Richard J. Noel

Expression of Human Endogenous Retrovirus Type K (HML-2) Is Activated by the Tat Protein of HIV-1

ABSTRACT Human endogenous retroviruses (HERVs) make up 8% of the human genome. The expression of HERV-K (HML-2), the family of HERVs that most recently entered the genome, is tightly regulated but becomes markedly increased after infection with HIV-1. To better understand the mechanisms involved in this activation, we explored the role of the HIV-1 Tat protein in inducing the expression of these endogenous retroviral genes. Administration of recombinant HIV-1 Tat protein caused a 13-fold increase in HERV-K (HML-2) gag RNA transcripts in Jurkat T cells and a 10-fold increase in primary lymphocytes, and the expression of the HERV-K (HML-2) rec and np9 oncogenes was also markedly increased. This activation was seen especially in lymphocytes and monocytic cells, the natural hosts for HIV-1 infection. Luciferase reporter gene assays demonstrated that the effect of Tat on HERV-K (HML-2) expression occurred at the level of the transcriptional promoter. The transcription factors NF-κB and NF-AT contribute to the Tat-induced activation of the promoter, as shown by chromatin immunoprecipitation assays, mutational analysis of the HERV-K (HML-2) long terminal repeat, and treatments with agents that inhibit NF-κB or NF-AT activation. These studies demonstrate that HIV-1 Tat plays an important role in activating expression of HERV-K (HML-2) in the setting of HIV-1 infection.

HIV-1 gp120 induces expression of IL-6 through a nuclear factor-kappa B-dependent mechanism: suppression by gp120 specific small interfering RNA.

In addition to its role in virus entry, HIV-1 gp120 has also been implicated in HIV-associated neurocognitive disorders. However, the mechanism(s) responsible for gp120-mediated neuroinflammation remain undefined. In view of increased levels of IL-6 in HIV-positive individuals with neurological manifestations, we sought to address whether gp120 is involved in IL-6 over-expression in astrocytes. Transfection of a human astrocyte cell line with a plasmid encoding gp120 resulted in increased expression of IL-6 at the levels of mRNA and protein by 51.3±2.1 and 11.6±2.2 fold respectively; this effect of gp120 on IL-6 expression was also demonstrated using primary human fetal astrocytes. A similar effect on IL-6 expression was observed when primary astrocytes were treated with gp120 protein derived from different strains of X4 and R5 tropic HIV-1. The induction of IL-6 could be abrogated by use of gp120-specific siRNA. Furthermore, this study showed that the NF-κB pathway is involved in gp120-mediated IL-6 over-expression, as IKK-2 and IKKβ inhibitors inhibited IL-6 expression by 56.5% and 60.8%, respectively. These results were also confirmed through the use of NF-κB specific siRNA. We also showed that gp120 could increase the phosphorylation of IκBα. Furthermore, gp120 transfection in the SVGA cells increased translocation of NF-κB from cytoplasm to nucleus. These results demonstrate that HIV-1 gp120-mediated over-expression of IL-6 in astrocytes is one mechanism responsible for neuroinflammation in HIV-infected individuals and this is mediated by the NF-κB pathway.

Increased Viral Replication in Simian Immunodeficiency Virus/Simian-HIV-Infected Macaques With Self-Administering Model of Chronic Alcohol Consumption

Alcohol abuse constitutes a major cohort among HIV-infected individuals. The precise effect of alcohol addiction on HIV pathogenesis remains inconclusive, however. This study was designed to determine the effect of alcohol dependence on virus replication and CD4 profiles in simian immunodeficiency virus/simian-HIV-infected rhesus macaques. A group of 3 male Indian rhesus macaques was adapted to a self-drinking model of alcohol consumption, whereas another group of 3 macaques was provided a Nutrasweet solution. After 7 weeks of alcohol consumption, the alcohol-dependent animals along with controls were intravenously inoculated with a mixture of SHIVKU, SHIV89.6P, and SIV/17E-Fr. These animals were followed for a period of 24 weeks for complete blood cell counts, CD4 cell profiles, and viral loads in the blood and cerebral compartments. The alcohol and control groups showed comparable peak viral loads in the blood. The plasma viral load in the alcohol group was 31- to 85-fold higher than that in the control group at weeks 18 through 24 after infection, however. The pattern of cerebrospinal fluid viral replication was also comparable during the acute phase; however, the virus continued to replicate in the brain of alcohol-dependent animals, whereas it became undetectable in the controls. The extent of CD4 cell loss in the alcohol group was significantly higher than that in the control animals at week 1 after infection.

