Latency Model Research Articles

Magnetoelectric Extracellular Vesicle Latency-Targeting (MELT) Nanotherapeutic for the Block-Lock-and-Kill HIV Eradication Strategy.

Human immunodeficiency virus (HIV) establishes latent infections in cellular reservoirs, including microglia. HC69 cells, a microglial model of HIV latency, contain an HIV promoter long terminal repeat (LTR)-GFP reporter and were used for testing the efficacy of a two-step magnetoelectric nanoparticle (MENP) and extracellular vesicle (xEV) latency-targeting (MELT) nanotherapeutic. GFP expression in HC69 at rest is low (GFPLo), and upon exposure to LTR, transcription-activating agents (i.e., TNF-α) are induced to be high expressing (GFPHi). The first step of MELT utilized ZL0580, an HIV Tat inhibitor loaded into EVs (80%) via incubation. ZL0580-EVs were taken up by GFPLo and blocked LTR transcriptional reactivation by 50% and were 90% less toxic than ZL0580 alone. The second step in MELT involved conjugation of monomethyl auristatin E (MMAE) to MENPs. HPLC measurements showed 80% MMAE attachment to MENPs. Flow cytometry-based measurements of the membrane potential indicated that the membranes of GFPHi HC69 were 60% more polarized than GFPLo HC69 cells. More MMAE-MENPs were internalized by GFPLo HC69. Using a mixed-cell blood-brain barrier (BBB) Transwell model, we demonstrated that 20% of MELT crossed the BBB, was taken up by HC69 cells, and reduced LTR reactivation by 10%. Overall, this study demonstrated that MELT can potentially be utilized as a nanotherapeutic to target HIV latency in microglia.

Rev-RRE activity modulates HIV-1 replication and latency reactivation: Implications for viral persistence and cure strategies.

The HIV-1 Rev-RRE regulatory axis plays a crucial role in viral replication by facilitating the nucleo-cytoplasmic export and expression of viral mRNAs with retained introns. In this study, we investigated the impact of variation in Rev-RRE functional activity on HIV-1 replication kinetics and reactivation from latency. Using a novel HIV-1 clone with an interchangeable Rev cassette, we engineered viruses with different Rev functional activities and demonstrated that higher Rev-RRE activity confers greater viral replication capacity while maintaining a constant level of Nef expression. In addition, a low Rev activity virus rapidly acquired a compensatory mutation in the RRE that significantly increased Rev-RRE activity and replication. In a latency model, proviruses with differing Rev-RRE activity levels varied in the efficiency of viral reactivation, affecting both initial viral release and subsequent replication kinetics. These results demonstrate that activity differences in the Rev-RRE axis among different viral isolates have important implications for HIV replication dynamics and persistence. Importantly, our findings indicate that bolstering Rev/RRE activity could be explored as part of latency reversal strategies in HIV cure efforts.

Optimized Convolutional Neural Network at the IoT edge for image detection using pruning and quantization

AbstractMost real-time computer vision applications heavily rely on Convolutional Neural Network (CNN) based models, for image classification and recognition. Due to the computationally and memory-intensive nature of the CNN model, it’s challenging to deploy on resource-constrained Internet of Things (IoT) devices to enable Edge intelligence for real-time decision-making. Edge intelligence requires minimum inference latency, memory footprint, and energy-efficient model. This work aims to develop an energy-efficient deep learning accelerator using a 3-stage pipeline: Training, Weight-pruning, and Quantization to reduce the model size and optimize the resources. First, we employ YOLOv3, a CNN architecture to detect objects in an image on the trained data. In addition, a sparse network of YOLO has been created by using pruning, which helps to improve the network’s performance and efficiency by reducing the computational requirements. Finally, we utilize 8-bit quantization to reduce the precision of the weights and activations, in a neural network. The evaluation of our proposed model shows that combining pruning and 8-bit quantization improves the efficiency and performance of the model. While pruning shows a decline of 80.39% in model parameters. The combination of 8-bit quantization results in an improvement in inference latency by 22.72% compared to existing SQuantization approach and a reduction of energy consumption by 29.41%.

