In quiescent memory CD4+ T cells, the transcription elongation factor P-TEFb is scarce because the mRNA encoding its cyclin T1 (CycT1) subunit is translationally repressed. A 2026 preprint (Mbonye et al.) showed that RasGRP1-selective DAG-indololactones reactivate latent HIV by driving posttranscriptional CycT1 synthesis through MEK-ERK1/2-mTORC1 signaling, with minimal T-cell activation and CD4 loss, and located CycT1 mRNA outside P bodies while CD69 transcripts reside within them. The problem is the mechanistic core the preprint leaves open: identify the subcellular ribonucleoprotein compartment holding translationally repressed CycT1 mRNA (P bodies, the cytoplasmic messenger-RNA decay foci, excluded; stress granules or other phase-separated condensates hypothesized), the RNA-level machinery (microRNAs, RNA-binding proteins, cis-acting elements) whose repression is selectively relieved by ERK-activated mTORC1, and the ERK-dependent, mTORC1/RSK-independent effector - mitogen-activated protein kinase-interacting kinase (MNK) being the untested candidate - that drives CD69 translation.

Background

Antiretroviral therapy suppresses HIV replication but cannot eradicate the virus because a small population of long-lived memory CD4+ T cells carries transcriptionally latent, replication-competent proviruses. Reversing this latency ('shock and kill') requires latency-reversing agents (LRAs) that provoke viral gene expression without globally activating T cells. A decisive barrier in quiescent CD4+ T cells is the scarcity of P-TEFb (positive transcription elongation factor b), a heterodimer of the CDK9 kinase and cyclin T1 (CycT1, encoded by CCNT1) that the viral transactivator Tat recruits to proviral HIV to license RNA polymerase II elongation. In actively cycling cells P-TEFb is abundant and largely sequestered in the inhibitory 7SK small nuclear ribonucleoprotein complex; in resting memory CD4+ T cells, however, P-TEFb is limiting mainly because CycT1 is under dual posttranscriptional control: translational repression of CycT1 mRNA and proteasomal degradation of the protein by the Siah1/2 ubiquitin ligases. The repression arm has been partially mapped: microRNAs miR-27b (directly, through a 3' untranslated region (3'UTR) site), miR-29b, miR-150, and miR-223 repress CycT1 in resting CD4+ T cells (Chiang, Sung and Rice, 2012); the RNA-binding protein NF90 controls CycT1 translation through the 3'UTR independently of microRNAs in monocytic cells (Hoque et al., 2011); and miR-198 represses CycT1 in monocytes (Sung and Rice, 2009). Quiescent T cells more generally hold a pool of roughly 250 essential mRNAs in a translationally repressed but ribosome-poised state (Wolf et al., 2020; Ricciardi et al., 2018), so the machinery that keeps specific transcripts silent is a general feature of T-cell quiescence. Where individual repressed mRNAs physically reside matters because translationally silent messages are typically compartmentalized in cytoplasmic ribonucleoprotein (RNP) condensates: P bodies (conserved cytoplasmic foci enriched in decapping and 5'-to-3' decay factors) and stress granules (phase-separated assemblies of silent mRNA, translation initiation factors, and stalled ribosomes, in which localized translation can nevertheless occur). On the signaling side, diacylglycerol (DAG)-mimicking agonists of C1 domains (the DAG-binding zinc-finger regulatory modules of protein kinase C and RasGRP1) - natural products such as ingenol, prostratin, and bryostatin - potently reactivate latent HIV but act on protein kinase C (PKC), causing global T-cell activation and PKC-dependent phosphorylation, internalization, and degradation of the CD4 receptor. Mbonye et al. (2021) established that these agents induce active P-TEFb (coordinated CycT1 expression with CDK9 phosphorylation at Ser175) and reverse latency not through PKC but through RasGRP1, a DAG-binding Ras guanine nucleotide exchange factor that activates the Ras-Raf-MEK-ERK1/2 mitogen-activated protein kinase cascade, together with phosphoinositide 3-kinase (PI3K)-mTORC2-AKT signaling to mTORC1 (mechanistic target of rapamycin complex 1, the kinase complex that broadly stimulates protein synthesis). Synthetic DAG-indololactones (built by adding an indole at the sn-2 position of the DAG-lactone AJH836; Garcia et al., 2014; Elhalem et al., 2017) bind RasGRP1 over PKC with up to ~60-fold selectivity. The 2026 preprint (Mbonye et al., bioRxiv 2026.01.14.699500) showed that such compounds (e.g., 2A127, 2C78) induce P-TEFb posttranscriptionally via MEK-ERK1/2-mTORC1, synergize with histone deacetylase inhibitors (HDACi, which relieve epigenetic repression at the proviral promoter) to reactivate latent HIV in the QUECEL (quiescent effector cell latency) primary Th17 model of latent HIV and in memory CD4+ T cells from treated people with HIV, spare CD4, and barely induce activation markers. Its RNA fluorescence in situ hybridization (RNA FISH) data place repressed CycT1 mRNA in cytoplasmic regions distinct from P bodies, whereas CD69 (an early activation marker whose transcripts are also posttranscriptionally induced) colocalizes with P bodies. ERK is known to promote translation through at least three arms: inactivation of the TSC1/TSC2 complex (a GTPase-activating-protein complex that restrains mTORC1) leading to mTORC1 activation; p90 ribosomal S6 kinase (RSK); and the MNK (mitogen-activated protein kinase-interacting kinase) family, which phosphorylate the cap-binding translation initiation factor eIF4E (Ueda et al., 2004). Pharmacological epistasis in the preprint showed that DAG-indololactone-driven latency reversal and P-TEFb induction require mTORC1 but not RSK, while CD69/CD25 induction by C1-domain agonists (ingenol, prostratin, and the DAG-lactone AJH836) is ERK-dependent yet resistant to combined mTORC1 and RSK inhibition - leaving an unidentified ERK effector, with MNK the leading untested candidate.

Problem Statement

In primary quiescent memory CD4+ T cells, determine the posttranscriptional circuit that keeps cyclin T1 (CCNT1) mRNA translationally repressed and is disarmed upon RasGRP1 activation. This comprises three jointly linked questions: (i) Compartment - identify the subcellular ribonucleoprotein structure in which translationally repressed CycT1 mRNA resides; P bodies are excluded by the existing RNA FISH data (unlike CD69 transcripts), leaving stress granules or other cytoplasmic phase-separated condensates as the candidates. (ii) Machinery - identify the RNA-level repressive machinery acting on CycT1 mRNA in the quiescent state (the principal contributing factors - the microRNAs and RNA-binding proteins already documented for CCNT1, such as miR-27b and NF90, together with any additional factor shown to contribute functionally - and the cis-acting 3'UTR elements involved) and establish the molecular mechanism by which ERK-activated mTORC1 signaling relieves CycT1 mRNA translational repression. (iii) Effector assignment - identify the ERK-dependent, mTORC1/RSK-independent kinase that drives CD69 translation upon C1-domain agonist challenge; in particular, determine whether DAG-indololactones activate MNK1/2 and whether MNK-mediated eIF4E phosphorylation accounts for CD69 translation, and determine whether the effector so assigned also contributes to the ERK-dependent relief of CycT1 repression in (ii). These mechanisms are to be determined in primary human memory CD4+ T cells (and, where relevant, the QUECEL primary latency model) on the basis of the cited evidence and must remain consistent with the prior observations that DAG-indololactone-driven P-TEFb induction and HIV reactivation are mTORC1-dependent and RSK-independent.

References

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