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.
Drug abuse alone has been shown to cause epigenetic changes in brain tissue that have been shown to play roles in addictive behaviors. In conjunction with HIV-1 infection, it can cause epigenetic changes at the viral promoter that can result in altered gene expression, and exacerbate disease progression overall. This review entails an in-depth look at research conducted on the epigenetic effects of three of the most widely abused drugs (cannabinoids, opioids, and cocaine), with a particular focus on the mechanisms through which these drugs interact with HIV-1 infection at the viral promoter. Here we discuss the impact of this interplay on disease progression from the point of view of the nature of gene regulation at the level of chromatin accessibility, chromatin remodeling, and nucleosome repositioning. Given the importance of chromatin remodeling and DNA methylation in controlling the retroviral promoter, and the high susceptibility of the drug abusing population of individuals to HIV infection, it would be beneficial to understand the way in which the host genome is modified and regulated by drugs of abuse.
drug abuse; epigenetics; chromatin remodeling; HIV-1; LTR
Approximately 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.
The 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.
The 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.
These 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.
μ-opioid receptor (MOR-1); DAMGO; Human immunodeficiency virus type 1 (HIV-1); Bone marrow; CXCR4
The adaptation of human immunodeficiency virus type-1 (HIV-1) to an array of physiologic niches is advantaged by the plasticity of the viral genome, encoded proteins, and promoter. CXCR4-utilizing (X4) viruses preferentially, but not universally, infect CD4+ T cells, generating high levels of virus within activated HIV-1-infected T cells that can be detected in regional lymph nodes and peripheral blood. By comparison, the CCR5-utilizing (R5) viruses have a greater preference for cells of the monocyte-macrophage lineage; however, while R5 viruses also display a propensity to enter and replicate in T cells, they infect a smaller percentage of CD4+ T cells in comparison to X4 viruses. Additionally, R5 viruses have been associated with viral transmission and CNS disease and are also more prevalent during HIV-1 disease. Specific adaptive changes associated with X4 and R5 viruses were identified in co-linear viral sequences beyond the Env-V3. The in silico position-specific scoring matrix (PSSM) algorithm was used to define distinct groups of X4 and R5 sequences based solely on sequences in Env-V3. Bioinformatic tools were used to identify genetic signatures involving specific protein domains or long terminal repeat (LTR) transcription factor sites within co-linear viral protein R (Vpr), trans-activator of transcription (Tat), or LTR sequences that were preferentially associated with X4 or R5 Env-V3 sequences. A number of differential amino acid and nucleotide changes were identified across the co-linear Vpr, Tat, and LTR sequences, suggesting the presence of specific genetic signatures that preferentially associate with X4 or R5 viruses. Investigation of the genetic relatedness between X4 and R5 viruses utilizing phylogenetic analyses of complete sequences could not be used to definitively and uniquely identify groups of R5 or X4 sequences; in contrast, differences in the genetic diversities between X4 and R5 were readily identified within these co-linear sequences in HIV-1-infected patients.
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.
Extracellular human immunodeficiency virus type type 1 (HIV-1) viral protein R (Vpr) is a pleiotropic protein accomplishing several functions within the viral life cycle. While Vpr has been described extensively as an intracellular protein, very little is known about its role as an extracellular protein. In fact, HIV-1 Vpr has been detected in the blood, serum, and cerebrospinal fluid of HIV-1-infected patients, with concentrations increasingly higher in late-stage disease. To determine the role exogenous Vpr plays in HIV-associated central nervous system dysfunction, primary human fetal astrocytes were exposed to recombinant Vpr and a time- and dose-dependent decrease was demonstrated in two fundamental intracellular metabolites (ATP and glutathione (GSH)). Additionally, exposure to exogenous Vpr led to increased caspase activity and secretion of proinflammatory cytokines IL-6 and IL-8 and chemoattractants, monocyte chemotactic protein-1 and migration inhibition factor. Extracellular Vpr also dampened the glycolytic pathway through impairment of GAPDH activity, causing a decline in the levels of ATP. The reduction in intracellular ATP increased reactive oxygen species buildup, decreasing GSH concentrations, which affected several genes in the oxidative stress pathway. In addition, exposure of the SK-N-SH neuroblastoma cell line to conditioned medium from exogenous Vpr-treated astrocytes decreased synthesis of GSH, leading to their apoptosis. These observations point to a role that Vpr plays in altering astrocytic metabolism and indirectly affecting neuronal survival. We propose a model that may explain some of the neurological damage and therefore neurocognitive impairment observed during the course of HIV-1 disease.
