Resistance to the Tat Inhibitor Didehydro-Cortistatin A Is Mediated by Heightened Basal HIV-1 Transcription.

Human immunodeficiency virus type 1 (HIV-1) Tat binds the viral RNA structure transactivation-responsive element (TAR) and recruits transcriptional cofactors, amplifying viral mRNA expression. The Tat inhibitor didehydro-cortistatin A (dCA) promotes a state of persistent latency, refractory to viral reactivation. Here we investigated mechanisms of HIV-1 resistance to dCA in vitro Mutations in Tat and TAR were not identified, consistent with the high level of conservation of these elements. Instead, viruses resistant to dCA developed higher Tat-independent basal transcription. We identified a combination of mutations in the HIV-1 promoter that increased basal transcriptional activity and modifications in viral Nef and Vpr proteins that increased NF-κB activity. Importantly, these variants are unlikely to enter latency due to accrued transcriptional fitness and loss of sensitivity to Tat feedback loop regulation. Furthermore, cells infected with these variants become more susceptible to cytopathic effects and immune-mediated clearance. This is the first report of viral escape to a Tat inhibitor resulting in heightened Tat-independent activity, all while maintaining wild-type Tat and TAR.IMPORTANCE HIV-1 Tat enhances viral RNA transcription by binding to TAR and recruiting activating factors. Tat enhances its own transcription via a positive-feedback loop. Didehydro-cortistatin A (dCA) is a potent Tat inhibitor, reducing HIV-1 transcription and preventing viral rebound. dCA activity demonstrates the potential of the "block-and-lock" functional cure approaches. We investigated the viral genetic barrier to dCA resistance in vitro While mutations in Tat and TAR were not identified, mutations in the promoter and in the Nef and Vpr proteins promoted high Tat-independent activity. Promoter mutations increased the basal transcription, while Nef and Vpr mutations increased NF-κB nuclear translocation. This heightened transcriptional activity renders CD4+ T cells infected with these viruses more susceptible to cytotoxic T cell-mediated killing and to cell death by cytopathic effects. Results provide insights on drug resistance to a novel class of antiretrovirals and reveal novel aspects of viral transcriptional regulation.

The Tat inhibitor didehydro-cortistatin A suppresses SIV replication and reactivation.

The HIV-1 transactivation protein (Tat) binds the HIV mRNA transactivation responsive element (TAR), regulating transcription and reactivation from latency. Drugs against Tat are unfortunately not clinically available. We reported that didehydro-cortistatin A (dCA) inhibits HIV-1 Tat activity. In human CD4+ T cells isolated from aviremic individuals and in the humanized mouse model of latency, combining dCA with antiretroviral therapy accelerates HIV-1 suppression and delays viral rebound upon treatment interruption. This drug class is amenable to block-and-lock functional cure approaches, aimed at a durable state of latency. Simian immunodeficiency virus (SIV) infection of rhesus macaques (RhMs) is the best-characterized model for AIDS research. Here, we demonstrate, using in vitro and cell-based assays, that dCA directly binds to SIV Tat's basic domain. dCA specifically inhibits SIV Tat binding to TAR, but not a Tat-Rev fusion protein, which activates transcription when Rev binds to its cognate RNA binding site replacing the apical region of TAR. Tat-TAR inhibition results in loss of RNA polymerase II recruitment to the SIV promoter. Importantly, dCA potently inhibits SIV reactivation from latently infected Hut78 cells and from primary CD4+ T cells explanted from SIVmac239-infected RhMs. In sum, dCA's remarkable breadth of activity encourages SIV-infected RhM use for dCA preclinical evaluation.-Mediouni, S., Kessing, C. F., Jablonski, J. A., Thenin-Houssier, S., Clementz, M., Kovach, M. D., Mousseau, G., de Vera, I.M.S., Li, C., Kojetin, D. J., Evans, D. T., Valente, S. T. The Tat inhibitor didehydro-cortistatin A suppresses SIV replication and reactivation.

Didehydro-Cortistatin A Inhibits HIV-1 by Specifically Binding to the Unstructured Basic Region of Tat.

The intrinsically disordered HIV-1 Tat protein binds the viral RNA transactivation response structure (TAR), which recruits transcriptional cofactors, amplifying viral mRNA expression. Limited Tat transactivation correlates with HIV-1 latency. Unfortunately, Tat inhibitors are not clinically available. The small molecule didehydro-cortistatin A (dCA) inhibits Tat, locking HIV-1 in persistent latency, blocking viral rebound. We generated chemical derivatives of dCA that rationalized molecular docking of dCA to an active and specific Tat conformer. These revealed the importance of the cycloheptene ring and the isoquinoline nitrogen's positioning in the interaction with specific residues of Tat's basic domain. These features are distinct from the ones required for inhibition of cyclin-dependent kinase 8 (CDK8), the only other known ligand of dCA. Besides, we demonstrated that dCA activity on HIV-1 transcription is independent of CDK8. The binding of dCA to Tat with nanomolar affinity alters the local protein environment, rendering Tat more resistant to proteolytic digestion. dCA thus locks a transient conformer of Tat, specifically blocking functions dependent of its basic domain, namely the Tat-TAR interaction; while proteins with similar basic patches are unaffected by dCA. Our results improve our knowledge of the mode of action of dCA and support structure-based design strategies targeting Tat, to help advance development of dCA, as well as novel Tat inhibitors.IMPORTANCE Tat activates virus production, and limited Tat transactivation correlates with HIV-1 latency. The Tat inhibitor dCA locks HIV in persistent latency. This drug class enables block-and-lock functional cure approaches, aimed at reducing residual viremia during therapy and limiting viral rebound. dCA may also have additional therapeutic benefits since Tat is also neurotoxic. Unfortunately, Tat inhibitors are not clinically available. We generated chemical derivatives and rationalized binding to an active and specific Tat conformer. dCA features required for Tat inhibition are distinct from features needed for inhibition of cyclin-dependent kinase 8 (CDK8), the only other known target of dCA. Furthermore, knockdown of CDK8 did not impact dCA's activity on HIV-1 transcription. Binding of dCA to Tat's basic domain altered the local protein environment and rendered Tat more resistant to proteolytic digestion. dCA locks a transient conformer of Tat, blocking functions dependent on its basic domain, namely its ability to amplify viral transcription. Our results define dCA's mode of action, support structure-based-design strategies targeting Tat, and provide valuable information for drug development around the dCA pharmacophore.

