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The RNA-Binding Proteins SRP14 and HMGB3 Control HIV-1 Tat mRNA Processing and Translation During HIV-1 Latency.

ABSTRACT: HIV-1 Tat protein is essential for virus production. RNA-binding proteins that facilitate Tat production may be absent or downregulated in resting CD4⁺ T-cells, the main reservoir of latent HIV in people with HIV (PWH) on antiretroviral therapy (ART). In this study, we examined the role of Tat RNA-binding proteins on the expression of Tat and control of latent and productive infection. Affinity purification coupled with mass spectrometry analysis was used to detect binding partners of MS2-tagged tat mRNA in a T cell-line model of HIV latency. The effect of knockdown and overexpression of the proteins of interest on Tat transactivation and translation was assessed by luciferase-based reporter assays and infections with a dual color HIV reporter virus. Out of the 243 interactions identified, knockdown of SRP14 (Signal Recognition Particle 14) negatively affected tat mRNA processing and translation as well as Tat-mediated transactivation, which led to an increase in latent infection. On the other hand, knockdown of HMGB3 (High Mobility Group Box 3) resulted in an increase in Tat transactivation and translation as well as an increase in productive infection. Footprinting experiments revealed that SRP14 and HMGB3 proteins bind to TIM-TAM, a conserved RNA sequence-structure in tat mRNA that functions as a Tat IRES modulator of tat mRNA. Overexpression of SRP14 in resting CD4⁺ T-cells from patients on ART was sufficient to reverse HIV-1 latency and induce virus production. The role of SRP14 and HMGB3 proteins in controlling HIV Tat expression during latency will be further assessed as potential drug targets.

SUBMITTER: Khoury G

PROVIDER: S-EPMC8236859 | biostudies-literature |

REPOSITORIES: biostudies-literature

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The RNA-binding proteins SRP14 and HMGB3 control HIV-1 Tat mRNA processing and translation during HIV-1 latency

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2021-06-14 | PXD025782 | Pride

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Long noncoding RNA NRON contributes to HIV-1 latency by specifically inducing tat protein degradation.

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Project description:HIV infection remains incurable due to the long-term persistence of latently infected cells, capable of refueling viremia upon antiretroviral therapy interruption. Shock-and-kill strategies aim to purge this latent reservoir by inducing the expression of viral genes, making infected cells visible for cleanup by the immune system. Here, we present a Tat mRNA formulation that, when combined with panobinostat, enables latency reversal at levels >3-fold higher than those reached with the most potent mitogens. We demonstrate that this mRNA formulation does not alter the transcriptome or phenotype of CD4 T cells, enabling the ex vivo assessment of infected cells upon latency reversal in a near-native state. Taking advantage of this characteristic, we show transcriptomic and proteomic differences between infected cells upon latency reversal and non-infected cells, including the upregulation of the cytotoxic protein granzyme A and a novel long non-coding RNA. Overall, we demonstrate that a Tat mRNA formulation enables potent reactivation of latent HIV, presenting a valuable research tool for studying the inducible HIV reservoir, as well as paving the way to a more effective shock-and-kill functional cure.

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Project description:HIV-1 latency results from a combination of tightly regulated molecular processes that act at distinct steps of HIV-1 gene expression. In an effort to elucidate the molecular players that govern viral latency, we previously performed a dCas9 chromatin immunoprecipitation coupled with mass spectrometry (Catchet-MS) and identified proteins bound differentially to the latent LTR that putatively promote HIV-1 latency. Here we characterize the Catchet-MS identified PCI domain-containing 2 (PCID2) protein to play a dual role in promoting HIV-1 latency by enforcing both HIV-1 transcription repression as well as post-transcriptional blocks. PCID2 bound the latent HIV-1 LTR and repressed transcription initiation during latency. Depletion of PCID2 remodeled the chromatin landscape at the HIV-1 promoter and resulted in transcriptional activation and reversal of latency. Immunoprecipitation coupled to Mass Spectrometry identified the PCID2 interacting proteins to include members of the spliceosome and other splicing regulators, including negative regulators of viral RNA alternative splicing. PCID2 depletion resulted in over-splicing of intron-containing HIV-1 RNA and mis-regulated expression of vRNA splice variants. Finally, consistent with its role in NXF1-mediated nascent RNA nucleocytoplasmic export as part of the TREX2 complex, PCID2 modulates export of completely spliced vRNA. In summary, we demonstrate that PCID2 is a previously unidentified factor involved in HIV-1 latency regulation which plays a dual role in blocking HIV-1 gene expression by acting on transcription initiation as well as viral RNA processing.

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