HIV-1 Tat-Mediated Induction of CCL5 in Astrocytes Involves NF-κB, AP-1, C/EBPα and C/EBPγ Transcription Factors and JAK, PI3K/Akt and p38 MAPK Signaling Pathways

The incidence of HIV-associated neurological disorders (HAND) has increased during recent years even though the highly active antiretroviral therapy (HAART) has significantly curtailed the virus replication and increased the life expectancy among HIV-1 infected individuals. These neurological deficits have been attributed to HIV proteins including HIV-1 Tat. HIV-1 Tat is known to up-regulate CCL5 expression in mouse astrocytes, but the mechanism of up-regulation is not known. The present study was undertaken with the objective of determining the mechanism(s) underlying HIV-1 Tat-mediated expression of CCL5 in astrocytes. SVGA astrocytes were transiently transfected with a plasmid encoding Tat, and expression of CCL5 was studied at the mRNA and protein levels using real time RT-PCR and multiplex cytokine bead array, respectively. HIV-1 Tat showed a time-dependent increase in the CCL5 expression with peak mRNA and protein levels, observed at 1 h and 48 h post-transfection, respectively. In order to explore the mechanism(s), pharmacological inhibitors and siRNA against different pathway(s) were used. Pre-treatment with SC514 (NF-κB inhibitor), LY294002 (PI3K inhibitor), AG490 (JAK2 inhibitor) and Janex-1 (JAK3 inhibitor) showed partial reduction of the Tat-mediated induction of CCL5 suggesting involvement of JAK, PI3K/Akt and NF-κB in CCL5 expression. These results were further confirmed by knockdown of the respective genes using siRNA. Furthermore, p38 MAPK was found to be involved since the knockdown of p38δ but not other isoforms showed partial reduction in CCL5 induction. This was further confirmed at transcriptional level that AP-1, C/EBPα and C/EBPγ were involved in CCL5 up-regulation.

Human immunodeficiency virus type 1 viral protein R (Vpr) induces CCL5 expression in astrocytes via PI3K and MAPK signaling pathways

BackgroundNeurocognitive impairments remain prevalent in HIV-1 infected individuals despite current antiretroviral therapies. It is increasingly becoming evident that astrocytes play a critical role in HIV-1 neuropathogenesis through the production of proinflammatory cytokines/chemokines. HIV-1 viral protein R (Vpr) plays an important role in neuronal dysfunction; however, its role in neuroinflammation is not well characterized. The major objective of this study was to determine the effect of Vpr in induction of proinflammatory chemokine CCL5 in astrocytes and to define the underlying mechanism(s).MethodsSVGA astrocytes were either mock transfected or were transfected with a plasmid encoding HIV-1 Vpr, and the cells were harvested at different time intervals. The mRNA level of CCL5 expression was quantified using real-time RT-PCR, and cell culture supernatants were assayed for CCL5 protein concentration. Immunocytochemistry was performed on HIV-1 Vpr transfected astrocytes to check CCL5 expression. Various signaling mechanisms such as p38 MAPK, PI3K/Akt, NF-κB and AP-1 were explored using specific chemical inhibitors and siRNAs.ResultsHIV-1 Vpr transfected astrocytes exhibited time-dependent induction of CCL5 as compared to mock-transfected astrocytes at both the mRNA and protein level. Immunostained images of astrocytes transfected with HIV-1 Vpr also showed much higher accumulation of CCL5 in comparison to untransfected and mock-transfected astrocytes. Pre-treatment with NF-κB (SC514) and PI3K/Akt (LY294002) inhibitor partially abrogated CCL5 mRNA and protein expression levels as opposed to untreated controls after HIV-1 Vpr transfection. Specific siRNAs against p50 and p65 subunits of NF-κB, p38δ MAPK, Akt-2 and Akt-3, and AP-1 transcription factor substantially inhibited the production of CCL5 in HIV-1 Vpr transfected astrocytes.ConclusionThese results demonstrate the ability of HIV-1 Vpr to induce CCL5 in astrocytes in a time-dependent manner. Furthermore, this effect was observed to be mediated by transcription factors NF-κB and AP-1 and involved the p38-MAPK and PI3K/Akt pathway.

Morphine and Rapid Disease Progression in Nonhuman Primate Model of AIDS: Inverse Correlation Between Disease Progression and Virus Evolution

HIV and simian immunodeficiency virus (SIV) have a formidable capacity for mutation and adaptation, a characteristic that has contributed to the extensive genetic variability. Evolutionary pressures imposed within the host and the viral capacity to mutate lead to the generation of such variants. To date, very little information is available regarding the evolution of HIV with drug abuse as a cofounding factor. Using our macaque model of drug dependency and AIDS, we have investigated the dynamics of SIV mutations in the genes tat, vpr, envelope, and nef. The results presented in this review, from our laboratory and others, contribute to the overall understanding of how drugs of abuse might influence immune selective pressure contribution to variation in different SIV genes. Additionally, the studies presented could help enlighten the development of HIV vaccines that take into consideration viral diversity.

