NSC95397 is a Novel HIV Latency Reversing Agent.

The latent viral reservoir represents one of the major barriers of curing HIV. 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 can reactivate latently integrated virus and further our understanding of the mechanisms driving HIV 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 can act in novel pathways and synergize with known LRAs. In this study, we identified a promising LRA, NSC95397, from a screen of ~4250 compounds in J-Lat cell lines. We validated that NSC95397 reactivates latent viral transcription and protein expression from cells with unique integration events. Cotreating cells with NSC95397 and known LRAs demonstrated that NSC95397 has the potential to synergize with different drugs, such prostratin, a PKC agonist, and SAHA, an HDAC inhibitor. By looking at multiple common markers of open chromatin, we show that NSC95397 does not increase open chromatin globally. Bulk RNA sequencing revealed that NSC95397 does not greatly change cellular transcription. Instead, NSC95397 downregulates many pathways key to metabolism, cell growth, and DNA repair - highlighting the potential of these pathways in regulating HIV latency. Overall, we identified NSC95397 as a novel LRA that does not 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 latency.

Viral Modulation of the DNA Damage Response and Innate Immunity: Two Sides of the Same Coin.

The DDR consists of multiple pathways that sense, signal, and respond to anomalous DNA. To promote efficient replication, viruses have evolved to engage and even modulate the DDR. In this review, we will discuss a select set of diverse viruses and the range of mechanisms they evolved to interact with the DDR and some of the subsequent cellular consequences. There is a dichotomy in that the DDR can be both beneficial for viruses yet antiviral. We will also review the connection between the DDR and innate immunity. Previously believed to be disparate cellular functions, more recent research is emerging that links these processes. Furthermore, we will discuss some discrepancies in the literature that we propose can be remedied by utilizing more consistent DDR-focused assays. By doing so, we hope to obtain a much clearer understanding of how broadly these mechanisms and phenotypes are conserved among all viruses. This is crucial for human health since understanding how viruses manipulate the DDR presents an important and tractable target for antiviral therapies.

Inflammasome activation leads to cDC1-independent cross-priming of CD8 T cells by epithelial cell-derived antigen.

The innate immune system detects pathogens and initiates adaptive immune responses. Inflammasomes are central components of the innate immune system, but whether inflammasomes provide sufficient signals to activate adaptive immunity is unclear. In intestinal epithelial cells (IECs), inflammasomes activate a lytic form of cell death called pyroptosis, leading to epithelial cell expulsion and the release of cytokines. Here, we employed a genetic system to show that simultaneous antigen expression and inflammasome activation specifically in IECs is sufficient to activate CD8+ T cells. By genetic elimination of direct T cell priming by IECs, we found that IEC-derived antigens were cross-presented to CD8+ T cells. However, cross-presentation of IEC-derived antigen to CD8+ T cells only partially depended on IEC pyroptosis. In the absence of inflammasome activation, cross-priming of CD8+ T cells required Batf3+ dendritic cells (conventional type one dendritic cells [cDC1]), whereas cross-priming in the presence of inflammasome activation required a Zbtb46+ but Batf3-independent cDC population. These data suggest the existence of parallel inflammasome-dependent and inflammasome-independent pathways for cross-presentation of IEC-derived antigens.

HIV Vpr Modulates the Host DNA Damage Response at Two Independent Steps to Damage DNA and Repress Double-Strand DNA Break Repair.

The DNA damage response (DDR) is a signaling cascade that is vital to ensuring the fidelity of the host genome in the presence of genotoxic stress. Growing evidence has emphasized the importance of both activation and repression of the host DDR by diverse DNA and RNA viruses. Previous work has shown that HIV-1 is also capable of engaging the host DDR, primarily through the conserved accessory protein Vpr. However, the extent of this engagement has remained unclear. Here, we show that HIV-1 and HIV-2 Vpr directly induce DNA damage and stall DNA replication, leading to the activation of several markers of double- and single-strand DNA breaks. Despite causing damage and activating the DDR, we found that Vpr represses the repair of double-strand breaks (DSB) by inhibiting homologous recombination (HR) and nonhomologous end joining (NHEJ). Mutational analyses of Vpr revealed that DNA damage and DDR activation are independent from repression of HR and Vpr-mediated cell cycle arrest. Moreover, we show that repression of HR does not require cell cycle arrest but instead may precede this long-standing enigmatic Vpr phenotype. Together, our data uncover that Vpr globally modulates the host DDR at at least two independent steps, offering novel insight into the primary functions of lentiviral Vpr and the roles of the DNA damage response in lentiviral replication.IMPORTANCE 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.