Protein delivery of cell-penetrating zinc-finger activators stimulates latent HIV-1 infected cells

Abstract Despite efforts to develop effective treatments for eradicating HIV-1, a cure has not yet been achieved. Whereas antiretroviral drugs target actively replicating virus, latent non-replicative forms persist during treatment. Pharmacological strategies that reactivate latent HIV-1 and expose cellular reservoirs to antiretroviral therapy and host immune system have so far been unsuccessful, often triggering severe side-effects mainly due to systemic immune activation. Here, we present an alternative approach for stimulating latent HIV-1 expression via direct protein delivery of cell-penetrating zinc-finger activators (ZFAs). Cys2-His2 zinc-fingers fused to a transcription activation domain were engineered to recognize the HIV-1 promoter and induce targeted viral transcription. Following conjugation with multiple positively charged nuclear localization signal (NLS) repeats, protein delivery of a single zinc-finger activator (3NLS-PBS1-VP64) efficiently internalized HIV-1 latently infected T-lymphocytes and specifically stimulated viral expression. We show that short-term treatment with this ZFA protein induces higher levels of viral reactivation in cell line models of HIV-1 latency than those observed with gene delivery. Our work establishes protein delivery of ZFA as a novel and safe approach towards eradication of HIV-1 reservoirs.