scholarly journals Expression of MDM2 in Macrophages Promotes the Early Postentry Steps of HIV-1 Infection through Inhibition of p53

2019 ◽  
Vol 93 (7) ◽  
Author(s):  
Yann Breton ◽  
Vincent Desrosiers ◽  
Michel Ouellet ◽  
Alexandre Deshiere ◽  
Cynthia Torresilla ◽  
...  
Keyword(s):  
Infected Individual ◽  
Human Monocyte ◽  
Careful Analysis ◽  
Binding Activity ◽  
Individual Identification ◽  
Small Interfering Rnas ◽  
Viral Reservoirs ◽  
Cellular Environment ◽  
Hiv 1

ABSTRACT The molecular basis for HIV-1 susceptibility in primary human monocyte-derived macrophages (MDMs) was previously evaluated by comparing the transcriptome of infected and bystander populations. Careful analysis of the data suggested that the ubiquitin ligase MDM2 acted as a positive regulator of HIV-1 replication in MDMs. In this study, MDM2 silencing through transcript-specific small interfering RNAs in MDMs induced a reduction in HIV-1 reverse transcription and integration along with an increase in the expression of p53-induced genes, including CDKN1A. Experiments with Nutlin-3, a pharmacological inhibitor of MDM2 p53-binding activity, showed a similar effect on HIV-1 infection, suggesting that the observed restriction in HIV-1 production results from the release/activation of p53 and not the absence of MDM2 per se. Knockdown and inhibition of MDM2 also both correlate with a decrease in the Thr592-phosphorylated inactive form of SAMHD1. The expression level of MDM2 and the p53 activation status are therefore important factors in the overall susceptibility of macrophages to HIV-1 infection, bringing a new understanding of signaling events controlling the process of virus replication in this cell type. IMPORTANCE Macrophages, with their long life span in vivo and their resistance to HIV-1-mediated cytopathic effect, might serve as viral reservoirs, contributing to virus persistence in an infected individual. Identification of host factors that increase the overall susceptibility of macrophages to HIV-1 might provide new therapeutic targets for the efficient control of viral replication in these cells and limit the formation of reservoirs in exposed individuals. In this study, we demonstrate the importance of p53 regulation by MDM2, which creates a cellular environment more favorable to the early steps of HIV-1 replication. Moreover, we show that p53 stabilization reduces virus infection in human macrophages, highlighting the important role of p53 in antiviral immunity.

HIV-1 Nef activates STAT1 in human monocytes/macrophages through the release of soluble factors

Blood ◽  
2001 ◽  
Vol 98 (9) ◽  
pp. 2752-2761 ◽  
Author(s):  
Maurizio Federico ◽  
Zulema Percario ◽  
Eleonora Olivetta ◽  
Gianna Fiorucci ◽  
Claudia Muratori ◽  
...  
Keyword(s):  
Hiv Infection ◽  
Intracellular Signaling ◽  
Human Monocyte ◽  
Binding Activity ◽  
Human Monocytes ◽  
Soluble Factors ◽  
In Vivo Models ◽  
Wide Range ◽  
Hiv 1

AbstractMonocytes/macrophages play a predominant role in the immunologic network by secreting and reacting to a wide range of soluble factors. Human immunodeficiency virus (HIV) infection leads to deep immunologic dysfunctions, also as a consequence of alterations in the pattern of cytokine release. Recent studies on in vivo models demonstrated that the expression of HIV Nef alone mimics many pathogenetic effects of HIV infection. In particular, Nef expression in monocytes/macrophages has been correlated with remarkable modifications in the pattern of secreted soluble factors, suggesting that the interaction of Nef with monocytes/macrophages plays a role in the pathogenesis of acquired immunodeficiency syndrome (AIDS). This study sought to define possible alterations in intracellular signaling induced by Nef in monocytes/macrophages. Results demonstrate that HIV-1 Nef specifically activates both α and β isoforms of the signal transducer and activator of transcription 1 (STAT1). This was observed both by infecting human monocyte-derived macrophages (MDMs) with HIV-1 deletion mutants, and by exploiting the ability of MDMs to internalize soluble, recombinant Nef protein (rNef). STAT1-α activation occurs on phosphorylation of both C-terminal Tyr701 and Ser727 and leads to a strong binding activity. Nef-dependent STAT1 activation is followed by increased expression of both STAT1 and interferon regulatory factor-1, a transcription factor transcriptionally regulated by STAT1 activation. It was also established that Nef-induced STAT1- α/β activation occurs through the secretion of soluble factors. Taken together, the results indicate that HIV-1 Nef could interfere with STAT1-governed intracellular signaling in human monocytes/macrophages.

