Sustained Induction of NF- B Is Required for Efficient Expression of Latent Human Immunodeficiency Virus Type 1

Gladstone Institute of Virology and Immunology, University of California San Francisco, 1650 Owens Street, San Francisco, CA 94158, USA.
Journal of Virology (Impact Factor: 4.44). 07/2007; 81(11):6043-56. DOI: 10.1128/JVI.02074-06
Source: PubMed


Cells harboring infectious, but transcriptionally latent, human immunodeficiency virus type 1 (HIV-1) proviruses currently pose an insurmountable barrier to viral eradication in infected patients. To better understand the molecular basis for HIV-1 latency, we used the J-Lat model of postintegration HIV-1 latency to assess the kinetic relationship between the induction of NF-kappaB and the activation of latent HIV-1 gene expression. Chromatin immunoprecipitation analyses revealed an oscillating pattern of RelA recruitment to the HIV-1 long terminal repeat (LTR) during continuous tumor necrosis factor alpha (TNF-alpha) stimulation. RNA polymerase II (Pol II) recruitment to the HIV-1 LTR closely mirrored RelA binding. Transient stimulation of cells with TNF-alpha for 15 min induced only a single round of RelA and RNA Pol II binding and failed to induce robust expression of latent HIV-1. Efficient formation of elongated HIV-1 transcripts required sustained induction by NF-kappaB, which promoted de novo synthesis of Tat. Cyclin-dependent kinase 9 (CDK9) and serine-2-phosphorylated RNA Pol II were rapidly recruited to the HIV-1 LTR after NF-kappaB induction; however, these elongating polymerase complexes were progressively dephosphorylated in the absence of Tat. Okadaic acid promoted sustained serine-2 phosphorylation of the C-terminal domain of RNA Pol II and stimulated efficient transcriptional elongation and HIV-1 expression in the absence of Tat. These findings underscore important differences between NF-kappaB and Tat stimulation of RNA Pol II elongation. While NF-kappaB binding to the HIV-1 LTR induces serial waves of efficient RNA Pol II initiation, elongation is impaired by the action of an okadaic acid-sensitive phosphatase that dephosphorylates the C-terminal domain of RNA Pol II. Conversely, the action of this phosphatase is overcome in the presence of Tat, promoting very efficient RNA Pol II elongation.

