The Intact Non-Inducible Latent HIV-1 Reservoir is Established In an In Vitro Primary TCM Cell Model of Latency.

The establishment of HIV-1 latency has hindered an HIV-1 cure. "Shock and Kill" strategies to target this reservoir aim to induce the latent provirus with latency reversing agents (LRAs). However, recent studies have shown that the majority of the intact HIV-1 viral reservoir found in ART-suppressed HIV infected individuals is not inducible. We sought to understand whether this non-inducible reservoir is established, and thus able to be studied, in an in vitro primary TCM model of latency. Furthermore, we wanted to expand this model system to include R5-tropic and non-B subtype viruses. To that end, we generated our TCM model of latency with an R5 subtype B virus, AD8 and an R5 subtype C virus, MJ4. Our results demonstrate that both intact and defective proviruses are generated in this model. Less than 50% of intact proviruses are inducible regardless of viral strain in the context of maximal stimulation through the TCR or with different clinically relevant LRAs including the HDAC inhibitors SAHA and MS-275, the PKC agonist Ingenol 3,20-dibenzoate or the SMAC mimetic AZD-5582. Our findings suggest that current LRA strategies are insufficient to effectively reactivate intact latent HIV-1 proviruses in primary CD4 TCM cells and that the mechanisms involved in the generation of the non-inducible HIV-1 reservoir can be studied using this primary in vitro model.Importance: HIV-1 establishes a latent reservoir that persists under antiretroviral therapy. Antiretroviral therapy is able to stop the spread of the virus and the progression of the disease but does not target this latent reservoir. If antiretroviral therapy is stopped, the virus is able to resume replication and the disease progresses. Recently, it has been demonstrated that most of the latent reservoir capable of generating replication competent virus cannot be induced in the laboratory setting. However, the mechanisms that influence the generation of this intact and non-inducible latent reservoir are still under investigation. Here we demonstrate the generation of defective, intact and intact non-inducible latent HIV-1 in a TCM model of latency using different HIV-1 strains. Thus, the mechanisms which control inducibility can be studied using this primary cell model of latency, which may accelerate our understanding of the latent reservoir and the development of curative strategies.

Human splice factors contribute to latent HIV infection in primary cell models and blood CD4+ T cells from ART-treated individuals.

It is unclear what mechanisms govern latent HIV infection in vivo or in primary cell models. To investigate these questions, we compared the HIV and cellular transcription profile in three primary cell models and peripheral CD4+ T cells from HIV-infected ART-suppressed individuals using RT-ddPCR and RNA-seq. All primary cell models recapitulated the block to HIV multiple splicing seen in cells from ART-suppressed individuals, suggesting that this may be a key feature of HIV latency in primary CD4+ T cells. Blocks to HIV transcriptional initiation and elongation were observed more variably among models. A common set of 234 cellular genes, including members of the minor spliceosome pathway, was differentially expressed between unstimulated and activated cells from primary cell models and ART-suppressed individuals, suggesting these genes may play a role in the blocks to HIV transcription and splicing underlying latent infection. These genes may represent new targets for therapies designed to reactivate or silence latently-infected cells.

Structure-Activity Relationship Analysis of Benzotriazine Analogues as HIV-1 Latency-Reversing Agents.

"Shock and kill" therapeutic strategies toward HIV eradication are based on the transcriptional activation of latent HIV with a latency-reversing agent (LRA) and the consequent killing of the reactivated cell by either the cytopathic effect of HIV or an arm of the immune system. We have recently found several benzotriazole and benzotriazine analogues that have the ability to reactivate latent HIV by inhibiting signal transducer and activator of transcription 5 (STAT5) SUMOylation and promoting STAT5 binding to the HIV long terminal repeat and increasing its transcriptional activity. To understand the essential structural groups required for biological activity of these molecules, we performed a systematic analysis of >40 analogues. First, we characterized the essential motifs within these molecules that are required for their biological activity. Second, we identified three benzotriazine analogues with similar activity. We demonstrated that these three compounds are able to increase STAT5 phosphorylation and transcriptional activity. All active analogues reactivate latent HIV in a primary cell model of latency and enhance the ability of interleukin-15 to reactivate latent HIV in cells isolated from aviremic participants. Third, this family of compounds also promote immune effector functions in vitro in the absence of toxicity or global immune activation. Finally, initial studies in mice suggest lack of acute toxicity in vivo A better understanding of the biological activity of these compounds will help in the design of improved LRAs that work via inhibition of STAT5 SUMOylation.

