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Selective cell death in HIV-1-infected cells by DDX3 inhibitors leads to depletion of the inducible reservoir.

ABSTRACT: An innovative approach to eliminate HIV-1-infected cells emerging out of latency, the major hurdle to HIV-1 cure, is to pharmacologically reactivate viral expression and concomitantly trigger intracellular pro-apoptotic pathways in order to selectively induce cell death (ICD) of infected cells, without reliance on the extracellular immune system. In this work, we demonstrate the effect of DDX3 inhibitors on selectively inducing cell death in latent HIV-1-infected cell lines, primary CD4+ T cells and in CD4+ T cells from cART-suppressed people living with HIV-1 (PLWHIV). We used single-cell FISH-Flow technology to characterise the contribution of viral RNA to inducing cell death. The pharmacological targeting of DDX3 induced HIV-1 RNA expression, resulting in phosphorylation of IRF3 and upregulation of IFNβ. DDX3 inhibition also resulted in the downregulation of BIRC5, critical to cell survival during HIV-1 infection, and selectively induced apoptosis in viral RNA-expressing CD4+ T cells but not bystander cells. DDX3 inhibitor treatment of CD4+ T cells from PLWHIV resulted in an approximately 50% reduction of the inducible latent HIV-1 reservoir by quantitation of HIV-1 RNA, by FISH-Flow, RT-qPCR and TILDA. This study provides proof of concept for pharmacological reversal of latency coupled to induction of apoptosis towards the elimination of the inducible reservoir.

SUBMITTER: Rao S

PROVIDER: S-EPMC8087668 | biostudies-literature | 2021 Apr

REPOSITORIES: biostudies-literature

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Selective cell death in HIV-1-infected cells by DDX3 inhibitors leads to depletion of the inducible reservoir.

Rao Shringar S Lungu Cynthia C Crespo Raquel R Steijaert Thijs H TH Gorska Alicja A Palstra Robert-Jan RJ Prins Henrieke A B HAB van Ijcken Wilfred W Mueller Yvonne M YM van Kampen Jeroen J A JJA Verbon Annelies A Katsikis Peter D PD Boucher Charles A B CAB Rokx Casper C Gruters Rob A RA Mahmoudi Tokameh T

Nature communications 20210430 1

An innovative approach to eliminate HIV-1-infected cells emerging out of latency, the major hurdle to HIV-1 cure, is to pharmacologically reactivate viral expression and concomitantly trigger intracellular pro-apoptotic pathways in order to selectively induce cell death (ICD) of infected cells, without reliance on the extracellular immune system. In this work, we demonstrate the effect of DDX3 inhibitors on selectively inducing cell death in latent HIV-1-infected cell lines, primary CD4+ T cells ...[more]

PMID: 33931637

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Project description:Quantifying latently infected cells is critical to evaluate the efficacy of therapeutic strategies aimed at reducing the size of the long-lived viral reservoir, but the low frequency of these cells makes this very challenging.We developed TILDA (Tat/rev Induced Limiting Dilution Assay) to measure the frequency of cells with inducible multiply-spliced HIV RNA, as these transcripts are usually absent in latently infected cells but induced upon viral reactivation. TILDA requires less than a million cells, does not require RNA extraction and can be completed in two days.In suppressed individuals on ART, we found the median frequency of latently infected CD4 + T cells as estimated by TILDA to be 24 cells/million, which was 48 times more than the frequency measured by the quantitative viral outgrowth assay, and 6-27 times less than the frequencies of cells harbouring viral DNA measured by PCR-based assays. TILDA measurements strongly correlated with most HIV DNA assays. The size of the latent reservoir measured by TILDA was lower in subjects who initiated ART during the early compared to late stage of infection (p = 0.011). In untreated HIV disease, the frequency of CD4 + cells carrying latent but inducible HIV largely exceeded the frequency of actively producing cells, demonstrating that the majority of infected cells are transcriptionally silent even in the absence of ART.Our results suggest that TILDA is a reproducible and sensitive approach to measure the frequency of productively and latently infected cells in clinical settings. We demonstrate that the latent reservoir represents a substantial fraction of all infected cells prior to ART initiation.In this manuscript, we describe the development of a novel assay that measures the magnitude of the latent HIV reservoir, the main barrier to HIV eradication. This novel assay, termed TILDA for Tat/rev Induced Limiting Dilution Assay, requires only 10 ml of blood, does not necessitate extraction of viral nucleic acids, is highly reproducible, covers a wide dynamic range of reservoir sizes and can be completed in two days. As such, TILDA may represent an alternative to existing assays used to evaluate the efficacy of therapeutic strategies aimed at reducing the size of the latent HIV reservoir.

Project description:Despite significant advances in the treatment of human immunodeficiency virus type-1 (HIV) infection, antiretroviral therapy only suppresses viral replication but is unable to eliminate infection. Thus, discontinuation of antiretrovirals results in viral reactivation and disease progression. A major reservoir of HIV latent infection resides in resting central memory CD4+ T cells (TCM) that escape clearance by current therapeutic regimens and will require novel strategies for elimination. Here, we evaluated the therapeutic potential of autophagy-inducing peptides, Tat-Beclin 1 and Tat-vFLIP-α2, which can induce a novel Na+/K+-ATPase dependent form of cell death (autosis), to kill latently HIV-infected TCM while preventing virologic rebound. In this study, we encapsulated autophagy inducing peptides into biodegradable lipid-coated hybrid PLGA (poly lactic-co-glycolic acid) nanoparticles for controlled intracellular delivery. A single dose of nanopeptides was found to eliminate latent HIV infection in an in vitro primary model of HIV latency and ex vivo using resting CD4+ T cells obtained from peripheral blood mononuclear cells of HIV-infected patients on antiretroviral with fully suppressed virus for greater than 12 months. Notably, increased LC3B lipidation, SQSTM1/p62 degradation and Na+/K+-ATPase activity characteristic of autosis, were detected in nanopeptide treated latently HIV-infected cells compared to untreated uninfected or infected cells. Nanopeptide-induced cell death could be reversed by knockdown of autophagy proteins, ATG5 and ATG7, and inhibition or knockdown of Na+/K+-ATPase. Importantly, viral rebound was not detected following the induction of the Na+/K+-ATPase dependent form of cell death induced by the Tat-Beclin 1 and Tat-vFLIP-α2 nanopeptides. These findings provide a novel strategy to eradicate HIV latently infected resting memory CD4+ T cells, the major reservoir of HIV latency, through the induction of Na+/K+-ATPase dependent autophagy, while preventing reactivation of virus and new infection of uninfected bystander cells.

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Selective cell death in HIV-1-infected cells by DDX3 inhibitors leads to depletion of the inducible reservoir.

Publications

Selective cell death in HIV-1-infected cells by DDX3 inhibitors leads to depletion of the inducible reservoir.

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