on interactions between the G/C box array of the HIV-1 LTR and the Sp family of transcription factors. The effect of monocytic differentiation on Sp factor binding and transactivation has been examined with respect to the HIV-1 LTR. Primary monocyte-derived macrophages (MDM), as well as monoblastic (U-937 and THP-1) and myelomonocytic (HL-60) cell lines were utilized in both the absence and presence of chemical differentiating agents to model selected aspects of monocytic differentiation. The binding of Sp1, full-length Sp3, and truncated Sp3 to a high affinity HIV-1 Sp element was examined utilizing electrophoretic mobility shift analyses. Sp1 binding increased relative to the sum of full-length and truncated Sp3 binding following PMA-induced monocytic differentiation in the cell lines. Sp binding ratios obtained with nuclear extracts from PMA-induced cell lines were also shown to correlate with those derived from studies performed with extracts from primary MDMs. This Sp binding phenotype was shown to alter the transcriptional activation generated by the HIV-1 G/C box array. from 2005 International Meeting of The Institute of Human Virology Baltimore, USA, 29 August – 2 September 2005

Use of HIV-1 LTR Sequence Variants as Prognostic Indicators of HIV Dementia

To date, no prognostic viral markers exist for the onset of human immunodeficiency virus type 1 (HIV-1)-associated dementia (HIVD). The long terminal repeat (LTR) regulates HIV-1 viral gene expression via its interaction with multiple viral and host factors, including CCAAT/ enhancer binding protein (C/EBP) and Sp transcription factor families. We have examined sequence variation at C/EBP sites I and II, and Sp sites I, II, and III in peripheral blood (PB)-derived LTRs from HIV-1-infected patients for potential signature sequences. The 3T configuration of C/ EBP site I (C to T at position 3) and 5T configuration of Sp site III (C to T at position 5) were the only variants examined that were found in low frequencies in PB-derived LTRs derived from patients at early stages of HIV-1 disease, and at increasing frequencies in patients representing later stages of disease. Sequence variation at these sites was also examined in LTRs derived from autopsied brain tissue of patients both with and without HIVD. The 3T C/ EBP site I was identified in 25% of brain-derived LTRs from patients with HIVD, but was absent in patients without HIVD. This suggests that 3T C/EBP site I, and possibly 5T Sp site III may prove valuable in assessing the likelihood of an HIV-1-infected individual developing HIVD. from 2005 International Meeting of The Institute of Human Virology Baltimore, USA, 29 August – 2 September 2005