adenosine triphosphate; astrocytic-neuronal network; glutathione; glyceraldehyde 3-phosphate dehydrogenase; HIV-1 viral protein R
HIV-1 gene expression is driven by the long terminal repeat (LTR), which contains many binding sites shown to interact with an array of host and viral factors. Selective pressures within the host as well as the low fidelity of reverse transcriptase lead to changes in the relative prevalence of genetic variants within the HIV-1 genome, including the LTR, resulting in viral quasispecies that can be differentially regulated and can potentially establish niches within specific cell types and tissues.
Utilizing flow cytometry and electromobility shift assays, specific single-nucleotide sequence polymorphisms (SNPs) were shown to alter both the phenotype of LTR-driven transcription and reactivation. Additional studies also demonstrated differential loading of transcription factors to probes derived from the double-variant LTR as compared to probes from the wild type.
This study has identified specific SNPs within CCAAT/enhancer binding protein (C/EBP) site I and Sp site III (3 T, C-to-T change at position 3, and 5 T, C-to-T change at position 5 of the binding site, respectively) that alter LTR-driven gene transcription and may alter the course of viral latency and reactivation. The HIV-1 LAI LTRs containing the SNPs of interest were coupled to a plasmid encoding green fluorescent protein (GFP), and polyclonal HIV-1 LTR-GFP stable cell lines utilizing bone marrow progenitor, T, and monocytic cell lines were constructed and utilized to explore the LTR phenotype associated with these genotypic changes.
Although the 3 T and 5 T SNPs have been shown to be low-affinity binding sites, the fact that they can still result in effective HIV-1 LTR-driven gene expression, particularly within the TF-1 cell line, has suggested that the low binding site affinities associated with the 3 T C/EBP site I and 5 T Sp site III are potentially compensated for by the interaction of nuclear factor-κB with its corresponding binding sites under selected physiological and cellular conditions. Additionally, tumor necrosis factor-α and Tat can enhance basal transcription of each SNP-specific HIV-1 LTR; however, differential regulation of the LTR is both SNP- and cell type-specific.
HIV-1 genetics; Viral transcription; Integration; Viral latency; Single-nucleotide polymorphisms; C/EBP; Sp
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.
Aging is a complicated process characterized by a progressive loss of homeostasis, which results in an increased vulnerability to multiple diseases. HIV-1-infected patients demonstrate a premature aging phenotype and develop certain age-related diseases earlier in their lifespan than what is seen in the general population. Age-related comorbidities may include the development of bone disease, metabolic disorders, neurologic impairment and immunosenescence. Age also appears to have an effect on traditional markers of HIV-1 disease progression, including CD4+ T-cell count and viral load. These effects are not only a consequence of HIV-1 infection, but in many cases, are also linked to antiretroviral therapy. This review summarizes the complex interplay between HIV-1 infection and aging, and the impact that aging has on markers of HIV-1 disease.