scholarly journals An ultrasensitive planar array p24 Gag ELISA to detect individual HIV-1 viral particles and infected cells

2021 ◽  
Author(s):  
Alberto Bosque ◽  
Callie Levinger ◽  
J Natalie Howard ◽  
Pingtao Tang ◽  
Amit Joshi
Keyword(s):  
Biological Fluids ◽  
Low Frequency ◽  
Viral Reservoirs ◽  
Hiv Persistence ◽  
Viral Particles ◽  
Planar Array ◽  
Infected Cells ◽  
Major Loss ◽  
Hiv 1

Abstract Human Immunodeficiency virus-1 (HIV-1) persistence in the presence of antiretroviral therapy (ART) has halted the development of curative strategies. Measuring HIV persistence is complex due to the low frequency of cells containing virus in vivo. Most of the commercially available assays to date measure nucleic acid. These assays have the advantage of being highly sensitive and allow for the analysis of sequence diversity, intactness of the HIV genome or evaluation of diverse RNA species. However, these assays are limited in evaluating translational competent viral reservoirs. In here, we developed an ultrasensitive p24 ELISA that uses the SimoaTM planar array technology that can detect as low as a single HIV-1 particle and a single HIV-1 infected cell. Furthermore, the assay is optimized to measure very low levels of p24 in different biological fluids without a major loss of sensitivity or reproducibility. Our results demonstrate that the ‘homebrew' planar p24 ELISA immunoassay is a broadly applicable new tool to evaluate HIV persistence in diverse biological fluids.

scholarly journals HIV-1 can infect northern pig-tailed macaques ( Macaca leonina ) and form viral reservoirs in vivo

2017 ◽  
Vol 62 (19) ◽  
pp. 1315-1324 ◽  
Author(s):  
Wei Pang ◽  
Gao-Hong Zhang ◽  
Jin Jiang ◽  
Hong-Yi Zheng ◽  
Lin-Tao Zhang ◽  
...  
Keyword(s):  
Viral Reservoirs ◽  
Macaca Leonina ◽  
Hiv 1

scholarly journals HIV-1 and microglia: EcoHIV and HIV-1 transgenic rats

2020 ◽  
Author(s):  
Hailong Li ◽  
Kristen A. McLaurin ◽  
Jessica M. Illenberger ◽  
Charles F. Mactutus ◽  
Rosemarie M. Booze
Keyword(s):  
Distribution Pattern ◽  
Biological System ◽  
Cell Type ◽  
Viral Reservoirs ◽  
Major Cell Type ◽  
Key Aspects ◽  
The Brain ◽  
Hiv 1

ABSTRACTThe persistence of HIV-1 viral reservoirs in the brain, despite treatment with combination antiretroviral therapy (cART), remains a critical roadblock for the development of a novel cure strategy for HIV-1. To enhance our understanding of viral reservoirs, two complementary studies were conducted to 1) evaluate the HIV-1 mRNA neuroanatomical distribution pattern and major cell type expressing HIV-1 mRNA in the HIV-1 transgenic (Tg) rat (i.e., under conditions of latent infection), and 2) to validate our findings by developing and critically testing a novel biological system to model active HIV-1 infection in the rat. First, a restricted, region-specific HIV-1 mRNA distribution pattern was observed in the HIV-1 Tg rat. Microglia were the predominant cell type expressing HIV-1 mRNA in the HIV-1 Tg rat. Second, we developed and critically tested a novel biological system to model key aspects of HIV-1 by infusing F344/N control rats with chimeric HIV (EcoHIV). In vitro, primary cultured microglia were treated with EcoHIV revealing prominent expression within 24 hours of infection. In vivo, EcoHIV expression was observed seven days after stereotaxic injections. Following EcoHIV infection, microglia were the major cell type expressing HIV-1 mRNA, results which are consistent with observations in the HIV-1 Tg rat. Within eight weeks of infection, EcoHIV rats exhibited neurocognitive impairments, synaptic dysfunction, which may result from activation of the NogoA-NgR3/PirB-RhoA signaling pathway, and neuroinflammation. Collectively, these studies enhance our understanding of HIV-1 viral reservoirs in the brain and offer a novel biological system to model HIV-associated neurocognitive disorders and associated comorbidities (i.e., drug abuse) in rats.

