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

Abstract

Lentiviral accessory genes enhance replication through diverse mechanisms. HIV-1 accessory protein Vpr modulates the host DNA damage response (DDR) at multiple steps through DNA damage, cell cycle arrest, the degradation of host proteins, and both the 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 cell cycle arrest and other DDR phenotypes including host protein degradation and repression of DDR. RNA-sequencing of cells expressing Vpr or Vpr mutants identified that Vpr alters cellular transcription through mechanisms both dependent and independent of cell cycle arrest. In tissue-cultured U2OS cells and primary human monocyte-derived macrophages (MDMs), Vpr-induced DNA damage activates the ATM-NEMO pathway and alters cellular transcription via NF-{kappa}B/RelA signaling. HIV-1 infection of primary MDMs validated Vpr-dependent NF-{kappa}B transcriptional activation during infection. Both virion delivered and de novo expressed Vpr induced DNA damage and activated ATM-NEMO dependent NF-{kappa}B transcription, suggesting that engagement of the DDR and transcriptional reprogramming can occur during early and late stages of viral replication. Together, our data identifies a mechanism by which Vpr activates NF-{kappa}B through DNA damage and the ATM-NEMO pathway, which occur independent of cell cycle arrest. We propose this is essential to overcoming restrictive environments, such as in macrophages, to enhance viral transcription and replication. IMPORTANCEThe HIV accessory protein Vpr is multi-functional and required for viral replication in vivo, yet how Vpr enhances viral replication is unknown. Emerging literature suggests that a conserved function of Vpr is engagement of the host DNA damage response (DDR). For example, Vpr activates DDR signaling, causes DDR-dependent cell cycle arrest, promotes degradation of various DDR proteins, and alters cellular consequences of DDR activation. However, a central understanding of how these phenotypes connect and how they affect HIV-infected cells remains unknown. Here, we found that Vpr-induced DNA damage alters the host transcriptome by activating an essential transcription pathway, NF-{kappa}B. This occurs early during infection of primary human immune cells, suggesting NF-{kappa}B activation and transcriptome remodeling are important for establishing productive HIV-1 infection. Together, our study provides novel insights into how Vpr alters the host environment through the DDR, and what roles Vpr and the DDR play to enhance HIV replication.