Development of a latency model for HIV-1 subtype C and the impact of long terminal repeat element genetic variation on latency reversal.

Sub-Saharan Africa accounts for almost 70% of people living with HIV (PLWH) worldwide, with the greatest numbers centred in South Africa where 98% of infections are caused by subtype C (HIV-1C). However, HIV-1 subtype B (HIV-1B), prevalent in Europe and North America, has been the focus of most cure research and testing despite making up only 12% of HIV-1 infections globally. Development of latency models for non-subtype B viruses is a necessary step to address this disproportionate focus. Furthermore, the impact of genetic variation between viral subtypes, specifically within the long terminal repeat (LTR) element of the viral transcriptional promoter on latency reversal, remains unclear. To address this scientific gap, we constructed a minimal genome retroviral vector expressing HIV-1C consensus transactivator of transcription protein (Tat) and green fluorescent protein (GFP) under the control of either HIV-1C consensus LTR (C731CC) or the transmitted/founder (T/F) LTRs derived from PLWH (CT/F731CC), produced corresponding LTR pseudotyped viruses using a vesicular stomatitis virus (VSV-G) pseudotyped Envelope vector and the pCMVΔR8.91 packaging vector containing HIV-1 accessory and rev genes. Viruses produced in this way were used to infect Jurkat E6 and primary CD4+ T cells in vitro. By enriching for latently infected cells, and treating them with different latency reversing agents, we developed an HIV-1C latency model that demonstrated that the HIV-1C consensus LTR has lower reactivation potential compared to its HIV-1B counterpart. Furthermore, HIV-1C T/F LTR pseudotyped proviral genetic variants exhibited a heterogenous reactivation response which was modulated by host cell (genetic) variation. Our data suggests that genetic variation both within and between HIV-1 subtypes influences latency reversal. Future studies should investigate the specific role of variation in host cellular environment on reactivation differences.

NSC95397 Is a Novel HIV-1 Latency-Reversing Agent.

The latent viral reservoir represents one of the major barriers to curing HIV-1. Focus on the "kick and kill" (also called "shock and kill") approach, in which virus expression is reactivated, and 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 that 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, shows potential for synergy with known LRAs, and may act through novel pathways not previously recognized for their ability to modulate HIV-1 latency.

Lytic promoter activity during herpes simplex virus latency is dependent on genome location.

Herpes simplex virus 1 (HSV-1) is a significant pathogen that establishes lifelong latent infections with intermittent episodes of resumed disease. In mouse models of HSV infection, sporadic low-level lytic gene expression has been detected during latency in the absence of reactivation events that lead to production of new viruses. This viral activity during latency has been reported using a sensitive Cre-marking model for several lytic gene promoters placed in one location in the HSV-1 genome. Here, we extend these findings in the same model by examining first, the activity of an ectopic lytic gene promoter in several places in the genome and second, whether any promoters might be active in their natural context. We found that Cre expression was detected during latency from ectopic and native promoters, but only in locations near the ends of the unique long genome segment. This location is significant because it is in close proximity to the region from which latency-associated transcripts (LATs) are derived. These results show that native HSV-1 lytic gene promoters can produce protein products during latency, but that this activity is only detectable when they are located close to the LAT locus.IMPORTANCEHSV is a significant human pathogen and the best studied model of mammalian virus latency. Traditionally, the active (lytic) and inactive (latent) phases of infection were considered to be distinct, but the notion of latency being entirely quiescent is evolving due to the detection of some lytic gene expression during latency. Here, we add to this literature by finding that the activity can be found for native lytic gene promoters as well as for constructs placed ectopically in the HSV genome. However, this activity was only detectable when these promoters were located close by a region known to be transcriptionally active during latency. These data have implications for our understanding of HSV gene regulation during latency and the extent to which transcriptionally active regions are insulated from adjacent parts of the viral genome.

Epitranscriptomic m6A modifications during reactivation of HIV-1 latency in CD4+ T cells.

