There has been extraordinary progress in understanding the roles of lentiviral accessory proteins in antagonizing host antiviral defense proteins. However, the precise primary function of the accessory gene Vpr remains elusive. Here we suggest that engagement with the DNA damage response is an important function of primate lentiviral Vpr proteins because of its conserved function among diverse lentiviral lineages. In contrast, we show that, for HIV-1, HIV-2, and related Vpr isolates and orthologs, there is a lack of correlation between DNA damage response activation and interaction with the host SLX4 protein complex of structure specific endonucleases; some Vpr proteins are able to interact with SLX4, but the majority are not. Using the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 method to knock out SLX4, we formally showed that HIV-1 and HIV-2 Vpr orthologs can still activate the DNA damage response and cell cycle arrest in the absence of SLX4. Together, our data suggest that activation of the DNA damage response, but not SLX4 interaction, is conserved and therefore indicative of an important function of Vpr. Our data also indicate that Vpr activates the DNA damage response through an SLX4-independent mechanism that remains uncharacterized. IMPORTANCE HIV-1 and HIV-2 belong to a family of viruses called lentiviruses that infect at least 40 primate species, including humans. Lentiviruses have been circulating in primates for at least 5 million years. In order to better fight HIV, we must understand the viral and host factors necessary for infection, adaptation, and transmission of these viruses. Using the natural variation of HIV-1, HIV-2, and related lentiviruses, we have investigated the role of the DNA damage response in the viral life cycle. We have found that the ability of lentiviruses to activate the DNA damage response is largely conserved. However, we also found that the SLX4 host factor is not required for this activation, as was previously proposed. This indicates that the DNA damage response is an important player in the viral life cycle, and yet the mechanism(s) by which HIV-1, HIV-2, and other primate lentiviruses engage the DNA damage response is still unknown.

The DNA damage response (DDR) is a signaling cascade that safeguards the genome from genotoxic agents, including human pathogens. However, the DDR has also been utilized by many pathogens, such as human immunodeficiency virus (HIV), to enhance infection. To properly treat HIV-positive individuals, we must understand how the virus usurps our own cellular processes. Here, we have found that an important yet poorly understood gene in HIV, Vpr, targets the DDR at two unique steps: it causes damage and activates DDR signaling, and it represses the ability of cells to repair this damage, which we hypothesize is central to the primary function of Vpr. In clarifying these important functions of Vpr, our work highlights the multiple ways human pathogens engage the DDR and further suggests that modulation of the DDR is a novel way to help in the fight against HIV.

Lentiviruses encode a number of multi-functional accessory proteins, however, the primary role of the accessory protein Vpr remains unclear. As Vpr engages the host DNA damage response (DDR) at multiple steps, modulation of the DDR is considered central to the function(s) of Vpr. Vpr activates ataxia telangiectasia and Rad3 (ATR)-mediated DDR signaling, resulting in cell cycle arrest. However, the cellular consequences of Vpr-induced DNA damage, and the connection of Vpr-induced DNA damage to other Vpr functions, are unknown. Here, we determined that HIV-1 Vpr-induced DNA damage activates the ATM-NF-κB essential modulator (NEMO) pathway and alters cellular transcription via NF-κB/RelA. Through RNA-sequencing (RNA-seq) of cells expressing Vpr or mutants that separate the ability of Vpr to induce DNA damage from other DDR phenotypes, we identified that Vpr alters the transcriptome independent of cell cycle arrest. In tissue-cultured U2OS cells and primary human monocyte-derived macrophages (MDMs), we showed Vpr activates both ataxia telangiectasia mutated (ATM) and NF-κB/RelA signaling cascades. While inhibition of NEMO did not affect Vpr-induced DNA damage, it prevented NF-κB activation by Vpr, highlighting the importance of NEMO in Vpr-mediated transcriptional reprogramming. Virion-delivered Vpr was sufficient to induce DNA damage and activate ATM-NEMO dependent NF-κB transcription, suggesting that engagement of the DDR and transcriptional changes can occur early during viral replication. Together, our data uncover cellular consequences of Vpr-induced DNA damage and provide a mechanism for how Vpr activates NF-κB through DNA damage and ATM-NEMO signaling, which occur independent of cell cycle arrest. We propose this is essential to overcoming restrictive environments, such as in macrophages, to enhance viral replication. IMPORTANCE: The 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 the 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-κB. This occurs early during the infection of primary human immune cells, suggesting NF-κB 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.

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-κB/RelA signaling. HIV-1 infection of primary MDMs validated Vpr-dependent NF-κB transcriptional activation during infection. Both virion delivered and de novo expressed Vpr induced DNA damage and activated ATM-NEMO dependent NF-κB 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-κB 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. IMPORTANCE: The 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-κB. This occurs early during infection of primary human immune cells, suggesting NF-κB 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.

The latent viral reservoir represents one of the major barriers of curing HIV-1. Focus on the “kick and kill” approach, in which virus expression is reactivated then cells producing virus are selectively depleted, has led to the discovery of many latency reversing agents (LRAs) that have furthered our understanding of the mechanisms driving HIV-1 latency and latency reversal. Thus far, individual compounds have yet to be robust enough to work as a therapy, highlighting the importance of identifying new compounds that target novel pathways and synergize with known LRAs. In this study, we identified a promising LRA, NSC95397, from a screen of ∼4250 compounds. We validated that NSC95397 reactivates latent viral transcription and protein expression from cells with unique integration events and across different latency models. Co-treating cells with NSC95397 and known LRAs demonstrated that NSC95397 synergizes with different drugs under both standard normoxic and physiological hypoxic conditions. NSC95397 does not globally increase open chromatin, and bulk RNA sequencing revealed NSC95397 does not greatly increase cellular transcription. Instead, NSC95397 downregulates pathways key to metabolism, cell growth, and DNA repair – highlighting the potential of these pathways in regulating HIV-1 latency. Overall, we identified NSC95397 as a novel LRA that does not largely alter global transcription, that shows potential for synergy with known LRAs, and that may act through novel pathways not previously recognized for their ability to modulate HIV-1 latency. Importance: One of the largest barriers to curing HIV-1 is the latent viral reservoir – this is when the virus incorporates itself into long-lived cells in the body, ready to reactivate and re-seed infection. Destroying dormant HIV-1 is one potential pathway to a cure, yet no therapeutics have been discovered to work well in patients. In our study, we identified a compound, NSC95397, that can awaken dormant HIV-1 on its own through novel mechanisms not previously linked to HIV-1 latency. Moreover, NSC95397 improves the abilities of previously identified compounds to reactivate latent HIV-1. Thus, our study has identified a compound that can help towards the better understanding of an HIV-1 cure.