A macrophage-cell model of HIV latency reveals the unusual importance of the bromodomain axis.

BACKGROUND: Although macrophages are now recognized as an essential part of the HIV latent reservoir, whether and how viral latency is established and reactivated in these cell types is poorly understood. To understand the fundamental mechanisms of viral latency in macrophages, there is an urgent need to develop latency models amenable to genetic manipulations and screening for appropriate latency-reversing agents (LRAs). Given that differentiated THP-1 cells resemble monocyte-derived macrophages in HIV replication mechanisms, we set out to establish a macrophage cell model for HIV latency using THP-1 cells. METHODS: We created single-cell clones of THP-1 cells infected with a single copy of the dual-labeled HIVGKO in which a codon switched eGFP (csGFP) is under the control of the HIV-1 5' LTR promoter, and a monomeric Kusabira orange 2 (mKO2) under the control of cellular elongation factor one alpha promoter (EF1α). Latently infected cells are csGFP-, mKO2+, while cells with actively replicating HIV (or reactivated virus) are csGFP+,mKO2+. After sorting for latently infected cells, each of the THP-1 clones with unique integration sites for HIV was differentiated into macrophage-like cells with phorbol 12-myristate 13-acetate (PMA) and treated with established LRAs to stimulate HIV reactivation. Monocyte-derived macrophages (MDMs) harboring single copies of HIVGKO were used to confirm our findings. RESULTS: We obtained clones of THP-1 cells with latently infected HIV with unique integration sites. When the differentiated THP-1 or primary MDMs cells were treated with various LRAs, the bromodomain inhibitors JQ1 and I-BET151 were the most potent compounds. Knockdown of BRD4, the target of JQ1, resulted in increased reactivation, thus confirming the pharmacological effect. The DYRK1A inhibitor Harmine and lipopolysaccharide (LPS) also showed significant reactivation across all three MDM donors. Remarkably, LRAs like PMA/ionomycin, bryostatin-1, and histone deacetylase inhibitors known to potently reactivate latent HIV in CD4 + T cells showed little activity in macrophages. CONCLUSIONS: Our results indicate that this model could be used to screen for appropriate LRAs for macrophages and show that HIV latency and reactivation mechanisms in macrophages may be distinct from those of CD4 + T cells.

Glucocorticoid receptor and specificity protein 1 (Sp1) or Sp3, but not the antibiotic Mithramycin A, stimulates human alphaherpesvirus 1 (HSV-1) replication.

Following acute human alphaherpesvirus 1 (HSV-1) infection of oral-facial mucosal surfaces, sensory neurons in trigeminal ganglia (TG) are important sites for life-long latency. Neurons in the central nervous system, including brainstem, also harbor viral genomes during latency. Periodically, certain cellular stressors trigger reactivation from latency, which can lead to recurrent HSV-1 disease: herpes labialis, herpes stromal keratitis, and encephalitis for example. Activation of the glucocorticoid receptor (GR) by stressful stimuli enhances HSV-1 gene expression, replication, and explant-induced reactivation. GR and certain stress-induced Krüppel like factors (KLF) cooperatively transactivate cis-regulatory modules (CRM) that drive expression of viral transcriptional regulatory proteins (ICP0, ICP4, and ICP27). These CRMs lack GR response elements (GRE); however, specificity protein 1 (Sp1) binding sites are crucial for GR and KLF15 or KLF4 mediated transactivation. Hence, we tested whether Sp1 or Sp3 regulate viral replication and transactivation of the ICP0 promoter. During early stages of explant-induced reactivation from latency, the number of Sp3+ TG neurons were significantly higher relative to TG from latently infected mice. Conversely, Sp1+ TG neurons were only increased in females, but not male mice, during explant-induced reactivation. Sp1 siRNA significantly reduced HSV-1 replication in cultured mouse (Neuro-2A) and monkey (CV-1) cells. Mithramycin A, an antibiotic that has anti-tumor activity preferentially interacts with GC-rich DNA, including Sp1 binding sites, significantly reduced HSV-1 replication indicating it has antiviral activity. GR and Sp1 or Sp3 transactivated the HSV-1 ICP0 promoter in Neuro-2A and CV-1 cells confirming these transcription factors enhance viral replication and gene expression.