RNA m6A modification is important for regulating gene expression and innate immune responses to HIV-1 infection. However, the functional significance of m6A modification during HIV-1 latency reactivation is unknown. To address this important question, in this study, we used established cellular models of HIV-1 latency, m6A-specific sequencing at single-base resolution, and functional assays. We demonstrate that HIV-1 latency reversal leads to increased levels of cellular m6A modification, correlates with cellular m6A levels, and is dependent on the catalytic activity of the m6A methyltransferase enzyme. We also identified cellular genes that are differentially m6A-modified during HIV-1 reactivation, as well as the sites of m6A within HIV-1 RNA. Our novel findings point toward a significant role for m6A modification in HIV-1 latency reversal.

Optimization of a lentivirus-mediated gene therapy targeting HIV-1 RNA to eliminate HIV-1-infected cells

Persistence of HIV-1 in cellular reservoirs results in lifelong infection, with cure achieved only in rare cases through ablation of marrow-derived cells. We report on optimization of an approach that could potentially be aimed at eliminating these reservoirs, hijacking the HIV-1 alternative splicing process to functionalize the herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) cell suicide system through targeted RNA trans-splicing at the HIV-1 D4 donor site. AUG1-deficient HSVtk therapeutic pre-mRNA was designed to gain an in-frame start codon from HIV-1 tat1. D4-targeting lentiviral vectors were produced and used to transduce HIV-1-expressing cells, where trans-spliced HIV-1 tat/HSVtk mRNA was successfully detected. However, translation of catalytically active HSVtk polypeptides from internal AUGs in HSVtk ΔAUG1 caused GCV-mediated cytotoxicity in uninfected cells. Modifying these sites in the D4 opt 2 lentiviral vector effectively mitigated this major off-target effect. Promoter choice was optimized for increased transgene expression. Affinity for HIV-1 RNA predicted in silico correlated with the propensity of opt 2 payloads to induce HIV-1 RNA trans-splicing and killing of HIV-1-expressing cells with no significant effect on uninfected cells. Following latency reversing agent (LRA) optimization and treatment, 45% of lymphocytes in an HIV-1-infected latency model could be eliminated with D4 opt 2/GCV. Further development would be warranted to exploit this approach.

Release of P-TEFb from the Super Elongation Complex promotes HIV-1 latency reversal.

The persistence of HIV-1 in long-lived latent reservoirs during suppressive antiretroviral therapy (ART) remains one of the principal barriers to a functional cure. Blocks to transcriptional elongation play a central role in maintaining the latent state, and several latency reversal strategies focus on the release of positive transcription elongation factor b (P-TEFb) from sequestration by negative regulatory complexes, such as the 7SK complex and BRD4. Another major cellular reservoir of P-TEFb is in Super Elongation Complexes (SECs), which play broad regulatory roles in host gene expression. Still, it is unknown if the release of P-TEFb from SECs is a viable latency reversal strategy. Here, we demonstrate that the SEC is not required for HIV-1 replication in primary CD4+ T cells and that a small molecular inhibitor of the P-TEFb/SEC interaction (termed KL-2) increases viral transcription. KL-2 acts synergistically with other latency reversing agents (LRAs) to reactivate viral transcription in several cell line models of latency in a manner that is, at least in part, dependent on the viral Tat protein. Finally, we demonstrate that KL-2 enhances viral reactivation in peripheral blood mononuclear cells (PBMCs) from people living with HIV (PLWH) on suppressive ART, most notably in combination with inhibitor of apoptosis protein antagonists (IAPi). Taken together, these results suggest that the release of P-TEFb from cellular SECs may be a novel route for HIV-1 latency reactivation.

Analysis of the effect of HDAC inhibitors on the formation of the HIV reservoir.

