Modeling HIV-1 Infection in CNS via Infected Monocytes Using Immunocompetent Brain Organoids.

The development of 3D-organoid models has revolutionized the way diseases are studied. Recently, our brain organoid model has been shown to recapitulate in in vitro the human brain cytoarchitecture originally encountered in HIV-1 neuropathogenesis, allowing downstream applications. Infected monocytes, macrophages, and microglia are critically important immune cells for infection and dissemination of HIV-1 throughout brain during acute and chronic phase of the disease. Once in the brain parenchyma, long-lived infected monocytes/macrophages along with resident microglia contribute to the establishment of CNS latency in people with HIV (PWH). Hence, it is important to better understand how HIV-1 enters and establishes infection and latency in CNS to further develop cure strategies. Here we detailed an accessible protocol to incorporate monocytes (infected and/or labeled) as a model of transmigration of peripheral monocytes into brain organoids that can be applied to characterize HIV-1 neuroinvasion and virus dissemination.

Downregulation of KLF5 by EBER1 via the ERK signaling pathway in EBV-positive nasopharyngeal carcinoma cells: implications for latent EBV infection.

Nasopharyngeal carcinoma (NPC) carcinogenesis and malignant transformation are intimately associated with Epstein-Barr virus (EBV) infection. A zinc-fingered transcription factor known as Krüppel-like factor 5 (KLF5) has been shown to be aberrantly expressed in a number of cancer types. However, little is known about the regulatory pathways and roles of KLF5 in EBV-positive NPC. Our study found that KLF5 expression was significantly lower in EBV-positive NPC than in EBV-negative NPC. Further investigation revealed that EBER1, which is encoded by EBV, down-regulates KLF5 via the extracellular signal-regulated kinase (ERK) signalling pathway. This down-regulation of KLF5 by EBER1 contributes to maintaining latent EBV infection in NPC. Furthermore, we uncovered the biological roles of KLF5 in NPC cells. Specifically, KLF5 may influence the cell cycle, prevent apoptosis, and encourage cell migration and proliferation - all of which have a generally pro-cancer impact. In conclusion, these findings offer novel strategies for EBV-positive NPC patients' antitumour treatment.

The nucleic acid binding protein SFPQ represses EBV lytic reactivation by promoting histone H1 expression.

Epstein-Barr virus (EBV) uses a biphasic lifecycle of latency and lytic reactivation to infect >95% of adults worldwide. Despite its central role in EBV persistence and oncogenesis, much remains unknown about how EBV latency is maintained. We used a human genome-wide CRISPR/Cas9 screen to identify that the nuclear protein SFPQ was critical for latency. SFPQ supported expression of linker histone H1, which stabilizes nucleosomes and regulates nuclear architecture, but has not been previously implicated in EBV gene regulation. H1 occupied latent EBV genomes, including the immediate early gene BZLF1 promoter. Upon reactivation, SFPQ was sequestered into sub-nuclear puncta, and EBV genomic H1 occupancy diminished. Enforced H1 expression blocked EBV reactivation upon SFPQ knockout, confirming it as necessary downstream of SFPQ. SFPQ knockout triggered reactivation of EBV in B and epithelial cells, as well as of Kaposi's sarcoma-associated herpesvirus in B cells, suggesting a conserved gamma-herpesvirus role. These findings highlight SFPQ as a major regulator of H1 expression and EBV latency.

Gene editing for latent herpes simplex virus infection reduces viral load and shedding in vivo.

Anti-HSV therapies are only suppressive because they do not eliminate latent HSV present in ganglionic neurons, the source of recurrent disease. We have developed a potentially curative approach against HSV infection, based on gene editing using HSV-specific meganucleases delivered by adeno-associated virus (AAV) vectors. Gene editing performed with two anti-HSV-1 meganucleases delivered by a combination of AAV9, AAV-Dj/8, and AAV-Rh10 can eliminate 90% or more of latent HSV DNA in mouse models of orofacial infection, and up to 97% of latent HSV DNA in mouse models of genital infection. Using a pharmacological approach to reactivate latent HSV-1, we demonstrate that ganglionic viral load reduction leads to a significant decrease of viral shedding in treated female mice. While therapy is well tolerated, in some instances, we observe hepatotoxicity at high doses and subtle histological evidence of neuronal injury without observable neurological signs or deficits. Simplification of the regimen through use of a single serotype (AAV9) delivering single meganuclease targeting a duplicated region of the HSV genome, dose reduction, and use of a neuron-specific promoter each results in improved tolerability while retaining efficacy. These results reinforce the curative potential of gene editing for HSV disease.

