Resting CD4+ T cells in people living with human immunodeficiency virus type (HIV) harbour integrated HIV-1 DNA that remains transcriptionally silent. These cells constitute the latent reservoir under antiretroviral therapy (ART). Reactivating this silent infection is key to curing HIV by exposing these cells to immune-mediated killing and the cytopathic effect of the virus.
Recently, our lab has discovered that interferon-alpha 8 (IFNα8) can reactivate silent HIV in CD4 T cells. However, the mechanism behind this reactivation is unknown. We have identified 3 pathways downstream of the IFNα receptor (IFNAR) that could mediate HIV transcription: JAK-STAT, CRKL-STAT5 and PI3K pathways. We used pharmacological inhibitors to elucidate which pathway is involved in IFNα-induced HIV reactivation. Briefly, memory CD4 T cells isolated from healthy donors were infected with HIVBaL. At 2 days post-infection (dpi) and 2 hours prior to IFNα treatment, cells were treated with either the IFNAR (Jak1 and Tyk2) inhibitors, JAK-STAT (STAT1 and 3) inhibitors, CRKL-STAT5 (STAT5) inhibitor, and PI3K (mTOR, NF-κB and PI3K) inhibitors. HIV replication was then assessed at 5 dpi by p24 measurement using flow cytometry.
Our data showed that p24 expression was reduced by inhibiting both IFNAR receptors, and STAT1. We did not observe p24 reduction when inhibiting all other proteins. This suggests that the JAK-STAT pathway and the STAT1 protein are potentially involved in HIV reactivation. Future studies using mass spectrometry will explore proteomic and phosphorylated protein changes associated with HIV reactivation.
These insights could guide the development of new latency-reversing agents (LRAs), shifting the therapeutic approach from lifelong ART to potential cure strategies.