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    • "One of the major factors that determine HIV-1 latency, i.e. silencing of integrated viral genome, is down regulation or repression of HIV-1 transcription. Suppressed expression of Tat, multiple transformations of a positive transcription elongation factor b (P-TEFb) (Bisgrove et al., 2007; Chen et al., 2004; Hargreaves et al., 2009; Yang et al., 2005), as well as such epigenetic mechanisms as deacetylation of lysine residues in LTR-bound histones by histone deacetylases (HDAC) (Tyagi et al., 2010), methylation of histone H3 (Pearson et al., 2008) and direct methylation of cytosine residues in two CpG islands flanking HIV-1 transcription start site (Kauder et al., 2009) are critical factors of the repression of LTR transcription (Furia et al., 2002; Williams et al., 2007). Thus, the activation of latent HIV-1 proviral genomes in the infected cells, that results in viral replication is critical, since the virus itself as well as the host immune response in combination with antiretroviral drugs ( " shock and kill " strategy) may eliminate the virus following reactivation. "
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    ABSTRACT: The highly active antiretroviral therapy reduces HIV-1 RNA in plasma to undetectable levels. However, the virus continues to persist in the long-lived resting CD4(+) T cells, macrophages and astrocytes which form a viral reservoir in infected individuals. Reactivation of viral transcription is critical since the host immune response in combination with antiretroviral therapy may eradicate the virus. Using the chronically HIV-1 infected T lymphoblastoid and monocytic cell lines, primary quiescent CD4(+) T cells and humanized mice infected with dual-tropic HIV-1 89.6, we examined the effect of various X-ray irradiation (IR) doses (used for HIV-related lymphoma treatment and lower doses) on HIV-1 transcription and viability of infected cells. Treatment of both T cells and monocytes with IR, a well-defined stress signal, led to increase of HIV-1 transcription, as evidenced by the presence of RNA polymerase II and reduction of HDAC1 and methyl transferase SUV39H1 on the HIV-1 promoter. This correlated with the increased GFP signal and elevated level of intracellular HIV-1 RNA in the IR-treated quiescent CD4(+) T cells infected with GFP-encoding HIV-1. Exposition of latently HIV-1infected monocytes treated with PKC agonist bryostatin 1 to IR enhanced transcription activation effect of this latency-reversing agent. Increased HIV-1 replication after IR correlated with higher cell death: the level of phosphorylated Ser46 in p53, responsible for apoptosis induction, was markedly higher in the HIV-1 infected cells following IR treatment. Exposure of HIV-1 infected humanized mice with undetectable viral RNA level to IR resulted in a significant increase of HIV-1 RNA in plasma, lung and brain tissues. Collectively, these data point to the use of low to moderate dose of IR alone or in combination with HIV-1 transcription activators as a potential application for the "Shock and Kill" strategy for latently HIV-1 infected cells. Copyright © 2015 Elsevier Inc. All rights reserved.
    Full-text · Article · Jul 2015 · Virology
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    • "The p50/p65 dimers initiate HIV-1 transcription by associating with p300, thereby increasing the accessibility of the LTR for the cellular RNA polymerase II (RNAPII) (Williams et al., 2006). Furthermore, p50/p65 dimers recruit the P-TEFb complex to increase the processivity of RNAPII and to support RNA elongation (Williams et al., 2007). "
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    ABSTRACT: NF-κB is essential for effective transcription of primate lentiviral genomes and also activates antiviral host genes. Here, we show that the early protein Nef of most primate lentiviruses enhances NF-κB activation. In contrast, the late protein Vpu of HIV-1 and its simian precursors inhibits activation of NF-κB, even in the presence of Nef. Although this effect of Vpu did not correlate with its ability to interact with β-TrCP, it involved the stabilization of IκB and reduced nuclear translocation of p65. Interestingly, however, Vpu did not affect casein kinase II-mediated phosphorylation of p65. Lack of Vpu was associated with increased NF-κB activation and induction of interferon and interferon-stimulated genes (ISGs) in HIV-1-infected T cells. Thus, HIV-1 and its simian precursors employ Nef to boost NF-κB activation early during the viral life cycle to initiate proviral transcription, while Vpu is used to downmodulate NF-κB-dependent expression of ISGs at later stages. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Full-text · Article · Jan 2015 · Cell Reports
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    • "The above findings further prompted us to look at the redox sensitive NF-κB expression [31], [32], which on activation translocate into the nucleus [33], where it binds to the Long Terminal Repeats [11] of the integrated HIV-1 provirus and induces its replication [34], [35], [36]. Interestingly, we observed that the presence of Mtb infection in HIV-1 individuals significantly increased NF-κB expression, thereby making the conditions favorable for HIV-1 replication. "
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    ABSTRACT: Understanding of the chronic immune activation, breakdown of immune defense and synergistic effect between HIV and Mycobacterium tuberculosis (Mtb) may provide essential information regarding key factors involved in the pathogenesis of HIV disease. In this study, we aimed to highlight a few of the immunological events that may influence and accelerate the progression of HIV disease in the presence of co-infecting Mtb. A cross-sectional study was performed on cohorts, including anti-tubercular therapy (ATT) naïve active pulmonary tuberculosis (PTB) patients, antiretroviral therapy (ART) naïve HIV-1 infected individuals at different stages of disease, ATT and ART naïve HIV-PTB co-infected individuals and healthy controls. A significantly higher T-regulatory cell (Treg) frequency coupled with the high FoxP3 expression in the CD4 T-cells indicated an immunosuppressive environment in the advance stage of HIV-1 infection. This is further substantiated by high HO-1 expression favoring TB co-infection. Functionally, this change in Treg frequency in HIV-1 infected individuals correlated well with suppression of T-cell proliferation. Mtb infection seems to facilitate the expansion of the Treg pool along with increased expression of FoxP3, specifically the variant-1, as evident from the data in HIV-1 co-infected as well as in patients with only PTB. A significantly lower expression of HO-1 in co-infected individuals compared to patients with only HIV-infection having comparable CD4 count correlated well with increased expression of CCR5 and CxCR4 as well as NF-κB and inflammatory cytokines IL-6 and TNF-α, which collectively may contribute to enhanced viral replication and increased cell death, hence faster disease progression in co-infected individuals.
    Full-text · Article · Sep 2014 · PLoS ONE
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