Michael R. Nonnemacher

Breaking down the barrier: The effects of HIV-1 on the blood–brain barrier

Human immunodeficiency virus type 1 (HIV-1) primarily infects CD4(+) T cells and cells of the monocyte-macrophage lineage, resulting in immunodeficiency in an infected patient. Along with this immune deficiency, HIV-1 has been linked to a number of neurological symptoms in the absence of opportunistic infections or other co-morbidities, suggesting that HIV-1 is able to cross the blood-brain barrier (BBB), enter the central nervous system (CNS), and cause neurocognitive impairment. HIV-1-infected monocyte-macrophages traverse the BBB and enter the CNS throughout the course of HIV-1 disease. Once in the brain, both free virus and virus-infected cells are able to infect neighboring resident microglia and astrocytes and possibly other cell types. HIV-1-infected cells in both the periphery and the CNS give rise to elevated levels of viral proteins, including gp120, Tat, and Nef, and of host inflammatory mediators such as cytokines and chemokines. It has been shown that the viral proteins may act alone or in concert with host cytokines and chemokines, affecting the integrity of the BBB. The pathological end point of these interactions may facilitate a positive feedback loop resulting in increased penetration of HIV into the CNS. It is proposed in this review that the dysregulation of the BBB during and after neuroinvasion is a critical component of the neuropathogenic process and that dysregulation of this protective barrier is caused by a combination of viral and host factors including secreted viral proteins, components of the inflammatory process, the aging process, therapeutics, and drug or alcohol abuse.

Regulation of HIV-1 transcription in cells of the monocyte-macrophage lineage

Human immunodeficiency virus type 1 (HIV-1) has been shown to replicate productively in cells of the monocyte-macrophage lineage, although replication occurs to a lesser extent than in infected T cells. As cells of the monocyte-macrophage lineage become differentiated and activated and subsequently travel to a variety of end organs, they become a source of infectious virus and secreted viral proteins and cellular products that likely initiate pathological consequences in a number of organ systems. During this process, alterations in a number of signaling pathways, including the level and functional properties of many cellular transcription factors, alter the course of HIV-1 long terminal repeat (LTR)-directed gene expression. This process ultimately results in events that contribute to the pathogenesis of HIV-1 infection. First, increased transcription leads to the upregulation of infectious virus production, and the increased production of viral proteins (gp120, Tat, Nef, and Vpr), which have additional activities as extracellular proteins. Increased viral production and the presence of toxic proteins lead to enhanced deregulation of cellular functions increasing the production of toxic cellular proteins and metabolites and the resulting organ-specific pathologic consequences such as neuroAIDS. This article reviews the structural and functional features of the cis-acting elements upstream and downstream of the transcriptional start site in the retroviral LTR. It also includes a discussion of the regulation of the retroviral LTR in the monocyte-macrophage lineage during virus infection of the bone marrow, the peripheral blood, the lymphoid tissues, and end organs such as the brain. The impact of genetic variation on LTR-directed transcription during the course of retrovirus disease is also reviewed.

Interaction between CCAAT/Enhancer Binding Protein and Cyclic AMP Response Element Binding Protein 1 Regulates Human Immunodeficiency Virus Type 1 Transcription in Cells of the Monocyte/Macrophage Lineage

ABSTRACT Recent observations have shown two CCAAT/enhancer binding protein (C/EBP) binding sites to be critically important for efficient human immunodeficiency virus type 1 (HIV-1) replication within cells of the monocyte/macrophage lineage, a cell type likely involved in transport of the virus to the brain. Additionally, sequence variation at C/EBP site I, which lies immediately upstream of the distal nuclear factor kappa B site and immediately downstream of a binding site for activating transcription factor (ATF)/cyclic AMP response element binding protein (CREB), has been shown to affect HIV-1 long terminal repeat (LTR) activity. Given that C/EBP proteins have been shown to interact with many other transcription factors including members of the ATF/CREB family, we proceeded to determine whether an adjacent ATF/CREB binding site could affect C/EBP protein binding to C/EBP site I. Electrophoretic mobility shift analyses indicated that selected ATF/CREB site variants assisted in the recruitment of C/EBP proteins to an adjacent, naturally occurring, low-affinity C/EBP site. This biophysical interaction appears to occur via at least two mechanisms. First, low amounts of CREB-1 and C/EBP appear to heterodimerize and bind to a site consisting of a half site from both the ATF/CREB and C/EBP binding sites. In addition, CREB-1 homodimers bind to the ATF/CREB site and recruit C/EBP dimers to their cognate weak binding sites. This interaction is reciprocal, since C/EBP dimer binding to a strong C/EBP site leads to enhanced CREB-1 recruitment to ATF/CREB sites that are weakly bound by CREB. Sequence variation at both C/EBP and ATF/CREB sites affects the molecular interactions involved in mediating both of these mechanisms. Most importantly, sequence variation at the ATF/CREB binding site affected basal LTR activity as well as LTR function following interleukin-6 stimulation, a treatment that leads to increases in C/EBP activation. Thus, HIV-1 LTR ATF/CREB binding site sequence variation may modulate cellular signaling at the viral promoter through the C/EBP pathway.

