Anupam Banerjee

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.

Transcriptional regulation of the chemokine co-receptor CCR5 by the cAMP/PKA/CREB pathway.

The cyclic adenosine monophosphate (cAMP)-dependent signaling pathway directs the expression of several genes involved in diverse neuroendocrine, immune, metabolic, and developmental pathways. The primary effectors of this pathway are members of the cAMP response element binding (CREB) family of transcription factors, in particular the CREB-1 and cAMP response element modulator (CREM). Both these genes encode alternative splice variants that serve as activators or repressors in a context- and position-specific manner. Although the β-chemokine receptor CC chemokine receptor 5 (CCR5) has been identified on progenitor cells in the bone marrow, the regulatory mechanisms orchestrating its expression are not fully understood. Previous reports have identified putative cAMP response elements in the CCR5 promoter and have described a suppressive role of cAMP in CCR5 expression. In this study, the CD34+CD4+CCR5+ human bone marrow progenitor cell line TF-1 was used to investigate the detailed kinetics of CCR5 transcription in response to the elevation of intracellular cAMP levels and the underlying molecular events. We hypothesize that CCR5 transcription follows an asymmetrical sinusoidal pattern in TF-1 cells that parallels a protein kinase A-dependent alternating change in the ratio of activator pCREB-1-α,Δ to repressor pCREM-α,β isoforms. However, elevated CCR5 mRNA levels do not correlate with enhancement in infectivity with respect to the R5 human immunodeficiency virus type 1 (HIV-1) strain. Our results lend critical insight into the precise mechanism governing the cAMP-CCR5 axis in progenitor cells and pose interesting questions regarding its functional role in HIV-1 infection.

Effect of μ-opioid agonist DAMGO on surface CXCR4 and HIV-1 replication in TF-1 human bone marrow progenitor cells.

BackgroundApproximately one-third of the AIDS cases in the United States have been attributed to the use of injected drugs, frequently involving the abuse of opioids. Consequently, it is critical to address whether opioid use directly contributes to altered susceptibility to HIV-1 beyond the increased risk of exposure. Previous in vitro and in vivo studies addressing the role of μ-opioid agonists in altering levels of the co-receptor CXCR4 and subsequent HIV-1 replication have yielded contrasting results. The bone marrow is believed to be a potential anatomical sanctuary for HIV-1.MethodsThe well-characterized CD34+CD38+ human bone marrow–derived hematopoietic progenitor cell line TF-1 was used as a model to investigate the effects of the μ-opioid receptor–specific peptide DAMGO (D-Ala2,N-Me-Phe4, Gly5-ol-enkephalin) on CXCR4 expression as well as infection of undifferentiated human hematopoietic progenitor cells.ResultsThe results revealed the presence of the μ-opioid receptor-1 isoform (MOR-1) on the surface of TF-1 cells. Furthermore, immunostaining revealed that the majority of TF-1 cells co-express MOR-1 and CXCR4, and a subpopulation of these double-positive cells express the two receptors in overlapping membrane domains. Three subpopulations of TF-1 cells were categorized based on their levels of surface CXCR4 expression, defined as non-, low-, and high-expressing. Flow cytometry indicated that treatment with DAMGO resulted in a shift in the relative proportion of CXCR4+ cells to the low-expressing phenotype. This result correlated with a >3-fold reduction in replication of the X4 HIV-1 strain IIIB, indicating a role for the CXCR4 high-expression subpopulation in sustaining infection within this progenitor cell line.ConclusionsThese experiments provide insight into the impact of μ-opioid exposure with respect to inhibition of viral replication in this human TF-1 bone marrow progenitor cell line model.

Functional Studies of CCAAT/Enhancer Binding Protein Site Located Downstream of the Transcriptional Start Site.

Previous studies have identified a CCAAT/enhancer binding protein (C/EBP) site located downstream of the transcriptional start site (DS3). The role of the DS3 element with respect to HIV-1 transactivation by Tat and viral replication has not been characterized. We have demonstrated that DS3 was a functional C/EBPβ binding site and mutation of this site to the C/EBP knockout DS3-9C variant showed lower HIV-1 long terminal repeat (LTR) transactivation by C/EBPβ. However, it was able to exhibit similar or even higher transcription levels by Tat compared to the parental LTR. C/EBPβ and Tat together further enhanced the transcription level of the parental LAI-LTR and DS3-9C LTR, with higher levels in the DS3-9C LTR. HIV molecular clone viruses carrying the DS3-9C variant LTR demonstrated a decreased replication capacity and delayed rate of replication. These results suggest that DS3 plays a role in virus transcriptional initiation and provides new insight into C/EBP regulation of HIV-1.

cAMP Signaling Enhances HIV-1 Long Terminal Repeat (LTR)-directed Transcription and Viral Replication in Bone Marrow Progenitor Cells

CD34+ hematopoietic progenitor cells have been shown to be susceptible to HIV-1 infection, possibly due to a low-level expression of CXCR4, a coreceptor for HIV-1 entry. Given these observations, we have explored the impact of forskolin on cell surface expression of CXCR4 in a cell line model (TF-1). The elevation of intracellular cyclic adenosine monophosphate (cAMP) by forskolin through adenylyl cyclase (AC) resulted in transcriptional upregulation of CXCR4 with a concomitant increase in replication of the CXCR4-utilizing HIV-1 strain IIIB. Transient expression analyses also demonstrated an increase in CXCR4-, CCR5-, and CXCR4-/CCR5-utilizing HIV-1 (LAI, YU2, and 89.6, respectively) promoter activity. Studies also implicated the protein kinase A (PKA) pathway and the downstream transcription factor CREB-1 in interfacing with cAMP response elements located in the CXCR4 and viral promoter. These observations suggest that the cAMP signaling pathway may serve as a regulator of CXCR4 levels and concomitantly of HIV-1 replication in bone marrow (BM) progenitor cells.

HIV Latency and Reactivation: Role in Neuropathogenesis

Human immunodeficiency virus type 1 (HIV-1) over the past 25 years has become a chronic disease in the developed world that is treatable by antiretroviral therapy but is incapable of being eradicated due to a subpopulation of cells that escape the immune system and therapies and become latently infected. This chapter discusses molecular mechanisms surrounding the development of latency, both pre- and post-integration of the proviral genome and the maintenance of latency, including the role of nucleotide levels, adenosine triphosphate (ATP), levels of certain host transcription factors (NF-κB and NFAT) as well as viral proteins (Vif and Tat), and the chromatin architecture. Also discussed are the cellular reservoirs involved in latency, including the memory CD4+ T cells, which have been proposed as one of the main latent cellular reservoirs, the monocyte–macrophage cell lineage and its role in both HIV-1 latency in the bone marrow, peripheral blood, and CNS, and other cells of the CNS such as the microglial cells and the astrocytes. Finally, the role of latency in neuropathogenesis and reseeding of the peripheral blood from these viral reservoirs are discussed.