Systems analysis of latent HIV reversal reveals altered stress kinase signaling and increased cell death in infected T cells

Viral latency remains the most significant obstacle to HIV eradication. Clinical strategies aim to purge the latent CD4+ T cell reservoir by activating viral expression to induce death, but are undercut by the inability to target latently infected cells. Here we explored the acute signaling response of latent HIV-infected CD4+ T cells to identify dynamic phosphorylation signatures that could be targeted for therapy. Stimulation with CD3/CD28, PMA/ionomycin, or latency reversing agents prostratin and SAHA, yielded increased phosphorylation of IκBα, ERK, p38, and JNK in HIV-infected cells across two in vitro latency models. Both latent infection and viral protein expression contributed to changes in perturbation-induced signaling. Data-driven statistical models calculated from the phosphorylation signatures successfully classified infected and uninfected cells and further identified signals that were functionally important for regulating cell death. Specifically, the stress kinase pathways p38 and JNK were modified in latently infected cells, and activation of p38 and JNK signaling by anisomycin resulted in increased cell death independent of HIV reactivation. Our findings suggest that altered phosphorylation signatures in infected T cells provide a novel strategy to more selectively target the latent reservoir to enhance eradication efforts.