The HIV reservoir is more dynamic than previously thought with around 70% of the latent reservoir originating from viruses circulating within 1 year of the initiation of antiretroviral therapy (ART). In an ex vivo model system of HIV latency, it was reported that early exposure to class I histone deacetylase (HDAC) inhibitors might prevent these more recently infected cells from entering a state of stable viral latency. This finding raises the possibility that co-administration of HDAC inhibitors at the time of ART initiation may prevent the establishment of much of the HIV reservoir. Here, we tested the effects of the HDAC inhibitors suberoylanilide hydroxamic acid (SAHA) and panobinostat co-administered at the time of ART initiation on the formation of the viral reservoir in HIV-infected humanized mice. As previously shown, SAHA and panobinostat were well tolerated in humanized mice. Unexpectedly, co-administration of SAHA resulted in an increase in the frequency of CD4+ cells carrying HIV DNA but did not alter the frequency of cell-associated HIV RNA in HIV-infected, ART-treated humanized mice. Co-administration of panobinostat did not alter levels of cell-associated HIV DNA or RNA. Our in vivo findings indicate that co-administration of HDAC inhibitors initiated at the same time of ART treatment does not prevent recently infected cells from entering latency.IMPORTANCECurrent antiretroviral therapy (ART) does not eradicate cells harboring replication-competent HIV reservoir. Withdrawal of ART inevitably results in a rapid viremia rebound. The HIV reservoir is more dynamic than previously thought. Early exposure to class I histone deacetylase (HDAC) inhibitors inhibit these more recently infected cells from entering a state of stable viral latency in an ex vivo model of latency, raising the possibility that co-administration of HDAC inhibitors at the time of ART initiation may reduce much of the HIV reservoir. Here, we tested the effects of the HDAC inhibitors suberoylanilide hydroxamic acid or panobinostat during ART initiation on the formation of the viral reservoir in HIV-infected humanized mice. Our in vivo study indicates that in contrast to in vitro observations, the co-administration of HDAC inhibitors at the same time of ART initiation does not prevent recently infected cells from entering latency.

Static Scheduling of Weight Programming for DNN Acceleration with Resource Constrained PIM

Most existing architectural studies on ReRAM-based processing-in-memory (PIM) DNN accelerators assume that all weights of the DNN can be mapped to the crossbar at once. However, these studies are over-idealized. ReRAM crossbar resources for calculation are limited because of technological limitations, so multiple weight mapping procedures are required during the inference process. In this article, we propose a static scheduling framework which generates the mapping between DNN weights and ReRAM cells with minimum runtime weight programming cost. We first build a ReRAM crossbar programming latency model by simultaneously considering the DNN weight patterns, ReRAM programming operations, and PIM architecture characteristics. Then, the model is used in the searching process to obtain an optimized weight-to-OU mapping table with minimum online programming latency. Finally, an OU scheduler is used to coordinate the activation sequences of OUs in the crossbars to perform the inference computation correctly. Evaluation results show the proposed framework significantly reduces the weight programming overhead and the overall inference latency for various DNN models with different input datasets.

On-Edge Deployment of Vision Transformers for Medical Diagnostics Using the Kvasir-Capsule Dataset

This paper aims to explore the possibility of utilizing vision transformers (ViTs) for on-edge medical diagnostics by experimenting with the Kvasir-Capsule image classification dataset, a large-scale image dataset of gastrointestinal diseases. Quantization techniques made available through TensorFlow Lite (TFLite), including post-training float-16 (F16) quantization and quantization-aware training (QAT), are applied to achieve reductions in model size, without compromising performance. The seven ViT models selected for this study are EfficientFormerV2S2, EfficientViT_B0, EfficientViT_M4, MobileViT_V2_050, MobileViT_V2_100, MobileViT_V2_175, and RepViT_M11. Three metrics are considered when analyzing a model: (i) F1-score, (ii) model size, and (iii) performance-to-size ratio, where performance is the F1-score and size is the model size in megabytes (MB). In terms of F1-score, we show that MobileViT_V2_175 with F16 quantization outperforms all other models with an F1-score of 0.9534. On the other hand, MobileViT_V2_050 trained using QAT was scaled down to a model size of 1.70 MB, making it the smallest model amongst the variations this paper examined. MobileViT_V2_050 also achieved the highest performance-to-size ratio of 41.25. Despite preferring smaller models for latency and memory concerns, medical diagnostics cannot afford poor-performing models. We conclude that MobileViT_V2_175 with F16 quantization is our best-performing model, with a small size of 27.47 MB, providing a benchmark for lightweight models on the Kvasir-Capsule dataset.