The sounds of silencing: dynamic epigenetic control of HIV latency.

PURPOSE OF REVIEW: This review highlights advances in understanding the epigenetic control mechanisms that regulate HIV-1 latency mechanisms in T-cells and microglial cells and describes the potential of current therapeutic approaches targeting the epigenetic machinery to eliminate or block the HIV-1 latent reservoir. RECENT FINDINGS: Large-scale unbiased CRISPR-Cas9 library-based screenings, coupled with biochemical studies, have comprehensively identified the epigenetic factors pivotal in regulating HIV-1 latency, paving the way for potential novel targets in therapeutic development. These studies also highlight how the bivalency observed at the HIV-1 5'LTR primes latent proviruses for rapid reactivation. SUMMARY: The HIV-1 latent is established very early during infection, and its persistence is the major obstacle to achieving an HIV-1 cure. Here, we present a succinct summary of the latest research findings, shedding light on the pivotal roles played by host epigenetic machinery in the control of HIV-1 latency. Newly uncovered mechanisms permitting rapid reversal of epigenetic restrictions upon viral reactivation highlight the formidable challenges of achieving enduring and irreversible epigenetic silencing of HIV-1.

New latency-promoting agents for a block-and-lock functional cure strategy.

PURPOSE OF REVIEW: Currently, HIV-infected patients are treated with antiretroviral therapy. However, when the treatment is interrupted, viral rebound occurs from latently infected cells. Therefore, scientists aim to develop an HIV-1 cure which eradicates or permanently silences the latent reservoir. RECENT FINDINGS: Previously, scientists focused on the shock-and-kill cure strategy, which aims to eradicate the latent reservoir using latency-reactivating agents. Limited success shifts the interest towards the block-and-lock cure approach, which aims to achieve a functional cure by "blocking" HIV-1 transcription and "locking" the provirus in a deep latent state, resistant to treatment-interruption. In this strategy, latency promoting agents are used to induce transcriptional silencing and alter the epigenetics environment at the HIV promotor. SUMMARY: For the block-and-lock cure strategy to succeed more investigation into the transcriptional and epigenetic regulation of HIV-1 gene expression is necessary to design optimal latency-promoting agents. In this review, we will discuss the latency promoting agents that have been described in literature during the past 2 years (2022-2023).

HIV-Tocky system to visualize proviral expression dynamics.

Determinants of HIV-1 latency establishment are yet to be elucidated. HIV reservoir comprises a rare fraction of infected cells that can survive host and virus-mediated killing. In vitro reporter models so far offered a feasible means to inspect this population, but with limited capabilities to dissect provirus silencing dynamics. Here, we describe a new HIV reporter model, HIV-Timer of cell kinetics and activity (HIV-Tocky) with dual fluorescence spontaneous shifting to reveal provirus silencing and reactivation dynamics. This unique feature allows, for the first time, identifying two latent populations: a directly latent, and a recently silenced subset, with the latter having integration features suggestive of stable latency. Our proposed model can help address the heterogeneous nature of HIV reservoirs and offers new possibilities for evaluating eradication strategies.

HIV-1 Transcription and Latency in the Spotlight.

At every integrated HIV-1 genome, there is a transcriptional cycle that ultimately shapes proviral fate [...].

HIV-1 Proviral Genome Engineering with CRISPR-Cas9 for Mechanistic Studies.

HIV-1 latency remains a barrier to a functional cure because of the ability of virtually silent yet inducible proviruses within reservoir cells to transcriptionally reactivate upon cell stimulation. HIV-1 reactivation occurs through the sequential action of host transcription factors (TFs) during the "host phase" and the viral TF Tat during the "viral phase", which together facilitate the positive feedback loop required for exponential transcription, replication, and pathogenesis. The sequential action of these TFs poses a challenge to precisely delineate the contributions of the host and viral phases of the transcriptional program to guide future mechanistic and therapeutic studies. To address this limitation, we devised a genome engineering approach to mutate tat and create a genetically matched pair of Jurkat T cell clones harboring HIV-1 at the same integration site with and without Tat expression. By comparing the transcriptional profile of both clones, the transition point between the host and viral phases was defined, providing a system that enables the temporal mechanistic interrogation of HIV-1 transcription prior to and after Tat synthesis. Importantly, this CRISPR method is broadly applicable to knockout individual viral proteins or genomic regulatory elements to delineate their contributions to various aspects of the viral life cycle and ultimately may facilitate therapeutic approaches in our race towards achieving a functional cure.

