HIV-1 Vpr-induced DNA damage activates NF-κB independent of cell cycle arrest and CRL4A DCAF1 engagement.

Lentiviral accessory genes enhance replication through diverse mechanisms. HIV-1 accessory protein Vpr modulates the host DNA damage response (DDR) at multiple steps through the degradation of host proteins, cell cycle arrest, DNA damage, and both activation and repression of DDR signaling. Vpr also alters host and viral transcription; however, the connection between Vpr-mediated DDR modulation and transcriptional activation remains unclear. Here, we determined the cellular consequences of Vpr-induced DNA damage using Vpr mutants that allow us to separate the ability of Vpr to induce DNA damage from CRL4A DCAF1 complex dependent phenotypes including cell cycle arrest, host protein degradation, and repression of DDR. In both tissue-cultured U2OS cells and primary human monocyte-derived macrophages (MDMs), we found that Vpr induces DNA breaks and activates DDR signaling in the absence of cell cycle arrest and CRL4A DCAF1 complex engagement. Moreover, through RNA-sequencing, we found that Vpr-induced DNA damage alters cellular transcription via activation of NF-κB/RelA signaling. NF-κB/RelA transcriptional activation was dependent on ATM-NEMO, as inhibition of NEMO resulted in loss of NF-κB transcriptional upregulation by Vpr. Furthermore, HIV-1 infection of primary MDMs validated NF-κB transcriptional activation during infection. Both virion delivered and de novo expressed Vpr induced DNA damage and activated NF-κB transcription, suggesting that engagement of the DDR can occur during early and late stages of viral replication. Together, our data support a model where Vpr-induced DNA damage activates NF-κB through the ATM-NEMO pathway, independent of cell cycle arrest and CRL4A DCAF1 engagement. We propose this is essential to overcoming restrictive environments, such as macrophages, to enhance viral transcription and replication.