The impact of Tat expression on the host transcriptome landscape as the cells transit through the successive phases of the latency cycle

We hypothesized that Tat may exert opposing effects on viral gene expression depending on its PTMs. Furthermore, we propose that Tat can actively regulate the expression of host factors responsible for these PTMs, thereby influencing the establishment of latency. To investigate this, we define a latency cycle, capturing the distinct stages of cellular transition through the OFF-ON-OFF phases of viral gene expression. Jurkat cells were infected with a dual-reporter viral construct co-expressing a short-lived d2EGFP and a long-lived mScarlet fluorescent protein fused to Tat. The extended half-life of the Tat-mScarlet fusion enabled us to track and stabilize Tat expression even during the LTR-OFF state, providing a means to assess differential host gene expression. In the defined latency cycle, prior to stimulation, infected cells were dual-negative for both reporters (DN: GFP? RFP?), indicative of a latent state. Upon activation of the LTR, cells transitioned to a dual-positive phase (DP: GFP? RFP?), followed by a single-positive phase (SP: GFP? RFP?), and eventually returned to the DN phase. These subsets correspond to distinct stages in the latency cycle: latent, transcriptionally active, early latent, and latent, respectively. We developed a doxycycline-inducible system to selectively activate the viral LTR, without stimulating the host cells. Upon doxycycline treatment, diTat is expressed, providing a controlled, pulse-like activation of the viral LTR and enabling the establishment of a latency cycle. The distinct phases of the latency cycle were sorted, and a transcriptomic analysis was conducted. Overall design: Samples are as follows: Day 0: P1 – DN (GFP? RFP?), representing the latent pre-induction state Day 3: P2 – DP (GFP? RFP?), active transcription phase; and P3 – SP (GFP? RFP?), early latent cells Control: rtTA3 (cells expressing rtTA3 protein), and diTat (cells with Tet-ON-Tat system)