Performance of binary MLC using real-time optical sensor feedback system.

The Radixact system (Accuray Inc., Sunnyvale, CA) is the latest platform release based on the TomoTherapy technology. The most recent system does not apply a leaf latency model correction after plan optimization to ensure the correct MLC leaf-open time (LOT) agreement between the TPS and machine delivery. The MLC uses optical sensors to measure the delivered LOTs in real-time and individual leaf-specific latency corrections are made to ensure agreement. The aim of this study was to assess the performance of the Radixact MLC with leaf-specific latency correction using the optical sensor's real-time feedback. Specifically, the study statistically evaluated the MLC LOT errors observed from 290 plan-specific quality assurance (PSQA) measurements. Repeatability testing was performed to quantify the uncertainty in the MLC feedback system delivery by analyzing>1300 delivered treatment fractions throughout the course of radiotherapy. The clinical impact was evaluated by estimating the resulting dose difference in the patient targets due to the measured plan latencies. Our study measured an average plan latency equal to 2.0±0.4 ms (0.6%±0.2%) for 290 PSQAs. Repeatability tests showed a mean standard deviation in plan latencies measuring 0.05 ms (0.02%). The deviation from the TPS in the mean target dose due to the plan latencies was estimated to be 0.0%±0.2% (range: -0.7%-1.1%). The current MLC system with real-time optical sensor feedback is capable of accurately delivering the TPS-generated sinograms. Repeatability test results showed that the system allows for high reliability in daily sinogram delivery. The MLC latency deviations were shown to have minimal clinical impact on the overall target dosimetry.

Rethinking the mobile edge for vehicular services

The growing connected car market requires mobile network operators (MNOs) to rethink their network architecture to deliver ultra-reliable low-latency communications. In response, Multi-Access Edge Computing (MEC) has emerged as a solution, enabling the deployment of computing resources at the network edge. For MNOs to tap into the potential benefits of MEC, they need to transform their networks accordingly. Consequently, the primary objective of this study is to design a realistic MEC architecture and corresponding optimal deployment strategy – deciding on the placement and configuration of computing resources – as opposed to prior studies focusing on MEC run-time management and orchestration (e.g., service placement, computation offloading, and user allocation). To cater to the heterogeneous demands of vehicular services, we propose a multi-tier MEC architecture aligned with 5G and Beyond-5G radio access network deployments. Therefore, we frame MEC deployment as an optimization problem within this architecture, assuming 3 MEC tiers. Our data-driven evaluation, grounded in realistic assumptions about network architecture, usage, latency, and cost models, relies on datasets from a major MNO in the UK. We show the benefits of adopting a 3-tier MEC architecture over single-tier (centralized or distributed) architectures for heterogeneous vehicular services, in terms of deployment cost, energy consumption, and robustness.

HIV-1 latency reversal and immune enhancing activity of IL-15 is not influenced by sex hormones.

The role of different biological variables including biological sex, age, and sex hormones in Human immunodeficiency virus (HIV) cure approaches is not well understood. The γc-cytokine IL-15 is a clinically relevant cytokine that promotes immune activation and mediates HIV reactivation from latency. In this work, we examined the interplay that biological sex, age, and sex hormones 17β-estradiol, progesterone, and testosterone may have on the biological activity of IL-15. We found that IL-15-mediated CD4+ T cell activation was higher in female donors than in male donors. This difference was abrogated at high 17β-estradiol concentration. Additionally, there was a positive correlation between age and both IL-15-mediated CD8+ T cell activation and IFN-γ production. In a primary cell model of latency, biological sex, age, or sex hormones did not influence the ability of IL-15 to reactivate latent HIV. Finally, 17β-estradiol did not consistently affect reactivation of translation-competent reservoirs in CD4+ T cells from people living with HIV who are antiretroviral therapy (ART) suppressed. Our study has found that biological sex and age, but not sex hormones, may influence some of the biological activities of IL-15. Understanding how different biological variables may affect HIV cure therapies will help us evaluate current and future clinical trials aimed toward HIV cure in diverse populations.

