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Systems analysis of latent HIV reversal reveals altered stress kinase signaling and increased cell death in infected T cells.

ABSTRACT: Viral latency remains the most significant obstacle to HIV eradication. Clinical strategies aim to purge the latent CD4+ T cell reservoir by activating viral expression to induce death, but are undercut by the inability to target latently infected cells. Here we explored the acute signaling response of latent HIV-infected CD4+ T cells to identify dynamic phosphorylation signatures that could be targeted for therapy. Stimulation with CD3/CD28, PMA/ionomycin, or latency reversing agents prostratin and SAHA, yielded increased phosphorylation of I?B?, ERK, p38, and JNK in HIV-infected cells across two in vitro latency models. Both latent infection and viral protein expression contributed to changes in perturbation-induced signaling. Data-driven statistical models calculated from the phosphorylation signatures successfully classified infected and uninfected cells and further identified signals that were functionally important for regulating cell death. Specifically, the stress kinase pathways p38 and JNK were modified in latently infected cells, and activation of p38 and JNK signaling by anisomycin resulted in increased cell death independent of HIV reactivation. Our findings suggest that altered phosphorylation signatures in infected T cells provide a novel strategy to more selectively target the latent reservoir to enhance eradication efforts.

SUBMITTER: Fong LE

PROVIDER: S-EPMC5701066 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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Systems analysis of latent HIV reversal reveals altered stress kinase signaling and increased cell death in infected T cells.

Fong Linda E LE Sulistijo Endah S ES Miller-Jensen Kathryn K

Scientific reports 20171123 1

Viral latency remains the most significant obstacle to HIV eradication. Clinical strategies aim to purge the latent CD4+ T cell reservoir by activating viral expression to induce death, but are undercut by the inability to target latently infected cells. Here we explored the acute signaling response of latent HIV-infected CD4+ T cells to identify dynamic phosphorylation signatures that could be targeted for therapy. Stimulation with CD3/CD28, PMA/ionomycin, or latency reversing agents prostratin ...[more]

PMID: 29170390

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Project description:The underlying mechanisms for increased neurodegeneration and neurocognitive deficits in HIV-infected people are unclear. Therefore, this study was aimed to investigate the mechanisms of increased neurodegeneration in 5-month old male HIV-1 Transgenic (Tg) rats compared to the age- and gender-matched wild-type (WT) by evaluating histological changes and biochemical parameters of the key proteins involved in the cell death signaling and apoptosis. Histological and immunohistochemical analyses revealed decreased neuronal cells with elevated astrogliosis in HIV-1 Tg rats compared to WT. Mechanistic studies revealed that increased levels of nitroxidative stress marker proteins such as NADPH-oxidase, cytochrome P450-2E1 (CYP2E1), inducible nitric oxide synthase (iNOS), the stress-activated mitogen-activated protein kinases such as JNK and p38K, activated cell-cycle dependent CDK5, hypoxia-inducible protein-1?, nitrated proteins, hyperphosphorylated tau, and amyloid plaques in HIV-Tg rats were consistently observed in HIV-1 Tg rats. Confocal microscopy and cell viability analyses showed that treatment with an antioxidant N-acetylcysteine or a specific inhibitor of iNOS 1400W significantly prevented the increased apoptosis of neuro-2A cells by HIV-1 Tat or gp120 protein, demonstrating the causal role of HIV-1 mediated nitroxidative stress and protein nitration in promoting neuronal cell death. Immunoprecipitation and immunoblot analysis confirmed nitration of Hsp90, evaluated as an example of nitrated proteins, suggesting possible involvement of nitrated proteins in neuronal damage. Further, activated p-JNK directly binds tau and phosphorylates multiple amino acids, suggesting an important role of p-JNK in tau hyperphosphorylation and tauopathy. These changes were accompanied with elevated levels of many apoptosis-related proteins Bax and cleaved (activated) caspase-3 as well as proinflammatory cytokines including TNF-?, IL-6 and MCP-1. Collectively, these results indicate that raised nitroxidative stress accompanied by elevated inflammation, cell death signaling pathway including activated p-JNK, C-terminal C99 amyloid fragment formation and tau hyperphosphorylation are responsible for increased apoptosis of neuronal cells and neurodegeneration in 5-month old HIV-Tg rats.

Project description:In vitro, disulfiram activated HIV transcription in a primary T-cell model of HIV latency and in a pilot clinical study increased plasma HIV RNA in individuals with adequate drug exposure. We assessed the effect of disulfiram on HIV transcription in a dose-escalation study.In this prospective dose-escalation study, to optimise disulfiram exposure we included adults with HIV on suppressive antiretroviral therapy, with plasma HIV RNA of less than 50 copies per mL and a CD4 cell count greater than 350 cells per ?L. Participants were allocated sequentially to one of three dosing groups (500 mg, 1000 mg, and 2000 mg) and received disulfiram daily for 3 days. Only the staff who did laboratory assays were masked to group assignment. The primary endpoint was change in cell-associated unspliced HIV RNA in CD4 cells. The primary analysis method was a negative binomial regression, with the number of copies as the outcome variable and the input total RNA or plasma volume as an exposure variable, which is equivalent to modelling copies or input. We used these models to estimate changes from before disulfiram to timepoints during and after disulfiram administration. This study is registered with ClinicalTrials.gov, number NCT01944371.Of 34 participants screened for eligibility at The Alfred Hospital (Melbourne, VIC, Australia), and San Francisco General Hospital (San Francisco, CA, USA), 30 people were enrolled between Sept 24, 2013, and March 31, 2014. The estimated fold increases in cell-associated unspliced HIV RNA from baseline were 1·7 (95% CI 1·3-2·2; p<0·0001) to the timepoint during disulfiram treatment and 2·1 (1·5-2·9; p<0·0001) to the timepoint after disulfiram in the 500 mg group; 1·9 (1·6-2·4; p<0·0001) and 2·5 (1·9-3·3; p<0·0001) in the 1000 mg group; and 1·6 (1·2-2·1; p=0·0026) and 2·1 (1·5-3·1; p=0·0001) in the 2000 mg group. No deaths occurred, and no serious adverse events were noted. Disulfiram was well tolerated at all doses.Short-term administration of disulfiram resulted in increases in cell-associated unspliced HIV RNA at all doses, consistent with activating HIV latency. Disulfiram may be suited for future studies of combination and prolonged therapy to activate latent HIV.The Foundation for AIDS Research (amfAR); National Institute of Allergy and Infectious Diseases, National Institutes of Health; Australian National Health and Medical Research Council.

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Systems analysis of latent HIV reversal reveals altered stress kinase signaling and increased cell death in infected T cells.

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Systems analysis of latent HIV reversal reveals altered stress kinase signaling and increased cell death in infected T cells.

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