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, p… Show more
“…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.…”
Section: Fish-flowmentioning
confidence: 99%
“…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 .…”
HIV-1 latency results from tightly regulated molecular processes that act at distinct steps of HIV-1 gene expression. To elucidate the molecular players that govern latency, we previously performed a dCas9-chromatin immunoprecipitation coupled with mass spectrometry (Catchet-MS) and identified the interactome of the latent HIV-1 LTR. Here we characterize the Catchet-MS-identified PCI domain-containing 2 (PCID2) protein, a component of the TREX2 complex, to play a dual role in promoting HIV-1 latency by enforcing both transcriptional repression and post-transcriptional blocks to HIV-1 gene expression. PCID2 bound the latent HIV-1 LTR and repressed transcription initiation during latency. Depletion of PCID2 remodelled the chromatin landscape at the HIV-1 promoter and resulted in transcriptional activation and reversal of latency. Immunoprecipitation coupled to Mass Spectrometry identified PCID2-interacting proteins to include members of the spliceosome, including negative viral RNA (vRNA) alternative splicing regulators, and PCID2 depletion resulted in over-splicing of intron-containing vRNA and misregulated expression of vRNA splice variants. We demonstrate that MCM3AP and DSS1, two other RNA-binding TREX2 complex subunits that comprise the dock of the complex also inhibit transcription initiation and viral RNA alternative splicing during latency and similarly to PCID2 function as prominent latency associated repressors of HIV-1 gene expression. Thus, PCID2 is a novel HIV-1 latency-promoting factor, which in context of the TREX2 sub-complex PCID2-DSS1-MCM3AP blocks transcription and dysregulates vRNA processing.
“…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.…”
Section: Fish-flowmentioning
confidence: 99%
“…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 .…”
HIV-1 latency results from tightly regulated molecular processes that act at distinct steps of HIV-1 gene expression. To elucidate the molecular players that govern latency, we previously performed a dCas9-chromatin immunoprecipitation coupled with mass spectrometry (Catchet-MS) and identified the interactome of the latent HIV-1 LTR. Here we characterize the Catchet-MS-identified PCI domain-containing 2 (PCID2) protein, a component of the TREX2 complex, to play a dual role in promoting HIV-1 latency by enforcing both transcriptional repression and post-transcriptional blocks to HIV-1 gene expression. PCID2 bound the latent HIV-1 LTR and repressed transcription initiation during latency. Depletion of PCID2 remodelled the chromatin landscape at the HIV-1 promoter and resulted in transcriptional activation and reversal of latency. Immunoprecipitation coupled to Mass Spectrometry identified PCID2-interacting proteins to include members of the spliceosome, including negative viral RNA (vRNA) alternative splicing regulators, and PCID2 depletion resulted in over-splicing of intron-containing vRNA and misregulated expression of vRNA splice variants. We demonstrate that MCM3AP and DSS1, two other RNA-binding TREX2 complex subunits that comprise the dock of the complex also inhibit transcription initiation and viral RNA alternative splicing during latency and similarly to PCID2 function as prominent latency associated repressors of HIV-1 gene expression. Thus, PCID2 is a novel HIV-1 latency-promoting factor, which in context of the TREX2 sub-complex PCID2-DSS1-MCM3AP blocks transcription and dysregulates vRNA processing.
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