scholarly journals Cytotoxicity assessment, inflammatory properties, and cellular uptake of Neutraplex lipid-based nanoparticles in THP-1 monocyte-derived macrophages

2017 ◽  
Vol 4 ◽  
pp. 184954351774625 ◽  
Author(s):  
Eric Berger ◽  
Dalibor Breznan ◽  
Sandra Stals ◽  
Viraj J Jasinghe ◽  
David Gonçalves ◽  
...  
Keyword(s):  
Targeted Delivery ◽  
Small Interfering Rna ◽  
Human Monocyte ◽  
Antiretroviral Drugs ◽  
Small Interfering Rnas ◽  
Lactate Dehydrogenase Release ◽  
Drug Concentrations ◽  
Interfering Rna ◽  
Hiv 1

Current antiretroviral drugs used to prevent or treat human immunodeficiency virus type 1 (HIV-1) infection are not able to eliminate the virus within tissues or cells where HIV establishes reservoirs. Hence, there is an urgent need to develop targeted delivery systems to enhance drug concentrations in these viral sanctuary sites. Macrophages are key players in HIV infection and contribute significantly to the cellular reservoirs of HIV because the virus can survive for prolonged periods in these cells. In the present work, we investigated the potential of the lipid-based Neutraplex nanosystem to deliver anti-HIV therapeutics in human macrophages using the human monocyte/macrophage cell line THP-1. Neutraplex nanoparticles as well as cationic and anionic Neutraplex nanolipoplexes (Neutraplex/small interfering RNA) were prepared and characterized by dynamic light scattering. Neutraplex nanoparticles showed low cytotoxicity in CellTiter-Blue reduction and lactate dehydrogenase release assays and were not found to have pro-inflammatory effects. In addition, confocal studies showed that the Neutraplex nanoparticles and nanolipoplexes are rapidly internalized into THP-1 macrophages and that they can escape the late endosome/lysosome compartment allowing the delivery of small interfering RNAs in the cytoplasm. Furthermore, HIV replication was inhibited in the in vitro TZM-bl infectivity assay when small interfering RNAs targeting CXCR4 co-receptor was delivered by Neutraplex nanoparticles compared to a random small interfering RNA sequence. This study demonstrates that the Neutraplex nanosystem has potential for further development as a delivery strategy to efficiently and safely enhance the transport of therapeutic molecules into human monocyte-derived macrophages in the aim of targeting HIV-1 in this cellular reservoir.

scholarly journals Protein Arginine N-methyltransferases 5 and 7 Promote HIV-1 Production

Viruses ◽  
2020 ◽  
Vol 12 (3) ◽  
pp. 355 ◽  
Author(s):  
Hironobu Murakami ◽  
Takehiro Suzuki ◽  
Kiyoto Tsuchiya ◽  
Hiroyuki Gatanaga ◽  
Manabu Taura ◽  
...  
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Virus Production ◽  
Human Monocyte ◽  
Wild Type ◽  
Viral Reservoirs ◽  
Immunodeficiency Virus ◽  
Current Therapies ◽  
Hiv 1