aging; comorbidities; disease progression; HIV-1; neurocognitive impairment
Previous investigations showing that polydisperse biguanide (PDBG) molecules have activity against human immunodeficiency virus type 1 (HIV-1) also suggested a relationship between PDBG biologic activity and the lengths of hydrocarbon linkers surrounding the positively charged biguanide unit. To better define structure-activity relationships, PDBG molecules with select linker lengths were evaluated for cytotoxicity, anti-HIV-1 activity, and in vivo toxicity. Results of the in vitro experiments demonstrated that increases in linker length (and, therefore, increases in compound lipophilicity) were generally associated with increases in cytotoxicity and antiviral activity against HIV-1. However, a relationship between linker length asymmetry and in vitro therapeutic index (TI) suggested structural specificity in the mechanism of action against HIV-1. Polyethylene hexamethylene biguanide (PEHMB; biguanide units spaced between alternating ethylene and hexamethylene linkers) was found to have the highest in vitro TI (CC50/IC50) among the compounds examined. Recent improvements in PEHMB synthesis and purification have yielded preparations of PEHMB with in vitro TI values of 266 and 7000 against HIV-1 strains BaL and IIIB, respectively. The minimal toxicity of PEHMB relative to polyhexamethylene biguanide (PHMB; biguanide units alternating with hexamethylene linkers) in a murine model of cervicovaginal microbicide toxicity was consistent with considerable differences in cytotoxicity between PEHMB and PHMB observed during in vitro experiments. These structure-activity investigations increase our understanding of PDBG molecules as agents with activity against HIV-1 and provide the foundation for further preclinical studies of PEHMB and other biguanide-based compounds as antiviral and microbicidal agents.
HIV-1; Biguanide; Antiviral
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 immuno-deficiency 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.
Opioids; HIV-1; Cyclic AMP (cAMP)
Herpes simplex virus type 1 (HSV-1) is capable of causing a latent infection in sensory neurons that lasts for the lifetime of the host. The primary infection is resolved following the induction of the innate immune response that controls replication of the virus until the adaptive immune response can clear the active infection. HSV-1-specific CD8+ T cells survey the ganglionic regions containing latently infected neurons and participate in preventing reactivation of HSV from latency. The long-term residence and migration dynamics of the T cells in the trigeminal ganglia appear to distinguish them from the traditional memory T cell subsets. Recently described tissue resident memory (TRM) T cells establish residence and survive for long periods in peripheral tissue compartments following antigen exposure. This review focuses on the immune system response to HSV-1 infection. Particular emphasis is placed on the evidence pointing to the HSV-1-specific CD8+ T cells in the trigeminal belonging to the TRM class of memory T cells and the role of TRM cells in virus infection, pathogenesis, latency, and disease.
Herpes simplex virus; Tissue resident memory T cells; Trigeminal ganglia latency; Reactivation; Recurrent disease
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) 293T 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.
ATP; glutathione; Vpr; oxidative stress; astrocytes; HIV-1-induced 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 aden-osine 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.
In May 2012, the Division of AIDS Research at the National Institute of Mental Health (NIMH) organized the “Global NeuroAIDS Roundtable” in conjunction with the 11th International Symposium on Neurovirology and the 2012 Conference on HIV in the Nervous System. The meeting was held in New York, NY, USA and brought together NIMH-funded investigators who are currently working on projects related to the neurological complications of AIDS (NeuroAIDS) in Africa, Asia, Eastern Europe, and Latin America in order to provide an opportunity to share their recent findings and discuss the challenges encountered within each country. The major goals of the roundtable were to evaluate HIV-associated neurocognitive impairment and determine if it may be directly attributable to distinct HIV subtypes or clades and to discuss the future priorities for global NeuroAIDS research. At the “Global NeuroAIDS Roundtable”, presentations of preliminary research indicated that HIV-associated neurocognitive impairment is prevalent in all countries examined regardless of which HIV clade is present in the region. The only clear-cut difference between HIV-1 clades was in relation to subtypes A and D in Uganda. However, a key point that emerged from the discussions was that there is an urgent need to standardize neurocognitive assessment methodologies across the globe before definitive conclusions can be drawn regarding the relationship between HIV clade diversity and neuropathogenesis. Future research directions were also discussed at the roundtable with particular emphasis on the potential of viral and host factor molecular interactions to impact the pathophysiology of HIV-associated neurocognitive disorders (HAND) from a global perspective.