The transcriptional transactivator Tat selectively regulates viral splicing.

HIV-1 gene expression requires both viral and cellular factors to control and coordinate transcription. While the viral factor Tat is known for its transcriptional transactivator properties, we present evidence for an unexpected function of Tat in viral splicing regulation. We used a series of HIV-1 reporter minigenes to demonstrate that Tat's role in splicing is dependent on the cellular co-transcriptional splicing activators Tat-SF1 and CA150. Surprisingly, we show that this Tat-mediated splicing function is independent from transcriptional activation. In the context of the full-length viral genome, this mechanism promotes an autoregulatory feedback that decreases expression of tat and favors expression of the env-specific mRNA. Our data demonstrate that Tat-mediated regulation of transcription and splicing can be uncoupled and suggest a mechanism for the involvement of specific transcriptional activators in splicing.

In vivo detection of RNA-binding protein interactions with cognate RNA sequences by fluorescence resonance energy transfer.

Expression of the nascent RNA transcript is regulated by its interaction with a number of proteins. The misregulation of such interactions can often result in impaired cellular functions that can lead to cancer and a number of diseases. Thus, our understanding of RNA-protein interactions within the cellular context is essential for the development of novel diagnostic and therapeutic tools. While there are many in vitro methods that analyze RNA-protein interactions in vivo approaches are scarce. Here we established a method based on fluorescence resonance energy transfer (FRET), which we term RNA-binding mediated FRET (RB-FRET), which determines RNA-protein interaction inside cells and tested it on hnRNP H protein binding to its cognate RNA. Using two different approaches, we provide evidence that RB-FRET is sensitive enough to detect specific RNA-protein interactions in the cell, providing a powerful tool to study spatial and temporal localization of specific RNA-protein complexes.

Role of cellular RNA processing factors in human immunodeficiency virus type 1 mRNA metabolism, replication, and infectivity.

Expression of the human immunodeficiency virus type 1 genome requires several cellular factors regulating transcription, alternative splicing, RNA stability, and intracellular localization of the viral transcripts. In vitro and ex vivo approaches have identified SR proteins and hnRNPs of the A/B and H subfamilies as cellular factors that regulate different aspects of viral mRNA metabolism. To understand the role of these protein families within the context of the full replicating virus, we altered the expression levels of hnRNPs H, F, 2H9, GRSF1, A1, A2, and A3 and SR proteins SC35, SF2, and SRp40 in HEK 293 cells transfected with the proviral clone pNL4-3. Quantitative and semiquantitative PCR analyses showed that overexpression as well as downregulation of these proteins disrupted the balance of alternatively spliced viral mRNAs and may alter viral transcription. Furthermore, expression of hnRNPs H, F, 2H9, A1, and A2 and SR proteins SF2 and SRp40 increased nuclear localization of the unspliced Gag/Pol mRNA, while the same factors increased the cytoplasmic localization of the partially spliced Env mRNA. We also report that overexpression of hnRNPs A1 and A2 and SR proteins SF2, SC35, and SRp40 causes a dramatic decrease in virion production. Finally, utilizing a reporter TZM-bl cell line, we show that virion infectivity may be also impacted by deregulation of expression of most SR proteins and hnRNPs. This work demonstrates that cellular factors regulating mRNA processing have wide-ranging effects on human immunodeficiency virus type 1 replication and should be considered novel therapeutic targets.

The secondary structure of the human immunodeficiency virus type 1 transcript modulates viral splicing and infectivity.

Splicing of human immunodeficiency virus type 1 (HIV-1) exon 6D is regulated by the presence of a complex splicing regulatory element (SRE) sequence that interacts with the splicing factors hnRNP H and SC35. In this work, we show that, in the context of the wild-type viral sequence, hnRNP H acts as a repressor of exon 6D inclusion independent of its binding to the SRE. However, hnRNP H binding to the SRE acts as an enhancer of exon 6D inclusion in the presence of a critical T-to-C mutation. These seemingly contrasting functional properties of hnRNP H appear to be caused by a change in the RNA secondary structure induced by the T-to-C mutation that affects the spatial location of bound hnRNP H with respect to the exon 6D splicing determinants. We propose a new regulatory mechanism mediated by RNA folding that may also explain the dual properties of hnRNP H in splicing regulation.