Lytic and Latent Genetic Diversity of the Epstein-Barr Virus Reveals Raji-Related Variants from Southeastern Brazil Associated with Recombination Markers.

Epstein-Barr virus (EBV) is a ubiquitous gammaherpesvirus etiologically associated with benign and malignant diseases. Since the pathogenic mechanisms of EBV are not fully understood, understanding EBV genetic diversity is an ongoing goal. Therefore, the present work describes the genetic diversity of the lytic gene BZLF1 in a sampling of 70 EBV-positive cases from southeastern Brazil. Additionally, together with the genetic regions previously characterized, the aim of the present study was to determine the impact of viral genetic factors that may influence EBV genetic diversity. Accordingly, the phylogenetic analysis of the BZLF1 indicated two main clades with high support, BZ-A and BZ-B (PP > 0.85). Thus, the BZ-A clade was the most diverse clade associated with the main polymorphisms investigated, including the haplotype Type 1 + V3 (p < 0.001). Furthermore, the multigene phylogenetic analysis (MLA) between BZLF1 and the oncogene LMP1 showed specific clusters, revealing haplotypic segregation that previous single-gene phylogenies from both genes failed to demonstrate. Surprisingly, the LMP1 Raji-related variant clusters were shown to be more diverse, associated with BZ-A/B and the Type 2/1 + V3 haplotypes. Finally, due to the high haplotypic diversity of the Raji-related variants, the number of DNA recombination-inducing motifs (DRIMs) was evaluated within the different clusters defined by the MLA. Similarly, the haplotype BZ-A + Raji was shown to harbor a greater number of DRIMs (p < 0.001). These results call attention to the high haplotype diversity of EBV in southeast Brazil and strengthen the hypothesis of the recombinant potential of South American Raji-related variants via the LMP1 oncogene.

Latent Epstein-Barr virus infection collaborates with Myc over-expression in normal human B cells to induce Burkitt-like Lymphomas in mice.

Epstein-Barr virus (EBV) is an important cause of human lymphomas, including Burkitt lymphoma (BL). EBV+ BLs are driven by Myc translocation and have stringent forms of viral latency that do not express either of the two major EBV oncoproteins, EBNA2 (which mimics Notch signaling) and LMP1 (which activates NF-κB signaling). Suppression of Myc-induced apoptosis, often through mutation of the TP53 (p53) gene or inhibition of pro-apoptotic BCL2L11 (BIM) gene expression, is required for development of Myc-driven BLs. EBV+ BLs contain fewer cellular mutations in apoptotic pathways compared to EBV-negative BLs, suggesting that latent EBV infection inhibits Myc-induced apoptosis. Here we use an EBNA2-deleted EBV virus (ΔEBNA2 EBV) to create the first in vivo model for EBV+ BL-like lymphomas derived from primary human B cells. We show that cord blood B cells infected with both ΔEBNA2 EBV and a Myc-expressing vector proliferate indefinitely on a CD40L/IL21 expressing feeder layer in vitro and cause rapid onset EBV+ BL-like tumors in NSG mice. These LMP1/EBNA2-negative Myc-driven lymphomas have wild type p53 and very low BIM, and express numerous germinal center B cell proteins (including TCF3, BACH2, Myb, CD10, CCDN3, and GCSAM) in the absence of BCL6 expression. Myc-induced activation of Myb mediates expression of many of these BL-associated proteins. We demonstrate that Myc blocks LMP1 expression both by inhibiting expression of cellular factors (STAT3 and Src) that activate LMP1 transcription and by increasing expression of proteins (DNMT3B and UHRF1) known to enhance DNA methylation of the LMP1 promoters in human BLs. These results show that latent EBV infection collaborates with Myc over-expression to induce BL-like human B-cell lymphomas in mice. As NF-κB signaling retards the growth of EBV-negative BLs, Myc-mediated repression of LMP1 may be essential for latent EBV infection and Myc translocation to collaboratively induce human BLs.

Germinal center cytokine driven epigenetic control of Epstein-Barr virus latency gene expression.

