35 research outputs found

    Mechanisms of HIV Transcriptional Regulation and Their Contribution to Latency

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    Long-lived latent HIV-infected cells lead to the rebound of virus replication following antiretroviral treatment interruption and present a major barrier to eliminating HIV infection. These latent reservoirs, which include quiescent memory T cells and tissue-resident macrophages, represent a subset of cells with decreased or inactive proviral transcription. HIV proviral transcription is regulated at multiple levels including transcription initiation, polymerase recruitment, transcription elongation, and chromatin organization. How these biochemical processes are coordinated and their potential role in repressing HIV transcription along with establishing and maintaining latency are reviewed

    Interleukin 2-inducible T cell kinase (ITK) facilitates efficient egress of HIV-1 by coordinating Gag distribution and actin organization

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    AbstractInterleukin 2-inducible T cell kinase (ITK) influences T cell signaling by coordinating actin polymerization and polarization as well as recruitment of kinases and adapter proteins. ITK regulates multiple steps of HIV-1 replication, including virion assembly and release. Fluorescent microscopy was used to examine the functional interactions between ITK and HIV-1 Gag during viral particle release. ITK and Gag colocalized at the plasma membrane and were concentrated at sites of F-actin accumulation and membrane lipid rafts in HIV-1 infected T cells. There was polarized staining of ITK, Gag, and actin towards sites of T cell conjugates. Small molecule inhibitors of ITK disrupted F-actin capping, perturbed Gag-ITK colocalization, inhibited virus like particle release, and reduced HIV replication in primary human CD4+ T cells. These data provide insight as to how ITK influences HIV-1 replication and suggest that targeting host factors that regulate HIV-1 egress provides an innovative strategy for controlling HIV infection

    Quantifying Lipid Contents in Enveloped Virus Particles with Plasmonic Nanoparticles

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    Phosphatidylserine (PS) and monosialotetrahexosylganglioside (GM1) are examples of two host-derived lipids in the membrane of enveloped virus particles that are known to contribute to virus attachment, uptake, and ultimately dissemination. A quantitative characterization of their contribution to the functionality of the virus requires information about their relative concentrations in the viral membrane. Here, a gold nanoparticle (NP) binding assay for probing relative PS and GM1 lipid concentrations in the outer leaflet of different HIV-1 and Ebola virus-like particles (VLPs) using sample sizes of less than 3 × 106 particles is introduced. The assay evaluates both scattering intensity and resonance wavelength, and determines relative NP densities through plasmon coupling as a measure for the target lipid concentrations in the NP-labeled VLP membrane. A correlation of the optical observables with absolute lipid contents is achieved by calibration of the plasmon coupling-based methodology with unilamellar liposomes of known PS or GM1 concentration. The performed studies reveal significant differences in the membrane of VLPs that assemble at different intracellular sites and pave the way to an optical quantification of lipid concentration in virus particles at physiological titers.NIH grants RO1CA138509 (B.M.R.), RO1A1064099 (S. G., and 1R56Al104393 (B.M.R. and S. G.; Ethan Edmonds support (CHE 1156666

    Strength of T cell signaling regulates HIV-1 replication and establishment of latency.

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    A major barrier to curing HIV-1 is the long-lived latent reservoir that supports re-emergence of HIV-1 upon treatment interruption. Targeting this reservoir will require mechanistic insights into the establishment and maintenance of HIV-1 latency. Whether T cell signaling at the time of HIV-1 infection influences productive replication or latency is not fully understood. We used a panel of chimeric antigen receptors (CARs) with different ligand binding affinities to induce a range of signaling strengths to model differential T cell receptor signaling at the time of HIV-1 infection. Stimulation of T cell lines or primary CD4+ T cells expressing chimeric antigen receptors supported HIV-1 infection regardless of affinity for ligand; however, only signaling by the highest affinity receptor facilitated HIV-1 expression. Activation of chimeric antigen receptors that had intermediate and low binding affinities did not support provirus transcription, suggesting that a minimal signal is required for optimal HIV-1 expression. In addition, strong signaling at the time of infection produced a latent population that was readily inducible, whereas latent cells generated in response to weaker signals were not easily reversed. Chromatin immunoprecipitation showed HIV-1 transcription was limited by transcriptional elongation and that robust signaling decreased the presence of negative elongation factor, a pausing factor, by more than 80%. These studies demonstrate that T cell signaling influences HIV-1 infection and the establishment of different subsets of latently infected cells, which may have implications for targeting the HIV-1 reservoir

    Identification of benzazole compounds that induce HIV-1 transcription.

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    Despite advances in antiretroviral therapy, HIV-1 infection remains incurable in patients and continues to present a significant public health burden worldwide. While a number of factors contribute to persistent HIV-1 infection in patients, the presence of a stable, long-lived reservoir of latent provirus represents a significant hurdle in realizing an effective cure. One potential strategy to eliminate HIV-1 reservoirs in patients is reactivation of latent provirus with latency reversing agents in combination with antiretroviral therapy, a strategy termed "shock and kill". This strategy has shown limited clinical effectiveness thus far, potentially due to limitations of the few therapeutics currently available. We have identified a novel class of benzazole compounds effective at inducing HIV-1 expression in several cellular models. These compounds do not act via histone deacetylase inhibition or T cell activation, and show specificity in activating HIV-1 in vitro. Initial exploration of structure-activity relationships and pharmaceutical properties indicates that these compounds represent a potential scaffold for development of more potent HIV-1 latency reversing agents

    Induction of HIV-1 expressing cells following treatment with latency reversing agents.

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    <p>ACH-2 cells were not treated or treated with a final concentration of 30 μM Cmpd 1 or Cmpd 2, 10 μM SAHA, 1 mM JQ1 or 10 ng/ml PMA for 16 h. Cells were stained using anti-HIV-Gag-PE and analyzed by flow cytometry. Numbers within the profiles represent the percentage of positive cells. These profiles are from a single experiment. B) Data from four independent experiments. Y-axis is mean of % p24 positive cells. * Cmpd 1 p-value >0.005 compared to other treatments using a two-tailed t-test.</p
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