Activation of the Anti-Oxidative Stress Response Reactivates Latent HIV-1 Through the Mitochondrial Antiviral Signaling Protein Isoform MiniMAVS.

The mitochondrial antiviral signaling protein (MAVS) is part of the cell's innate immune mechanism of defense. MAVS mRNA is bicistronic and can give rise to a full length-MAVS and a shorter isoform termed miniMAVS. In response to viral infections, viral RNA can be sensed by the cytosolic RNA sensors retinoic acid-inducible gene I (RIG-I) and/or melanoma differentiation-associated protein 5 (MDA5) and activate NF-κB through interaction with MAVS. MAVS can also sense cellular stress and activate an anti-oxidative stress (AOS) response through the activation of NF-κB. Because NF-κB is a main cellular transcription factor for HIV-1, we wanted to address what role MAVS plays in HIV-1 reactivation from latency in CD4 T cells. Our results indicate that RIG-I agonists required full length-MAVS whereas the AOS response induced by Dynasore through its catechol group can reactivate latent HIV-1 in a MAVS dependent manner through miniMAVS isoform. Furthermore, we uncover that PKC agonists, a class of latency-reversing agents, induce an AOS response in CD4 T cells and require miniMAVS to fully reactivate latent HIV-1. Our results indicate that the AOS response, through miniMAVS, can induce HIV-1 transcription in response to cellular stress and targeting this pathway adds to the repertoire of approaches to reactivate latent HIV-1 in 'shock-and-kill' strategies.

PLGA nanodepots co-encapsulating prostratin and anti-CD25 enhance primary natural killer cell antiviral and antitumor function.

Natural killer (NK) cells are attractive effector cells of the innate immune system against human immunodeficiency virus (HIV) and cancer. However, NK cell therapies are limited by the fact that target cells evade NK cells, for example, in latent reservoirs (in HIV) or through upregulation of inhibitory signals (in cancer). To address this limitation, we describe a biodegradable nanoparticle-based "priming" approach to enhance the cytotoxic efficacy of peripheral blood mononuclear cell-derived NK cells. We present poly(lactic-co-glycolic acid) (PLGA) nanodepots (NDs) that co-encapsulate prostratin, a latency-reversing agent, and anti-CD25 (aCD25), a cell surface binding antibody, to enhance primary NK cell function against HIV and cancer. We utilize a nanoemulsion synthesis scheme to encapsulate both prostratin and aCD25 within the PLGA NDs (termed Pro-aCD25-NDs). Physicochemical characterization studies of the NDs demonstrated that our synthesis scheme resulted in stable and monodisperse Pro-aCD25-NDs. The NDs successfully released both active prostratin and anti-CD25, and with controllable release kinetics. When Pro-aCD25-NDs were administered in an in vitro model of latent HIV and acute T cell leukemia using J-Lat 10.6 cells, the NDs were observed to prime J-Lat cells resulting in significantly increased NK cell-mediated cytotoxicity compared to free prostratin plus anti-CD25, and other controls. These findings demonstrate the feasibility of using our Pro-aCD25-NDs to prime target cells for enhancing the cytotoxicity of NK cells as antiviral or antitumor agents.

HIV-1 Latency and Latency Reversal: Does Subtype Matter?

Cells that are latently infected with HIV-1 preclude an HIV-1 cure, as antiretroviral therapy does not target this latent population. HIV-1 is highly genetically diverse, with over 10 subtypes and numerous recombinant forms circulating worldwide. In spite of this vast diversity, much of our understanding of latency and latency reversal is largely based on subtype B viruses. As such, most of the development of cure strategies targeting HIV-1 are solely based on subtype B. It is currently assumed that subtype does not influence the establishment or reactivation of latent viruses. However, this has not been conclusively proven one way or the other. A better understanding of the factors that influence HIV-1 latency in all viral subtypes will help develop therapeutic strategies that can be applied worldwide. Here, we review the latest literature on subtype-specific factors that affect viral replication, pathogenesis, and, most importantly, latency and its reversal.