Current therapies for human immunodeficiency virus type 1 (HIV-1) do not completely eliminate viral reservoirs in cells, such as macrophages. The HIV-1 accessory protein viral protein R (Vpr) promotes virus production in macrophages, and the maintenance of Vpr is essential for HIV-1 replication in these reservoir cells. We identified two novel Vpr-binding proteins, i.e., protein arginine N-methyltransferases (PRMTs) 5 and 7, using human monocyte-derived macrophages (MDMs). Both proteins found to be important for prevention of Vpr degradation by the proteasome; in the context of PRMT5 and PRMT7 knockdowns, degradation of Vpr could be prevented using a proteasome inhibitor. In MDMs infected with a wild-type strain, knockdown of PRMT5/PRMT7 and low expression of PRMT5 resulted in inefficient virus production like Vpr-deficient strain infections. Thus, our findings suggest that PRMT5 and PRMT7 support HIV-1 replication via maintenance of Vpr protein stability.

scholarly journals Inhibition of Human Immunodeficiency Virus Type 1 Replication in Primary Macrophages by Using Tat- or CCR5-Specific Small Interfering RNAs Expressed from a Lentivirus Vector

2003 ◽  
Vol 77 (22) ◽  
pp. 11964-11972 ◽  
Author(s):  
Ming-Ta M. Lee ◽  
Glen A. Coburn ◽  
Myra O. McClure ◽  
Bryan R. Cullen
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Viral Vectors ◽  
Expression Vectors ◽  
Small Interfering Rnas ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Although several groups have demonstrated that RNA interference, induced by transfection of small interfering RNA (siRNA) duplexes, can protect cells against a viral challenge in culture, this protection is transient. Here, we describe lentivirus expression vectors that can stably express siRNAs at levels sufficient to block virus replication. We have used these vectors to stably express siRNAs specific for the essential human immunodeficiency virus type 1 (HIV-1) Tat transcription factor or specific for a cellular coreceptor, CCR5, that is required for infection by the majority of primary HIV-1 isolates. These lentivirus vectors are shown to protect cells, including primary macrophages, against HIV-1 infection in culture by inducing selective degradation of their target mRNA species. These data suggest that it should be possible to block the expression of specific viral or cellular genes in vivo by using viral vectors to stably express the appropriate siRNAs.

scholarly journals Sustained Small Interfering RNA-Mediated HumanImmunodeficiency Virus Type 1 Inhibition in PrimaryMacrophages

2003 ◽  
Vol 77 (13) ◽  
pp. 7174-7181 ◽  
Author(s):  
Erwei Song ◽  
Sang-Kyung Lee ◽  
Derek M. Dykxhoorn ◽  
Carl Novina ◽  
Dong Zhang ◽  
...  
Keyword(s):  
Virus Type ◽  
Small Interfering Rnas ◽  
Viral Inhibition ◽  
Hiv Replication ◽  
Dividing Cells ◽  
Viral Target ◽  
Interfering Rna ◽  
Hiv 1

ABSTRACT Small interfering RNAs (siRNAs) can induce potent gene silencing by degradation of cognate mRNA. However, in dividing cells, the silencing lasts only 3 to 7 days, presumably because of siRNA dilution with cell division. Here, we investigated if sustained siRNA-mediated silencing of human immunodeficiency virus type 1 (HIV-1) is possible in terminally differentiated macrophages, which constitute an important reservoir of HIV in vivo. CCR5, the major HIV-1 coreceptor in macrophages, and the viral structural gene for p24 were targeted either singly or in combination. When transfected 2 days prior to infection, both CCR5 and p24 siRNAs effectively reduced HIV-1 infection for the entire 15-day period of observation, and combined targeting of both genes abolished infection. To investigate whether exogenously introduced siRNA is maintained stably in macrophages, we tested the kinetics of siRNA-mediated viral inhibition by initiating infections at various times (2 to 15 days) after transfection with CCR5 and p24 siRNAs. HIV suppression mediated by viral p24 siRNA progressively decreased and was lost by day 7 posttransfection. In contrast, viral inhibition by cellular CCR5 knockdown was sustained even when transfection preceded infection by 15 days, suggesting that the continued presence of target RNA may be needed for persistence of siRNA. The longer sustenance of CCR5 relative to p24 siRNA in uninfected macrophages was also confirmed by detection of internalized siRNA by modified Northern blot analysis. We also tested the potential of p24 siRNA to stably silence HIV in the setting of an established infection where the viral target gene is actively transcribed. Under these circumstances, long-term suppression of HIV replication could be achieved with p24 siRNA. Thus, siRNAs can induce potent and long-lasting HIV inhibition in nondividing cells such as macrophages.