Role of mu-opioids as cofactors in human immunodeficiency virus type 1 disease progression and neuropathogenesis

About one third of acquired immunodeficiency syndrome cases in the USA have been attributed to the use of injected addictive drugs, frequently involving opioids like heroin and morphine, establishing them as significant predisposing risk factors for contracting human immunodeficiency virus type 1 (HIV-1). Accumulating evidence from in vitro and in vivo experimental systems indicates that opioids act in concert with HIV-1 proteins to exacerbate dysregulation of neural and immune cell function and survival through diverse molecular mechanisms. In contrast, the impact of opioid exposure and withdrawal on the viral life cycle and HIV-1 disease progression itself is unclear, with conflicting reports emerging from the simian immunodeficiency virus and simian–human immunodeficiency virus infection models. However, these studies suggest a potential role of opioids in elevated viral production. Because human microglia, astrocytes, CD4+ T lymphocytes, and monocyte-derived macrophages express opioid receptors, it is likely that intracellular signaling events triggered by morphine facilitate enhancement of HIV-1 infection in these target cell populations. This review highlights the biochemical changes that accompany prolonged exposure to and withdrawal from morphine that synergize with HIV-1 proteins to disrupt normal cellular physiological functions especially within the central nervous system. More importantly, it collates evidence from epidemiological studies, animal models, and heterologous cell systems to propose a mechanistic link between such physiological adaptations and direct modulation of HIV-1 production. Understanding the opioid–HIV-1 interface at the molecular level is vitally important in designing better treatment strategies for HIV-1-infected patients who abuse opioids.

Impact of Tat Genetic Variation on HIV-1 Disease

The human immunodeficiency virus type 1 (HIV-1) promoter or long-terminal repeat (LTR) regulates viral gene expression by interacting with multiple viral and host factors. The viral transactivator protein Tat plays an important role in transcriptional activation of HIV-1 gene expression. Functional domains of Tat and its interaction with transactivation response element RNA and cellular transcription factors have been examined. Genetic variation within tat of different HIV-1 subtypes has been shown to affect the interaction of the viral transactivator with cellular and/or viral proteins, influencing the overall level of transcriptional activation as well as its action as a neurotoxic protein. Consequently, the genetic variability within tat may impact the molecular architecture of functional domains of the Tat protein that may impact HIV pathogenesis and disease. Tat as a therapeutic target for anti-HIV drugs has also been discussed.

Development of co-selected single nucleotide polymorphisms in the viral promoter precedes the onset of human immunodeficiency virus type 1-associated neurocognitive impairment

The long terminal repeat (LTR) regulates gene expression of HIV-1 by interacting with multiple host and viral factors. Cross-sectional studies in the pre-HAART era demonstrated that single nucleotide polymorphisms (SNPs) in peripheral blood-derived LTRs (a C-to-T change at position 3 of C/EBP site I (3T) and at position 5 of Sp site III (5T)) increased in frequency as disease severity increased. Additionally, the 3T variant correlated with HIV-1-associated dementia. LTR sequences derived by longitudinal sampling of peripheral blood from a single patient in the DrexelMed HIV/AIDS Genetic Analysis Cohort resulted in the detection of the 3T and 5T co-selected SNPs before the onset of neurologic impairment, demonstrating that these SNPs may be useful in predicting HIV-associated neurological complications. The relative fitness of the LTRs containing the 3T and/or 5T co-selected SNPs as they evolve in their native patient-derived LTR backbone structure demonstrated a spectrum of basal and Tat-mediated transcriptional activities using the IIIB-derived Tat and colinear Tat derived from the same molecular clone containing the 3T/5T LTR SNP. In silico predictions utilizing colinear envelope sequence suggested that the patient’s virus evolved from an X4 to an R5 swarm prior to the development of neurological complications and more advanced HIV disease. These results suggest that the HIV-1 genomic swarm may evolve during the course of disease in response to selective pressures that lead to changes in prevalence of specific polymorphisms in the LTR, env, and/or tat that could predict the onset of neurological disease and result in alterations in viral function.