Host-encoded CTCF regulates human cytomegalovirus latency via chromatin looping.

Human cytomegalovirus (HCMV) is a prevalent pathogen that establishes life-long latent infection in hematopoietic cells. While this infection is usually asymptomatic, immune dysregulation leads to viral reactivation, which can cause significant morbidity and mortality. However, the mechanisms underpinning reactivation remain incompletely understood. The HCMV major immediate early promoter (MIEP)/enhancer is a key factor in this process, as its transactivation from a repressed to active state helps drive viral gene transcription necessary for reactivation from latency. Numerous host transcription factors bind the MIE locus and recruit repressive chromatin modifiers, thus impeding virus reactivation. One such factor is CCCTC-binding protein (CTCF), a highly conserved host zinc finger protein that mediates chromatin conformation and nuclear architecture. However, the mechanisms by which CTCF contributes to HCMV latency were previously unexplored. Here, we confirm that CTCF binds two convergent sites within the MIE locus during latency in primary CD14+ monocytes, and following cellular differentiation, CTCF association is lost as the virus reactivates. While mutation of the MIE enhancer CTCF binding site does not impact viral lytic growth in fibroblasts, this mutant virus fails to maintain latency in myeloid cells. Furthermore, we show the two convergent CTCF binding sites allow looping to occur across the MIEP, supporting transcriptional repression during latency. Indeed, looping between the two sites diminishes during virus reactivation, concurrent with activation of MIE transcription. Taken together, our data reveal that three-dimensional chromatin looping aids in the regulation of HCMV latency and provides insight into promoter/enhancer regulation that may prove broadly applicable across biological systems.

A role of BPTF in viral oncogenicity delineated through studies of heritable Kaposi sarcoma.

Kaposi sarcoma (KS), caused by Herpesvirus-8 (HHV-8; KSHV), shows sporadic, endemic, and epidemic forms. While familial clustering of KS was previously recorded, the molecular basis of hereditary predilection to KS remains largely unknown. We demonstrate through genetic studies that a dominantly inherited missense mutation in BPTF segregates with a phenotype of classical KS in multiple immunocompetent individuals in two families. Using an rKSHV.219-infected CRISPR/cas9-model, we show that BPTFI2012T mutant cells exhibit higher latent-to-lytic ratio, decreased virion production, increased LANA staining, and latent phenotype in viral transcriptomics. RNA-sequencing demonstrated that KSHV infection dysregulated oncogenic-like response and P53 pathways, MAPK cascade, and blood vessel development pathways, consistent with KS. BPTFI2012T also enriched pathways of viral genome regulation and replication, immune response, and chemotaxis, including downregulation of IFI16, SHFL HLAs, TGFB1, and HSPA5, all previously associated with KSHV infection and tumorigenesis. Many of the differentially expressed genes are regulated by Rel-NF-κB, which regulates immune processes, cell survival, and proliferation and is pivotal to oncogenesis. We thus demonstrate BPTF mutation-mediated monogenic hereditary predilection of KSHV virus-induced oncogenesis, and suggest BPTF as a drug target.

Novel role of host protein SLC25A42 in the HIV-1 reactivation of latent HIV-1 provirus.