Glutamate metabolism and HIV-associated neurocognitive disorders

HIV-1 infection can lead to neurocognitive impairment collectively known as HIV-associated neurocognitive disorders (HAND). Although combined antiretroviral treatment (cART) has significantly ameliorated HIV’s morbidity and mortality, persistent neuroinflammation and neurocognitive dysfunction continue. This review focuses on the current clinical and molecular evidence of the viral and host factors that influence glutamate-mediated neurotoxicity and neuropathogenesis as an important underlying mechanism during the course of HAND development. In addition, discusses potential pharmacological strategies targeting the glutamatergic system that may help prevent and improve neurological outcomes in HIV-1-infected subjects.

Opiates, immune system, acquired immunodeficiency syndrome, and nonhuman primate model.

Both human immunodeficiency virus (HIV) and illicit drug addiction remain major health problems not only in the United States but all over globe. The effect of drug addiction on HIV/AIDS (acquired immunodeficiency syndrome) has been somewhat underexplored. However, in United States more than one fourth of HIV-positive individuals are injection drug users. Opiates are known to negatively affect the immune system, and therefore may have deleterious effects on progression of disease among HIV-infected individuals. This review discusses the effects of opiates on immune system as well as its effect on HIV replication and AIDS progression. In addition, the effects of opiates on disease progression in non-human primate model of AIDS is presented with at least one possible reason for rapid disease progression in multi-virus the challenge model.

Compartmentalization of drug resistance-associated mutations in a treatment-naive HIV-infected female

Development of a drug-resistant variant of HIV-1 has been one of the major concerns contributing to the transmission of the virus. A 40-year-old woman presented to the clinic with micosis and oral candidiasis. The subject was referred for HIV-1 diagnosis. Subsequent investigations revealed a very low CD4 T cell count (48 cell/microl blood) and high plasma HIV-1 RNA load (4.33 x 10(5) copy/ml). A 1.3-kb pol fragment was sequenced in virus collected from plasma and the vaginal compartment. Plasma virus had no mutation in reverse transcriptase and one mutation in protease (L63P). On the other hand vaginal virus contained L63P and M184V mutations in protease and reverse transcriptase, respectively. These mutations were accompanied by several other mutations in previously identified CTL epitopic regions of the two genes. In the absence of antiretroviral treatment, a drug-resistant mutant was thought to develop because of immune pressure. This is the first report describing the role of immune pressure in the development of a drug-resistant virus.

Astrocytic expression of HIV-1 viral protein R in the hippocampus causes chromatolysis, synaptic loss and memory impairment

BackgroundHIV-infected individuals are at an increased risk of developing neurological abnormalities. HIV induces neurotoxicity by host cellular factors and individual viral proteins. Some of these proteins including viral protein R (Vpr) promote immune activation and neuronal damage. Vpr is known to contribute to cell death of cultured rat hippocampal neurons and suppresses axonal growth. Behavioral studies are limited and suggest hyperactivity in the presence of Vpr. Thus Vpr may play a role in hippocampal loss of function. The purpose of this study is to determine the ability of HIV-1 Vpr production by astrocytes in the hippocampus to cause neurological deficits and memory impairments.MethodsWe tested the performance of rats in novel object and novel location tasks after hippocampal infusion with astrocytes expressing HIV-1 Vpr. Synaptic injury and morphological changes were measured by synaptophysin immunoreactivity and Nissl staining.ResultsVpr-infused rats showed impaired novel location and novel object recognition compared with control rats expressing green fluorescent protein (GFP). This impairment was correlated with a significant decrease in synaptophysin immunoreactivity in the hippocampal CA3 region, suggesting synaptic injury in HIV-1 Vpr-treated animals. In addition, Nissl staining showed morphological changes indicative of neuronal chromatolysis in the Vpr group. The Vpr-induced neuronal damage and synaptic loss suggest that neuronal dysfunction caused the spatial and recognition memory deficits found in the Vpr-infused animals.ConclusionsIn this study, we demonstrate that HIV-1 Vpr produced by astrocytes in the hippocampus impairs hippocampal-dependent learning. The data suggest Vpr is a neurotoxin with the potential to cause learning impairment in HIV-1 infected individuals even under conditions of limited viral replication.