Neuronal expression of herpes simplex virus-1 VP16 protein induces pseudorabies virus escape from silencing and reactivation.

Alpha herpesvirus (α-HV) particles enter their hosts from mucosal surfaces and efficiently maintain fast transport in peripheral nervous system (PNS) axons to establish infections in the peripheral ganglia. The path from axons to distant neuronal nuclei is challenging to dissect due to the difficulty of monitoring early events in a dispersed neuron culture model. We have established well-controlled, reproducible, and reactivateable latent infections in compartmented rodent neurons by infecting physically isolated axons with a small number of viral particles. This system not only recapitulates the physiological infection route but also facilitates independent treatment of isolated cell bodies or axons. Consequently, this system enables study not only of the stimuli that promote reactivation but also the factors that regulate the initial switch from productive to latent infection. Adeno-associated virus (AAV)-mediated expression of herpes simplex-1 (HSV-1) VP16 alone in neuronal cell bodies enabled the escape from silencing of incoming pseudorabies virus (PRV) genomes. Furthermore, the expression of HSV VP16 alone reactivated a latent PRV infection in this system. Surprisingly, the expression of PRV VP16 protein supported neither PRV escape from silencing nor reactivation. We compared transcription transactivation activity of both VP16 proteins in primary neurons by RNA sequencing and found that these homolog viral proteins produce different gene expression profiles. AAV-transduced HSV VP16 specifically induced the expression of proto-oncogenes including c-Jun and Pim2. In addition, HSV VP16 induces phosphorylation of c-Jun in neurons, and when this activity is inhibited, escape of PRV silencing is dramatically reduced.IMPORTANCEDuring latency, alpha herpesvirus genomes are silenced yet retain the capacity to reactivate. Currently, host and viral protein interactions that determine the establishment of latency, induce escape from genome silencing or reactivation are not completely understood. By using a compartmented neuronal culture model of latency, we investigated the effect of the viral transcriptional activator, VP16 on pseudorabies virus (PRV) escape from genome silencing. This model recapitulates the physiological infection route and enables the study of the stimuli that regulate the initial switch from a latent to productive infection. We investigated the neuronal transcriptional activation profiles of two homolog VP16 proteins (encoded by HSV-1 or PRV) and found distinct gene activation signatures leading to diverse infection outcomes. This study contributes to understanding of how alpha herpesvirus proteins modulate neuronal gene expression leading to the initiation of a productive or a latent infection.

Suppression of HSV-1 infection and viral reactivation by CRISPR-Cas9 gene editing in 2D and 3D culture models

Although our understanding of HSV-1 biology has been considerably enhanced, developing therapeutic strategies to eliminate HSV-1 in latently infected individuals remains a public health concern. Current antiviral drugs used for the treatment of HSV-1 complications are not specific and do not address latent infection. We recently developed a CRISPR/Cas9-based gene editing platform to specifically target the HSV-1 genome. In this study, we further used 2D Vero cell culture and 3D hiPSC-derived cerebral organoid (CO) models to assess the effectiveness of our editing constructs targeting viral ICP0 or ICP27 gene. We found that targeting the ICP0 or ICP27 gene with AAV2-CRISPR/Cas9 vectors in Vero cells drastically suppressed HSV-1 replication. In addition, we productively infected COs with HSV-1, characterized the viral replication kinetics, and established a viral latency model. Finally, we discovered that ICP0- or ICP27-targeting AAV2-CRISPR/Cas9 vector significantly reduced viral rebound in the COs that were latently infected with HSV-1. In summary, our results suggest that CRISPR/Cas9 gene editing of HSV-1 is an efficient therapeutic approach to eliminate the latent viral reservoir and treat HSV-1 associated complications.

Nanoparticle delivery of Tat synergizes with classical latency reversal agents to express HIV antigen targets.