Epstein-Barr virus (EBV) persistently infects 95% of adults worldwide and is associated with multiple human lymphomas that express characteristic EBV latency programs used by the virus to navigate the B-cell compartment. Upon primary infection, the EBV latency III program, comprised of six Epstein-Barr Nuclear Antigens (EBNA) and two Latent Membrane Protein (LMP) antigens, drives infected B-cells into germinal center (GC). By incompletely understood mechanisms, GC microenvironmental cues trigger the EBV genome to switch to the latency II program, comprised of EBNA1, LMP1 and LMP2A and observed in GC-derived Hodgkin lymphoma. To gain insights into pathways and epigenetic mechanisms that control EBV latency reprogramming as EBV-infected B-cells encounter microenvironmental cues, we characterized GC cytokine effects on EBV latency protein expression and on the EBV epigenome. We confirmed and extended prior studies highlighting GC cytokine effects in support of the latency II transition. The T-follicular helper cytokine interleukin 21 (IL-21), which is a major regulator of GC responses, and to a lesser extent IL-4 and IL-10, hyper-induced LMP1 expression, while repressing EBNA expression. However, follicular dendritic cell cytokines including IL-15 and IL-27 downmodulate EBNA but not LMP1 expression. CRISPR editing highlighted that STAT3 and STAT5 were necessary for cytokine mediated EBNA silencing via epigenetic effects at the EBV genomic C promoter. By contrast, STAT3 was instead necessary for LMP1 promoter epigenetic remodeling, including gain of activating histone chromatin marks and loss of repressive polycomb repressive complex silencing marks. Thus, EBV has evolved to coopt STAT signaling to oppositely regulate the epigenetic status of key viral genomic promoters in response to GC cytokine cues.

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.

Deciphering the Role of Epstein-Barr Virus Latent Membrane Protein 1 in Immune Modulation: A Multifaced Signalling Perspective.

The disruption of antiviral sensors and the evasion of immune defences by various tactics are hallmarks of EBV infection. One of the EBV latent gene products, LMP1, was shown to induce the activation of signalling pathways, such as NF-κB, MAPK (JNK, ERK1/2, p38), JAK/STAT and PI3K/Akt, via three subdomains of its C-terminal domain, regulating the expression of several cytokines responsible for modulation of the immune response and therefore promoting viral persistence. The aim of this review is to summarise the current knowledge on the EBV-mediated induction of immunomodulatory molecules by the activation of signal transduction pathways with a particular focus on LMP1-mediated mechanisms. A more detailed understanding of the cytokine biology molecular landscape in EBV infections could contribute to the more complete understanding of diseases associated with this virus.

Herpesvirus Infection of Endothelial Cells as a Systemic Pathological Axis in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome.

Understanding the pathophysiology of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is critical for advancing treatment options. This review explores the novel hypothesis that a herpesvirus infection of endothelial cells (ECs) may underlie ME/CFS symptomatology. We review evidence linking herpesviruses to persistent EC infection and the implications for endothelial dysfunction, encompassing blood flow regulation, coagulation, and cognitive impairment-symptoms consistent with ME/CFS and Long COVID. This paper provides a synthesis of current research on herpesvirus latency and reactivation, detailing the impact on ECs and subsequent systemic complications, including latent modulation and long-term maladaptation. We suggest that the chronicity of ME/CFS symptoms and the multisystemic nature of the disease may be partly attributable to herpesvirus-induced endothelial maladaptation. Our conclusions underscore the necessity for further investigation into the prevalence and load of herpesvirus infection within the ECs of ME/CFS patients. This review offers conceptual advances by proposing an endothelial infection model as a systemic mechanism contributing to ME/CFS, steering future research toward potentially unexplored avenues in understanding and treating this complex syndrome.