Extracellular human immunodeficiency virus type 1 viral protein R causes reductions in astrocytic ATP and glutathione levels compromising the antioxidant reservoir

Patients infected with human immunodeficiency virus type 1 (HIV-1) often display neurological complications in late stage disease and increased viral loads directly correlated with higher concentrations of extracellular HIV-1 viral protein r (Vpr) in the blood serum and cerebrospinal fluid. Additionally, HIV-1-infected patients with a low CD4+ T-lymphocyte count displayed lower concentrations of reduced glutathione (GSH), the main intracellular antioxidant molecule, and lower level of survival. To establish a correlation between increased concentrations of extracellular Vpr and an oxidative stress-induced phenotype, the U-87 MG astroglioma cell line has been used to determine the downstream effects induced by Vpr. Conditioned media obtained from the human endothelial kidney (HEK) 293 T cell line transfected either in the absence or presence of HIV-1 Vpr contained free Vpr. Exposure of U-87 MG to this conditioned media decreased intracellular levels of both adenosine triphosphate (ATP) and GSH. These observations were recapitulated using purified recombinant HIV-1 Vpr both in U-87 MG and primary human fetal astrocytes in a dose- and time-dependent manner. Vpr-induced oxidative stress could be partly restored by co-treatment with the antioxidant molecule N-acetyl-cysteine (NAC). In addition, free Vpr augmented production of reactive oxygen species due to an increase in the level of oxidized glutathione (GSSG). This event was almost entirely suppressed by treatment with an anti-Vpr antibody or co-treatment with NAC. These studies confirm a role of extracellular Vpr in impairing astrocytic levels of intracellular ATP and GSH. Studies are underway to better understand the intricate correlation between reductions in ATP and GSH metabolites and how they affect neuronal survival in end-stage disease.

Human Immunodeficiency Virus Viral Protein R as an Extracellular Protein in Neuropathogenesis

Numerous studies published in the past two decades have identified the viral protein R (Vpr) as one of the most versatile proteins in the life cycle of human immunodeficiency virus type 1 (HIV-1). In this regard, more than a thousand Vpr molecules are present in extracellular viral particles. Subsequent to viral entry, Vpr participates in early replicative events by assisting in viral genome nuclear import and, during the viral life cycle, by shuttling between the nucleus and the cytoplasm to accomplish its functions within the context of other replicative functions. Additionally, several studies have implicated Vpr as a proapoptotic protein because it promotes formation of permeability transition pores in mitochondria, which in turn affects transmembrane potential and adenosine triphosphate synthesis. Recent studies have identified Vpr as a virion-free protein in the serum and cerebrospinal fluid of patients infected with HIV-1 whose plasma viremia directly correlates with the extracellular concentration of Vpr. These observations pointed to a new role for Vpr as an additional weapon in the HIV-1 arsenal, involving the use of an extracellular protein to target and possibly inhibit HIV-1-uninfected bystander cells to enable them to escape immune surveillance. In addition, extracellular Vpr decreases adenosine triphosphate levels and affects the intracellular redox balance in neurons, ultimately causing their apoptosis. Herein, we review the role of Vpr as an extracellular protein and its downstream effects on cellular metabolism, functionality, and survival, with particular emphasis on how extracellular Vpr-induced oxidative stress might aggravate HIV-1-induced symptoms, thus affecting pathogenesis and disease progression.

CCAAT Enhancer Binding Protein and Nuclear Factor of Activated T Cells Regulate HIV-1 LTR via a Novel Conserved Downstream Site in Cells of the Monocyte-Macrophage Lineage

Transcriptional control of the human immunodeficiency virus type 1 (HIV-1) promoter, the long terminal repeat (LTR), is achieved by interactions with cis-acting elements present both upstream and downstream of the start site. In silico transcription factor binding analysis of the HIV-1 subtype B LTR sequences revealed a potential downstream CCAAT enhancer binding protein (C/EBP) binding site. This binding site (+158 to+172), designated DS3, was found to be conserved in 67% of 3,858 unique subtype B LTR sequences analyzed in terms of nucleotide sequence as well as physical location in the LTR. DS3 was found to be well represented in other subtypes as well. Interestingly, DS3 overlaps with a previously identified region that bind members of the nuclear factor of activated T cells (NFAT) family of proteins. NFATc2 exhibited a higher relative affinity for DS3 as compared with members of the C/EBP family (C/EBP α and β). DS3 was able to compete efficiently with the low-affinity upstream C/EBP binding site I with respect to C/EBP binding, suggesting utilization of both NFAT and C/EBP. Moreover, cyclosporine A treatment, which has been shown to prevent dephosphorylation and nuclear translocation of NFAT isoforms, resulted in enhanced C/EBPα binding. The interactions at DS3 were also validated in an integrated HIV-1 LTR in chronically infected U1 cells. A binding knockout of DS3 demonstrated reduced HIV-1 LTR-directed transcription under both basal and interleukin-6-stimulated conditions only in cells of the monocyte-macrophage lineage cells and not in cells of T-cell origin. Thus, the events at DS3 positively regulate the HIV-1 promoter in cells of the monocyte-macrophage lineage.

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.