Engagement of the DDR by viruses in a broadly conserved mechanism among viruses and understanding how viruses modulate the DDR has paved the way to understanding several basic biological processes in molecular biology. Despite this, the role of engaging and modulating the DDR in the context of HIV remains poorly understood. For many years it has been apparent that the viral accessory gene Vpr plays a role in engaging the DDR during the viral lifecycle, however, the molecular processes and phenotypes associated with modulation remain poorly understood. For my dissertation research, we examine the function of HIV Vpr in the context of the DDR. First, we explore how Vpr engages and modulates the DDR by determining distinct cellular phenotypes specific to Vpr function to generate a model. We next explore the chromatin-associated proteome of Vpr to understand the underlying molecular mechanisms governing modulation of the DDR. Altogether, this will broaden our understanding HIV molecular biology in the context of the DDR and at chromatin.

HIV-1 and HIV-2 are two evolutionarily district viruses that encode the accessory protein Vpr. Although many functions have been ascribed to HIV-1 and HIV-2 Vpr, the primary and conserved function of Vpr is unknown. Both HIV-1 and HIV-2 Vpr induce cell cycle arrest, modulate the DNA damage response (DDR), alter transcription, and engage the E3 ubiquitin ligase CRL4ADCAF1 complex, causing host protein degradation. Moreover, most of the phenotypes described for Vpr involve the ability of Vpr to modulate the DNA damage response (DDR). Here, we aimed to identify the conserved and divergent functions of HIV-1 and HIV-2 Vpr and determine how Vpr enhances viral replication.In Chapter 1, we wrote a review on viral modulation of DDR and the similarities between DDR and innate immunity. We report that many diverse viruses modulate the DDR at multiple steps to facilitate viral replication. Furthermore, we compare the DDR and innate immune response, as both pathways are responsible for sensing, signaling, and responding to aberrant nucleic acids. In Chapter 2, we found that HIV-1 Vpr induces DNA damage and activates ATM-signaling in the absence of cell cycle arrest and engagement with the CRL4ADCAF1 complex. These findings were novel as most functions described for accessory genes require host protein degradation. Yet, an emerging idea in the field is that accessory genes can function independently of co-opting E3 ubiquitin ligase complexes. Furthermore, in primary macrophages, we found that Vpr-induced DNA damage activates NF-κB transcription through the ATM-NEMO pathway. Lastly, we found virion-delivered and de novo expressed Vpr induces DNA damage and activates NF-κB. These data propose Vpr enhances viral replication by promoting proviral transcription through NF-κB. In Chapter 3, we found that HIV-2 Vpr induces DNA damage, activates DDR signaling, and promotes nuclear translocation of RelA, independent of CRL4ADCAF1 engagement and cell cycle arrest, as conserved by HIV-1 Vpr. However, HIV-2 Vpr does not upregulate NF-κB target genes that HIV-1 Vpr upregulates. These data suggest that there are both conserved and divergent mechanisms by which HIV-1 and HIV-2 Vpr modulate the DDR. In Chapter 4, we leveraged our understanding of viral modulation of the DDR to pose the hypothesis that DDR genes are rapidly evolving because they are in conflict with HIV-1. Here, we found that 14.6% of DDR genes show signatures of positive selection, and most of these are involved in homologous recombination. Through a CRISPR screen, we identified DDR genes that, when knocked out, altered HIV-1 infectivity. To investigate further, we focused on the candidate gene MUTYH and found that MUTYH protein levels are refractory to knock-down, overexpression, and interferon treatment. In Chapter 5, we found that HIV-1 and HIV-2 Vpr induce DNA breaks independent from cell cycle arrest. Furthermore, we found that Vpr inhibits homologous recombination repair and that repression requires the engagement of the CRL4ADCAF1 complex. These data propose that Vpr inhibits DNA repair through the degradation of an unknown host factor.