Despite the effectiveness of combination antiretroviral therapy, human immunodeficiency virus (HIV) infection remains incurable. To seek new strategies to overcome HIV type 1 (HIV-1) latency, one of the major barriers to HIV elimination, it is crucial to better understand how this state is maintained. Here, by means of an RNA interference screen employing an HIV-1 latency model using monocytic cell lines, we identified solute carrier family 25 member 42 (SLC25A42) as a potential host factor not previously known to affect HIV-1 latency. SLC25A42 knockdown resulted in increased HIV-1 expression, whereas forced expression of exogenous SLC25A42 suppressed it in SLC25A42-depleted cells. SLC25A42 depletion increased HIV-1 proviral transcriptional elongation but did not cause HIV-1 activation in an HIV-1 Tat-depleted latency model. This suggests that the role of SLC25A42 in HIV-1 transcription depends on HIV-1 Tat. Chromatin immunoprecipitation-qPCR analysis further revealed that SLC25A42 accumulated on or near the HIV-1 5' long terminal repeat promoter region of the HIV-1 provirus, suggesting a possible role in regulating HIV-1 Tat near this promoter region. These results indicate that SLC25A42 plays a novel role in HIV-1 latency maintenance in monocytic HIV-1 reservoirs.

Kaposi's sarcoma herpesvirus latency-associated nuclear antigen broadly regulates viral gene expression and is essential for lytic infection.

Kaposi's sarcoma herpesvirus (KSHV) is a leading cause of malignancy in AIDS and current therapies are limited. Like all herpesviruses, KSHV infection can be latent or lytic. KSHV latency-associated nuclear antigen (LANA) is essential for viral genome persistence during latent infection. LANA also maintains latency by antagonizing expression and function of the KSHV lytic switch protein, RTA. Here, we find LANA null KSHV is not capable of lytic replication, indicating a requirement for LANA. While LANA promoted both lytic and latent gene expression in cells partially permissive for lytic infection, it repressed expression in non-permissive cells. Importantly, forced RTA expression in non-permissive cells led to induction of lytic infection and LANA switched to promote, rather than repress, most lytic viral gene expression. When basal viral gene expression levels were high, LANA promoted expression, but repressed expression at low basal levels unless RTA expression was forcibly induced. LANA's effects were broad, but virus gene specific, extending to an engineered, recombinant viral GFP under control of host EF1α promoter, but not to host EF1α. Together, these results demonstrate that, in addition to its essential role in genome maintenance, LANA broadly regulates viral gene expression, and is required for high levels of lytic gene expression during lytic infection. Strategies that target LANA are expected to abolish KSHV infection.

Multifaceted roles for STAT3 in gammaherpesvirus latency revealed through in vivo B cell knockout models.

Cancers associated with the oncogenic gammaherpesviruses, Epstein-Barr virus and Kaposi sarcoma herpesvirus, are notable for their constitutive activation of the transcription factor signal transducer and activator of transcription 3 (STAT3). To better understand the role of STAT3 during gammaherpesvirus latency and the B cell response to infection, we used the model pathogen murine gammaherpesvirus 68 (MHV68). Genetic deletion of STAT3 in B cells of CD19cre/+Stat3f/f mice reduced peak MHV68 latency approximately sevenfold. However, infected CD19cre/+Stat3f/f mice exhibited disordered germinal centers and heightened virus-specific CD8 T cell responses compared to wild-type (WT) littermates. To circumvent the systemic immune alterations observed in the B cell-STAT3 knockout mice and more directly evaluate intrinsic roles for STAT3, we generated mixed bone marrow chimeric mice consisting of WT and STAT3 knockout B cells. We discovered a dramatic reduction in latency in STAT3 knockout B cells compared to their WT B cell counterparts in the same lymphoid organ. RNA sequencing of sorted germinal center B cells revealed that MHV68 infection shifts the gene signature toward proliferation and away from type I and type II IFN responses. Loss of STAT3 largely reversed the virus-driven transcriptional shift without impacting the viral gene expression program. STAT3 promoted B cell processes of the germinal center, including IL-21-stimulated downregulation of surface CD23 on B cells infected with MHV68 or EBV. Together, our data provide mechanistic insights into the role of STAT3 as a latency determinant in B cells for oncogenic gammaherpesviruses.IMPORTANCEThere are no directed therapies to the latency program of the human gammaherpesviruses, Epstein-Barr virus and Kaposi sarcoma herpesvirus. Activated host factor signal transducer and activator of transcription 3 (STAT3) is a hallmark of cancers caused by these viruses. We applied the murine gammaherpesvirus pathogen system to explore STAT3 function upon primary B cell infection in the host. Since STAT3 deletion in all CD19+ B cells of infected mice led to altered B and T cell responses, we generated chimeric mice with both normal and STAT3-deleted B cells. B cells lacking STAT3 failed to support virus latency compared to normal B cells from the same infected animal. Loss of STAT3 impaired B cell proliferation and differentiation and led to a striking upregulation of interferon-stimulated genes. These findings expand our understanding of STAT3-dependent processes that are key to its function as a pro-viral latency determinant for oncogenic gammaherpesviruses in B cells and may provide novel therapeutic targets.

