Induction of 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 DEAD-box polypeptide 3, X-Linked (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; pharmacological targeting of DDX3 reversed HIV-1 latency and selectively induced apoptosis in viral RNA-expressing CD4+ T cells from PLWHIV but not bystander cells. DDX3 inhibitor treatment of CD4+ T cells from PLWHIV in an in vitro culture model over five days resulted in an approximately 30% reduction of the inducible latent HIV-1 reservoir as determined by quantitation of CA HIV-1 RNA, by TILDA, as well as by FISH-Flow technology. RNA sequencing analysis revealed that while overall gene expression was minimally dysregulated, treatment of independent donor CD4+ T cells with DDX3 inhibitors led to significant downregulation of BIRC5 and HSPB1A, genes critical to cell survival during HIV-1 infection, providing mechanism for the observed selective cell death. Our data support the translation of DDX3 inhibitor class compounds into HIV-1 curative strategies and provide proof of concept for pharmacological reversal of latency coupled to induction of apoptosis towards elimination of the inducible reservoir.

PCID2 dysregulates transcription and viral RNA processing to promote HIV-1 latency

“…To analyze the dynamics of viral RNA (vRNA) and/or GFP-producing cells by FISH-Flow, five million control and PCID2-knockdown cells were collected, fixed, permeabilized and processed as described before 13 using the PrimeFlow RNA assay (ThermoFisher Scientific) according to manufacturer protocol. Viral RNA producing cells were detected by labelling HIV-1 unspliced mRNA with a set of 40 probe pairs against the GagPol region (Affymetrix eBioscience catalogue number GagPol HIV-1 VF10-10884) and RPL13A (Affymetrix eBioscience, catalogue number VA-13187) as a control diluted 1:5 in diluent provided in the kit and hybridized to the target HIV-1 vRNA for 2 hours at 40°C.…”

“…Further blocks to HIV-1 gene expression occur at a posttranscriptional level, including inhibition of viral RNA (vRNA) splicing, nucleocytoplasmic export, translation and trafficking (reviewed in 4 ). The contribution of these RNA processing mechanisms to the overall repression of HIV-1 gene expression has recently been characterized in both in vitro models of HIV-1 latency and CD4+ T cells obtained from people living with HIV-1 (PLWH) [10][11][12][13][14] , where it has been shown that blocks to HIV-1 gene expression occur largely at post-transcriptional levels. More recently, several unbiased and candidate studies, including our own, have successfully identified co-and post-transcriptional regulators of HIV-1 latency that control vRNA metabolism and either promote or inhibit viral gene expression 12,15,16 .…”