scholarly journals Microglial HIV-1 Expression: Role in HIV-1 Associated Neurocognitive Disorders

Viruses ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 924
Author(s):  
Hailong Li ◽  
Kristen A. McLaurin ◽  
Jessica M. Illenberger ◽  
Charles F. Mactutus ◽  
Rosemarie M. Booze
Keyword(s):  
Distribution Pattern ◽  
Biological System ◽  
Neurocognitive Disorders ◽  
Cell Type ◽  
Viral Reservoirs ◽  
Major Cell Type ◽  
The Brain ◽  
Hiv 1

The persistence of HIV-1 viral reservoirs in the brain, despite treatment with combination antiretroviral therapy (cART), remains a critical roadblock for the development of a novel cure strategy for HIV-1. To enhance our understanding of viral reservoirs, two complementary studies were conducted to (1) evaluate the HIV-1 mRNA distribution pattern and major cell type expressing HIV-1 mRNA in the HIV-1 transgenic (Tg) rat, and (2) validate our findings by developing and critically testing a novel biological system to model active HIV-1 infection in the rat. First, a restricted, region-specific HIV-1 mRNA distribution pattern was observed in the HIV-1 Tg rat. Microglia were the predominant cell type expressing HIV-1 mRNA in the HIV-1 Tg rat. Second, we developed and critically tested a novel biological system to model key aspects of HIV-1 by infusing F344/N control rats with chimeric HIV (EcoHIV). In vitro, primary cultured microglia were treated with EcoHIV revealing prominent expression within 24 h of infection. In vivo, EcoHIV expression was observed seven days after stereotaxic injections. Following EcoHIV infection, microglia were the major cell type expressing HIV-1 mRNA, results that are consistent with observations in the HIV-1 Tg rat. Within eight weeks of infection, EcoHIV rats exhibited neurocognitive impairments and synaptic dysfunction, which may result from activation of the NogoA-NgR3/PirB-RhoA signaling pathway and/or neuroinflammation. Collectively, these studies enhance our understanding of HIV-1 viral reservoirs in the brain and offer a novel biological system to model HIV-associated neurocognitive disorders and associated comorbidities (i.e., drug abuse) in rats.

scholarly journals An ultrasensitive planar array p24 Gag ELISA to detect HIV-1 in diverse biological matrixes

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Callie Levinger ◽  
JNatalie Howard ◽  
Jie Cheng ◽  
Pingtao Tang ◽  
Amit Joshi ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Limit Of Detection ◽  
Biological Fluids ◽  
Low Frequency ◽  
Viral Reservoirs ◽  
Hiv Persistence ◽  
Planar Array ◽  
Major Loss ◽  
Hiv 1

AbstractHuman immunodeficiency virus-1 (HIV-1) persistence in the presence of antiretroviral therapy (ART) has halted the development of curative strategies. Measuring HIV persistence is complex due to the low frequency of cells containing virus in vivo. Most of the commercially available assays to date measure nucleic acid. These assays have the advantage of being highly sensitive and allow for the analysis of sequence diversity, intactness of the HIV genome or evaluation of diverse RNA species. However, these assays are limited in evaluating translational competent viral reservoirs. In here, we developed an ultrasensitive p24 ELISA that uses the Simoa planar array technology that can detect HIV-1 virions and HIV-1 infected cell with limit of detection similar to nucleic acid assays. Furthermore, the assay is optimized to measure very low levels of p24 in different biological fluids without a major loss of sensitivity or reproducibility. Our results demonstrate that the ‘homebrew’ planar p24 ELISA immunoassay is a broadly applicable new tool to evaluate HIV persistence in diverse biological fluids and cells.

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