Background
Studies using the Jurkat T-cell model have identified numerous factors crucial for initiating HIV-1 transcription. However, a significant limitation of transformed cells is their inability to replicate the transitions between active effector cells and quiescent memory T cells, which are the key drivers of HIV latency in vivo.
Methods
We use refined primary cell models for HIV latency that accurately replicate the transitions between active effector cells and quiescent memory T cells. Our approach combines an HIV Cas-FISH immunofluorescence assay to trace changes in HIV DNA localization and immunofluorescence to identify key transcription factors. In parallel, the cells were analyzed by single-cell (sc) RNA-Seq, snRNA-Seq, AB-Seq, and ATAC-Seq, ensuring a comprehensive understanding of the process.
Results
As activated effector cells transition quiescence, HIV proviral DNA moves from the nuclear Intermediate Euchromatic Compartment (IEC) to the perinucleolar compartment (PNC). Strikingly, the localization of proviruses to the PNC was blocked by integrase inhibitor raltegravir, which showed that this was due to spatial genome rearrangements. Dramatic changes in histone methylation, cellular metabolism, and transcriptome changes accompany the transition into quiescence. snRNA-Seq and scATAC-Seq show that the subset of genes that are known HIV integration sites become more inaccessible when cells enter quiescence. During the reactivation of latently infected cells through the T-cell receptor, nascent viral mRNA transcripts emanating from proviruses in the PNC were detected. There was the rapid accumulation of the viral trans-activator Tat and its proviral reactivation regulatory partners, P-TEFb and 7SK snRNA, and RNA processing factors in large interchromatin granule clusters, which assembled near the provirus, and a shift of the proviruses away from the nucleolus. For all latency reversing agens (LRAs), there is a strong correlation between the extent of P-TEFb biogenesis and their potency. HIV proviruses in latently infected memory T cells from patients also accumulated in the PNC and showed identical patterns of nuclear rearrangements after cellular reactivation.
Conclusions
In contrast to transformed cells where proviruses are found primarily at the nuclear periphery, in primary memory T cells, P-TEFb highly restricts HIV transcription, and the nuclear architecture undergoes rearrangements that shape the transcriptional silencing and reactivation of proviral HIV.
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