Role of long noncoding RNA in regulating HIV infection-a comprehensive review.

A complete cure against human immunodeficiency virus (HIV) infection remains out of reach, as the virus persists in stable cell reservoirs that are resistant to antiretroviral therapy. The key to eliminating these reservoirs lies in deciphering the processes that govern viral gene expression and latency. However, while we comprehensively understand how host proteins influence HIV gene expression and viral latency, the emerging role of long noncoding RNAs (lncRNAs) in the context of T cell activation, HIV gene expression, and viral latency remain unexplored. This review dives into the evolving significance of lncRNAs and their impact on HIV gene expression and viral latency. We provide an overview of the current knowledge regarding how lncRNAs regulate HIV gene expression, categorizing them as either activators or inhibitors of viral gene expression and infectivity. Furthermore, we offer insights into the potential therapeutic applications of lncRNAs in combatting HIV. A deeper understanding of how lncRNAs modulate HIV gene transcription holds promise for developing novel RNA-based therapies to complement existing treatment strategies to eradicate HIV reservoirs.

Kaposi's sarcoma herpesvirus exploits the DNA damage response to circularize its genome.

To establish lifelong, latent infection, herpesviruses circularize their linear, double-stranded, DNA genomes through an unknown mechanism. Kaposi's sarcoma (KS) herpesvirus (KSHV), a gamma herpesvirus, is tightly linked with KS, primary effusion lymphoma, and multicentric Castleman's disease. KSHV persists in latently infected cells as a multi-copy, extrachromosomal episome. Here, we show the KSHV genome rapidly circularizes following infection, and viral protein expression is unnecessary for this process. The DNA damage response (DDR) kinases, ATM and DNA-PKcs, each exert roles, and absence of both severely compromises circularization and latency. These deficiencies were rescued by expression of ATM and DNA-PKcs, but not catalytically inactive mutants. In contrast, γH2AX did not function in KSHV circularization. The linear viral genomic ends resemble a DNA double strand break, and non-homologous DNA end joining (NHEJ) and homologous recombination (HR) reporters indicate both NHEJ and HR contribute to KSHV circularization. Last, we show, similar to KSHV, ATM and DNA-PKcs have roles in circularization of the alpha herpesvirus, herpes simplex virus-1 (HSV-1), while γH2AX does not. Therefore, the DDR mediates KSHV and HSV-1 circularization. This strategy may serve as a general herpesvirus mechanism to initiate latency, and its disruption may provide new opportunities for prevention of herpesvirus disease.

Targeting noncoding RNAs to reactivate or eliminate latent HIV reservoirs.

PURPOSE OF REVIEW: Expression of noncoding RNAs (ncRNAs) is more tissue and cell type-specific than expression of protein-coding genes. Understanding the mechanisms of action of ncRNAs and their roles in HIV replication and latency may inform targets for the latent HIV reservoir reactivation or elimination with high specificity to CD4 + T cells latently infected with HIV. RECENT FINDINGS: While the number of studies in the field of ncRNAs and HIV is limited, evidence points to complex interactions between different ncRNAs, protein-coding RNAs, and proteins. Latency-reversing agents modulate the expression of ncRNAs, with some effects being inhibitory for HIV reactivation. An important limitation of basic research on the ncRNA mechanisms of action is the reliance on cell lines. Because of cell type specificity, it is uncertain whether the ncRNAs function similarly in primary cells. SUMMARY: Comprehensive functional screens to uncover all ncRNAs that regulate HIV expression and the detailed exploration of their mechanisms of action in relevant cell types are needed to identify promising targets for HIV reservoir clearance. Classes of ncRNAs as a whole rather than individual ncRNAs might represent an attractive target for reservoir elimination. Compound screens for latency reversal should factor in the complexity of their effects on ncRNAs.

