Noncanonical-NF-κB activation and DDX3 inhibition reduces the HIV-1 reservoir by elimination of latently infected cells ex-vivo

Latency reversal and subsequent elimination of the human immunodeficiency virus-1 (HIV-1) reservoir using a combination of compounds with different mechanisms of action are considered a promising tool for HIV-1 cure. Here, we analyzed HIV-1 reservoir reduction by targeting the two host factors; inhibitor of apoptosis proteins (IAPs) and DEAD-box polypeptide 3 (DDX3) using a SMAC mimetic (SMACm) and DDX3 inhibitor (DDX3i), respectively. We observed that SMACm efficiently reactivated HIV-1 in a latency Jurkat model, which was further enhanced by DDX3 inhibition. Strikingly, this compound combination strongly decreased the proportion of latently as well as transcriptionally active infected cells in a T cell line model with a dual-reporter virus. To determine the efficacy of compounds to eradicate the HIV-1 reservoir in people living with HIV (PWH), a novel ex vivo HIV-1 reservoir reduction assay (HIVRRA) was developed. DDX3i and SMACm alone reduced the HIV-1 reservoir in peripheral blood mononuclear cells (PBMCs) from the majority of PWH, whereas notably, the SMACm/DDX3i combination reduced the HIV-1 reservoir even further with 53%-90% in all PWH analyzed, while uninfected bystander cells were not affected. Our data highlight that IAPs as well as factors involved in HIV-1 replication like DDX3 are excellent targets for HIV-1 cure strategies. We show for the first time that the combination of SMACm and DDX3i reverses viral latency and specifically eliminates the HIV-1-infected cells in vitro and ex vivo.