Limited cellular levels of the HIV transcriptional activator Tat are one contributor to proviral latency that might be targeted in HIV cure strategies. We recently demonstrated that lipid nanoparticles containing HIV tat mRNA induce HIV expression in primary CD4 T cells. Here, we sought to further characterize tat mRNA in the context of several benchmark latency reversal agents (LRAs), including inhibitor of apoptosis protein antagonists (IAPi), bromodomain and extra-Terminal motif inhibitors (BETi), and histone deacetylase inhibitors (HDACi). tat mRNA reversed latency across several different cell line models of HIV latency, an effect dependent on the TAR hairpin loop. Synergistic enhancement of tat mRNA activity was observed with IAPi, HDACi, and BETi, albeit to variable degrees. In primary CD4 T cells from durably suppressed people with HIV, tat mRNA profoundly increased the frequencies of elongated, multiply-spliced, and polyadenylated HIV transcripts, while having a lesser impact on TAR transcript frequencies. tat mRNAs alone resulted in variable HIV p24 protein induction across donors. However, tat mRNA in combination with IAPi, BETi, or HDACi markedly enhanced HIV RNA and protein expression without overt cytotoxicity or cellular activation. Notably, combination regimens approached or in some cases exceeded the latency reversal activity of maximal mitogenic T cell stimulation. Higher levels of tat mRNA-driven HIV p24 induction were observed in donors with larger mitogen-inducible HIV reservoirs, and expression increased with prolonged exposure time. Combination LRA strategies employing both small molecule inhibitors and Tat delivered to CD4 T cells are a promising approach to effectively target the HIV reservoir.

Viral and host network analysis of the human cytomegalovirus transcriptome in latency.

HCMV genes UL135 and UL138 play opposing roles regulating latency and reactivation in CD34+ human progenitor cells (HPCs). Using the THP-1 cell line model for latency and reactivation, we designed an RNA sequencing study to compare the transcriptional profile of HCMV infection in the presence and absence of these genes. The loss of UL138 results in elevated levels of viral gene expression and increased differentiation of cell populations that support HCMV gene expression and genome synthesis. The loss of UL135 results in diminished viral gene expression during an initial burst that occurs as latency is established and no expression of eleven viral genes from the ULb' region even following stimulation for differentiation and reactivation. Transcriptional network analysis revealed host transcription factors with potential to regulate the ULb' genes in coordination with pUL135. These results reveal roles for UL135 and UL138 in regulation of viral gene expression and potentially hematopoietic differentiation.

Monocyte to macrophage differentiation and changes in cellular redox homeostasis promote cell type-specific HIV latency reactivation

HIV latency regulation in monocytes and macrophages can vary according to signals directing differentiation, polarization, and function. To investigate these processes, we generated an HIV latency model in THP-1 monocytes and showed differential levels of HIV reactivation among clonal populations. Monocyte-to-macrophage differentiation of HIV-infected primary human CD14+ and THP-1 cells induced HIV reactivation and showed that virus production increased concomitant with macrophage differentiation. We applied the HIV-infected THP-1 monocyte-to-macrophage (MLat) model to assess the biological mechanisms regulating HIV latency dynamics during monocyte-to-macrophage differentiation. We pinpointed protein kinase C signaling pathway activation and Cyclin T1 upregulation as inherent differentiation mechanisms that regulate HIV latency reactivation. Macrophage polarization regulated latency, revealing proinflammatory M1 macrophages suppressed HIV reactivation while anti-inflammatory M2 macrophages promoted HIV reactivation. Because macrophages rely on reactive-oxygen species (ROS) to exert numerous cellular functions, we disrupted redox pathways and found that inhibitors of the thioredoxin (Trx) system acted as latency-promoting agents in T-cells and monocytes, but opposingly acted as latency-reversing agents in macrophages. We explored this mechanism with Auranofin, a clinical candidate for reducing HIV reservoirs, and demonstrated Trx reductase inhibition led to ROS induced NF-κB activity, which promoted HIV reactivation in macrophages, but not in T-cells and monocytes. Collectively, cell type-specific differences in HIV latency regulation could pose a barrier to HIV eradication strategies.