Kaposi's sarcoma-associated herpesvirus terminal repeat regulates inducible lytic gene promoters.

The Kaposi's sarcoma-associated herpesvirus (KSHV) genome consists of an approximately 140-kb unique coding region flanked by 30-40 copies of a 0.8-kb terminal repeat (TR) sequence. A gene enhancer recruits transcription-related enzymes by having arrays of transcription factor binding sites. Here, we show that KSHV TR possesses transcription regulatory function with latency-associated nuclear antigen (LANA). Cleavage under targets and release using nuclease demonstrated that TR fragments were occupied by LANA-interacting histone-modifying enzymes in naturally infected cells. The TR was enriched with histone H3K27 acetylation (H3K27Ac) and H3K4 tri-methylation (H3K4me3) modifications and also expressed nascent RNAs. The sites of H3K27Ac and H3K4me3 modifications were also conserved in the KSHV unique region among naturally infected primary effusion lymphoma cells. KSHV origin of lytic replication (Ori-Lyt) showed similar protein and histone modification occupancies with that of TR. In the Ori-Lyt region, the LANA and LANA-interacting proteins colocalized with an H3K27Ac-modified nucleosome along with paused RNA polymerase II. The KSHV transactivator KSHV replication and transcription activator (K-Rta) recruitment sites franked the LANA-bound nucleosome, and reactivation evicted the LANA-bound nucleosome. Including TR fragments in reporter plasmid enhanced inducible viral gene promoter activities independent of the orientations. In the presence of TR in reporter plasmids, K-Rta transactivation was drastically increased, while LANA acquired the promoter repression function. KSHV TR, therefore, functions as an enhancer for KSHV inducible genes. However, in contrast to cellular enhancers bound by multiple transcription factors, perhaps the KSHV enhancer is predominantly regulated by the LANA nuclear body.IMPORTANCEEnhancers are a crucial regulator of differential gene expression programs. Enhancers are the cis-regulatory sequences determining target genes' spatiotemporal and quantitative expression. Here, we show that Kaposi's sarcoma-associated herpesvirus (KSHV) terminal repeats fulfill the enhancer definition for KSHV inducible gene promoters. The KSHV enhancer is occupied by latency-associated nuclear antigen (LANA) and its interacting proteins, such as CHD4. Neighboring terminal repeat (TR) fragments to lytic gene promoters drastically enhanced KSHV replication and transcription activator and LANA transcription regulatory functions. This study, thus, proposes a new latency-lytic switch model in which TR accessibility to the KSHV gene promoters regulates viral inducible gene expression.

KSHV-encoded LANA bypasses transcriptional block through the stabilization of RNA Pol II in hypoxia.

IMPORTANCE: Hypoxia can induce the reactivation of Kaposi sarcoma-associated virus (KSHV), which necessitates the synthesis of critical structural proteins. Despite the unfavorable energetic conditions of hypoxia, KSHV utilizes mechanisms to prevent the degradation of essential cellular machinery required for successful reactivation. Our study provides new insights on strategies employed by KSHV-infected cells to maintain steady-state transcription by overcoming hypoxia-mediated metabolic stress to enable successful reactivation. Our discovery that the interaction of latency-associated nuclear antigen with HIF1α and NEDD4 inhibits its polyubiquitination activity, which blocks the degradation of RNA Pol II during hypoxia, is a significant contribution to our understanding of KSHV biology. This newfound knowledge provides new leads in the development of novel therapies for KSHV-associated diseases.

CD4+ T cell-targeting immunoliposomes for treatment of latent HIV reservoir.