Human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2); Acquired immunodeficiency syndrome (AIDS); HIV clade; NeuroAIDS; HIV-associated neurocognitive disorders (HAND); Neuropathogenesis
During the course of human immunodeficiency virus type 1 (HIV-1) disease, the virus has been shown to effectively escape the immune response with the subsequent establishment of latent viral reservoirs in specific cell populations within the peripheral blood (PB) and associated lymphoid tissues, bone marrow (BM), brain, and potentially other end organs. HIV-1, along with hepatitis B and C viruses (HBV and HCV), are known to share similar routes of transmission, including intravenous drug use, blood transfusions, sexual intercourse, and perinatal exposure. Substance abuse, including the use of opioids and cocaine, is a significant risk factor for exposure to HIV-1 and the development of acquired immune deficiency syndrome, as well as HBV and HCV exposure, infection, and disease. Thus, coinfection with HIV-1 and HBV or HCV is common and may be impacted by chronic substance abuse during the course of disease. HIV-1 impacts the natural course of HBV and HCV infection by accelerating the progression of HBV/HCV-associated liver disease toward end-stage cirrhosis and quantitative depletion of the CD4+ T-cell compartment. HBV or HCV coinfection with HIV-1 is also associated with increased mortality when compared to either infection alone. This review focuses on the impact of substance abuse and coinfection with HBV and HCV in the PB, BM, and brain on the HIV-1 pathogenic process as it relates to viral pathogenesis, disease progression, and the associated immune response during the course of this complex interplay. The impact of HIV-1 and substance abuse on hepatitis virus-induced disease is also a focal point.
bone marrow; brain; cocaine; HBV; HCV; HIV-1; opioids
Despite the success of highly active antiretroviral therapy in combating human immunodeficiency virus type 1 (HIV-1) infection, the virus still persists in viral reservoirs, often in a state of transcriptional silence. This review focuses on the HIV-1 protein and regulatory machinery and how expanding knowledge of the function of individual HIV-1-coded proteins has provided valuable insights into understanding HIV transcriptional regulation in selected susceptible cell types. Historically, Tat has been the most studied primary transactivator protein, but emerging knowledge of HIV-1 transcriptional regulation in cells of the monocyte–macrophage lineage has more recently established that a number of the HIV-1 accessory proteins like Vpr may directly or indirectly regulate the transcriptional process. The viral proteins Nef and matrix play important roles in modulating the cellular activation pathways to facilitate viral replication. These observations highlight the cross talk between the HIV-1 transcriptional machinery and cellular activation pathways. The review also discusses the proposed transcriptional regulation mechanisms that intersect with the pathways regulated by microRNAs and how development of the knowledge of chromatin biology has enhanced our understanding of key protein–protein and protein–DNA interactions that form the HIV-1 transcriptome. Finally, we discuss the potential pharmacological approaches to target viral persistence and enhance effective transcription to purge the virus in cellular reservoirs, especially within the central nervous system, and the novel therapeutics that are currently in various stages of development to achieve a much superior prognosis for the HIV-1-infected population.
Macrophage colony stimulating factor (M-CSF) is a cytokine that promotes monocyte differentiation and survival. When overexpressed, M-CSF contributes to pathology in a wide variety of diseases, including osteoporosis, obesity, certain human cancers, and in human immunodeficiency virus type 1 (HIV-1) infection, particularly with respect to monocyte/macrophage infection and the development of HIV-1 associated central nervous system disorders. In this study, our aim was to expand the current knowledge of M-CSF regulation, focusing on nuclear factor kappa B (NF-κB), a transcription factor playing a prominent role during inflammation and HIV-1 infection. Our results suggest that tumor necrosis factor alpha (TNF-α) promotes M-CSF secretion in primary macrophages and activates the −1310/+48 bp M-CSF promoter in Mono-Mac 1 cells. Inhibitors of the NF-κB pathway diminish this response. We identified four putative NF-κB and four CCAAT-enhancer-binding protein beta binding sites within the M-CSF promoter. Our findings, using promoter constructs mutated at individual NF-κB sites within the M-CSF promoter region, suggest that these sites are redundant with respect to NF-κB regulation. TNF-α treatment promoted NF-κB p65 binding to the M-CSF promoter in phorbol 12-myristate 13-acetate (PMA) treated U937 cells chronically infected with HIV-1 (U1 cells), but not in PMA treated uninfected U937 cells, suggesting that the presence of HIV-1 increases the NF-κB response. In conclusion, our findings demonstrate that NF-κB induces M-CSF expression on a promoter level via multiple functional NF-κB binding sites and that this pathway is likely relevant in HIV-1 infection of macrophages.