Active targeting nano-delivery is a promising approach to enhance therapeutic efficacy and specificity to the target cells. Liposomes (LPs) have been widely studied for the active targeting delivery due to their low toxicity, biodegradability, biocompatibility, and feasibility of surface medication to provide the interactions with cell receptors. One of the strategies is to functionalize the surface of LPs with monoclonal antibodies (mAbs) to obtain immunoliposomes (imLPs) that recognize specific receptors on target cells. Among several target cells, CD4+ T cells are known for playing a pivotal role in controlling the immune system in several diseases, including cancers, inflammatory diseases, and viral infections, particularly HIV-1. Here, we demonstrate two methods for conjugating αCD4 mAb with imLPs for specific targeting of CD4+ T cells that can harbor viral genome and serve as a predominant latent HIV reservoir. LPs conjugated with αCD4 mAb via neutravidin-biotin linkage were used for selectively targeting CD4+ J-Lat 10.6 cells. We demonstrate, via flow cytometry, the importance of the conjugation step, mAb density, and the presence of polyethylene glycol (PEG) for effective drug delivery to CD4+ T cells. The cellular uptake of imLPs is substantially higher if the imLPs are functionalized with the pre-conjugated αCD4 mAb-neutravidin complex. Furthermore, imLPs loaded with HIV-1 latency reversing agent, suberoylanilide hydroxamic acid (SAHA), could reactivate the J-Lat 10.6 cells, suggesting that the αCD4-imLPs could be potentially used as a targeted drug delivery system for HIV-1 latency reactivation or other CD4-targeted immunotherapies.

A distinct isoform of lymphoid enhancer binding factor 1 (LEF1) epigenetically restricts EBV reactivation to maintain viral latency.

As a human tumor virus, EBV is present as a latent infection in its associated malignancies where genetic and epigenetic changes have been shown to impede cellular differentiation and viral reactivation. We reported previously that levels of the Wnt signaling effector, lymphoid enhancer binding factor 1 (LEF1) increased following EBV epithelial infection and an epigenetic reprogramming event was maintained even after loss of the viral genome. Elevated LEF1 levels are also observed in nasopharyngeal carcinoma and Burkitt lymphoma. To determine the role played by LEF1 in the EBV life cycle, we used in silico analysis of EBV type 1 and 2 genomes to identify over 20 Wnt-response elements, which suggests that LEF1 may bind directly to the EBV genome and regulate the viral life cycle. Using CUT&RUN-seq, LEF1 was shown to bind the latent EBV genome at various sites encoding viral lytic products that included the immediate early transactivator BZLF1 and viral primase BSLF1 genes. The LEF1 gene encodes various long and short protein isoforms. siRNA depletion of specific LEF1 isoforms revealed that the alternative-promoter derived isoform with an N-terminal truncation (ΔN LEF1) transcriptionally repressed lytic genes associated with LEF1 binding. In addition, forced expression of the ΔN LEF1 isoform antagonized EBV reactivation. As LEF1 repression requires histone deacetylase activity through either recruitment of or direct intrinsic histone deacetylase activity, siRNA depletion of LEF1 resulted in increased histone 3 lysine 9 and lysine 27 acetylation at LEF1 binding sites and across the EBV genome. Taken together, these results indicate a novel role for LEF1 in maintaining EBV latency and restriction viral reactivation via repressive chromatin remodeling of critical lytic cycle factors.

Nucleolin Regulates the Expression of Kaposi's Sarcoma-Associated Herpesvirus' Latency-Associated Nuclear Antigen through G-Quadruplexes in the mRNA.

Kaposi's sarcoma-associated herpesvirus (KSHV) establishes life-long latent infection and is linked to several human malignancies. Latency-associated nuclear antigen (LANA) is highly expressed during latency, and is responsible for the replication and maintenance of the viral genome. The expression of LANA is regulated at transcriptional/translational levels through multiple mechanisms, including the secondary structures in the mRNA sequence. LANA mRNA has multiple G-quadruplexes (G4s) that are bound by multiple proteins to stabilize/destabilize these secondary structures for regulating LANA. In this manuscript, we demonstrate the role of Nucleolin (NCL) in regulating LANA expression through its interaction with G-quadruplexes of LANA mRNA. This interaction reduced LANA's protein expression through the sequestration of mRNA into the nucleus, demonstrated by the colocalization of G4-carrying mRNA with NCL. Furthermore, the downregulation of NCL, by way of a short hairpin, showed an increase in LANA translation following an alteration in the levels of LANA mRNA in the cytoplasm. Overall, the data presented in this manuscript showed that G-quadruplexes-mediated translational control could be regulated by NCL, which can be exploited for controlling KSHV latency.