The disappointing clinical failures of five topical vaginal microbicides have provided new insights into factors that impact microbicide safety and efficacy. Specifically, the greater risk for human immunodeficiency virus type 1 (HIV-1) acquisition associated with multiple uses of a nonoxynol-9 (N-9)-containing product has highlighted the importance of application frequency as a variable during pre-clinical microbicide development, particularly in animal model studies.
To evaluate an association between application frequency and N-9 toxicity, experiments were performed using a mouse model of cervicovaginal microbicide safety. In this model system, changes in cervical and vaginal epithelial integrity, cytokine release, and immune cell infiltration were assessed after single and multiple exposures to N-9.
After the initial application of N-9 (aqueous, 1%), considerable damage to the cervical epithelium (but not the vaginal epithelium) was observed as early as 10 min post-exposure and up to 8 h post-exposure. Subsequent daily exposures (up to 4 days) were characterized by diminished cervical toxicity relative to single exposures of like duration. Levels of pro-inflammatory cytokines released into the cervicovaginal lumen and the degree of CD14-positive immune cell infiltration proximal to the cervical epithelium were also dependent on the number of N-9 exposures.
Rather than causing cumulative cervical epithelial damage, repeated applications of N-9 were characterized by decreased sensitivity to N-9-associated toxicity and lower levels of immune cell recruitment. These results provide new insights into the failure of N-9-based microbicides and illustrate the importance of considering multiple exposure protocols in pre-clinical microbicide development strategies.
Microbicide; N-9; Cervix; Mouse; Toxicity
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.
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.
HIV; blood-brain barrier; Tat; Nef; gp120; drugs of abuse
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.
cAMP; CCR5; HIV-1
Continued efforts are being directed toward the development of microbicides that will be used to reduce or eliminate the risk of HIV-1 sexual transmission. Unfortunately, clinical trials involving polyanion-containing microbicide formulations, including Carraguard (λ-carrageenan [LC]) and Ushercell (cellulose sulfate [CS]) demonstrated that these products were ineffective and may have, in some circumstances, increased the risk of HIV-1 infection. These findings prompted reassessments of the in vitro activities of these agents to determine whether variables that can affect agent safety and efficacy had been overlooked during preclinical testing. One such variable is product retention and loss following topical application.
In the present studies involving an HIV-1-susceptible cell line and primary human immune cells, product loss was mimicked by introducing and then removing polyanionic compounds prior to HIV-1 infection. In these in vitro "washout" experiments, LC and CS significantly enhanced HIV-1 infection, despite potent antiviral activity when introduced simultaneously with the virus. The presence and magnitude of this effect were dependent on compound identity and concentration; target cell; interval between compound removal and virus challenge; and coreceptor usage. Levels of enhancement (relative to controls) were considerable, exceeding a 200% increase (CS) in P4-R5 MAGI cells and a 300% increase (LC) in human peripheral blood mononuclear cells.
These studies, which demonstrate significant increases in HIV-1 infection subsequent to application and removal of LC and CS, support plausible explanations for the failures of microbicides formulated from these compounds. Detailed studies are now underway to determine the mechanism responsible for this enhancement effect and to assess the potential contribution of this effect to the clinical failures of these agents.
AIDS; HIV-1; Microbicide; Polyanion; Carrageenan; Cellulose sulfate; Enhancement