SARS-CoV-2 superinfection in CD14+ monocytes with latent human cytomegalovirus (HCMV) promotes inflammatory cascade.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiologic agent of coronavirus disease 2019 (COVID-19), has posed significant challenges to global health. While much attention has been directed towards understanding the primary mechanisms of SARS-CoV-2 infection, emerging evidence suggests co-infections or superinfections with other viruses may contribute to increased morbidity and mortality, particularly in severe cases of COVID-19. Among viruses that have been reported in patients with SARS-CoV-2, seropositivity for Human cytomegalovirus (HCMV) is associated with increased COVID-19 risk and hospitalization. HCMV is a ubiquitous beta-herpesvirus with a seroprevalence of 60-90 % worldwide and one of the leading causes of mortality in immunocompromised individuals. The primary sites of latency for HCMV include CD14+ monocytes and CD34+ hematopoietic cells. In this study, we sought to investigate SARS-CoV-2 infection of CD14+ monocytes latently infected with HCMV. We demonstrate that CD14+ cells are susceptible and permissive to SARS-CoV-2 infection and detect subgenomic transcripts indicative of replication. To further investigate the molecular changes triggered by SARS-CoV-2 infection in HCMV-latent CD14+ monocytes, we conducted RNA sequencing coupled with bioinformatic differential gene analysis. The results revealed significant differences in cytokine-cytokine receptor interactions and inflammatory pathways in cells superinfected with replication-competent SARS-CoV-2 compared to the heat-inactivated and mock controls. Notably, there was a significant upregulation in transcripts associated with pro-inflammatory response factors and a decrease in anti-inflammatory factors. Taken together, these findings provide a basis for the heightened inflammatory response, offering potential avenues for targeted therapeutic interventions among HCMV-infected severe cases of COVID-19. SUMMARY: COVID-19 patients infected with secondary viruses have been associated with a higher prevalence of severe symptoms. Individuals seropositive for human cytomegalovirus (HCMV) infection are at an increased risk for severe COVID-19 disease and hospitalization. HCMV reactivation has been reported in severe COVID-19 cases with respiratory failure and could be the result of co-infection with SARS-CoV-2 and HCMV. In a cell culture model of superinfection, HCMV has previously been shown to increase infection of SARS-CoV-2 of epithelial cells by upregulating the human angiotensin-converting enzyme-2 (ACE2) receptor. In this study, we utilize CD14+ monocytes, a major cell type that harbors latent HCMV, to investigate co-infection of SARS-CoV-2 and HCMV. This study is a first step toward understanding the mechanism that may facilitate increased COVID-19 disease severity in patients infected with SARS-CoV-2 and HCMV.

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.

Differential carbonic anhydrase activities control EBV-induced B-cell transformation and lytic cycle reactivation.

Epstein-Barr virus (EBV) contributes to ~1% of all human cancers including several B-cell neoplasms. A characteristic feature of EBV life cycle is its ability to transform metabolically quiescent B-lymphocytes into hyperproliferating B-cell blasts with the establishment of viral latency, while intermittent lytic cycle induction is necessary for the production of progeny virus. Our RNA-Seq analyses of both latently infected naïve B-lymphocytes and transformed B-lymphocytes upon lytic cycle replication indicate a contrasting expression pattern of a membrane-associated carbonic anhydrase isoform CA9, an essential component for maintaining cell acid-base homeostasis. We show that while CA9 expression is transcriptionally activated during latent infection model, lytic cycle replication restrains its expression. Pharmacological inhibition of CA-activity using specific inhibitors retards EBV induced B-cell transformation, inhibits B-cells outgrowth and colony formation ability of transformed B-lymphocytes through lowering the intracellular pH, induction of cell apoptosis and facilitating degradation of CA9 transcripts. Reanalyses of ChIP-Seq data along with utilization of EBNA2 knockout virus, ectopic expression of EBNA2 and sh-RNA mediated knockdown of CA9 expression we further demonstrate that EBNA2 mediated CA9 transcriptional activation is essential for EBV latently infected B-cell survival. In contrast, during lytic cycle reactivation CA9 expression is transcriptionally suppressed by the key EBV lytic cycle transactivator, BZLF1 through its transactivation domain. Overall, our study highlights the dynamic alterations of CA9 expression and its activity in regulating pH homeostasis act as one of the major drivers for EBV induced B-cell transformation and subsequent B-cell lymphomagenesis.

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.

Targeting latent viral infection in EBV-associated lymphomas.

Epstein-Barr virus (EBV) contributes to the development of a significant subset of human lymphomas. As a herpes virus, EBV can transition between a lytic state which is required to establish infection and a latent state where a limited number of viral antigens are expressed which allows infected cells to escape immune surveillance. Three broad latency programs have been described which are defined by the expression of viral proteins RNA, with latency I being the most restrictive expressing only EBV nuclear antigen 1 (EBNA1) and EBV-encoded small RNAs (EBERs) and latency III expressing the full panel of latent viral genes including the latent membrane proteins 1 and 2 (LMP1/2), and EBNA 2, 3, and leader protein (LP) which induce a robust T-cell response. The therapeutic use of EBV-specific T-cells has advanced the treatment of EBV-associated lymphoma, however this approach is only effective against EBV-associated lymphomas that express the latency II or III program. Latency I tumors such as Burkitt lymphoma (BL) and a subset of diffuse large B-cell lymphomas (DLBCL) evade the host immune response to EBV and are resistant to EBV-specific T-cell therapies. Thus, strategies for inducing a switch from the latency I to the latency II or III program in EBV+ tumors are being investigated as mechanisms to sensitize tumors to T-cell mediated killing. Here, we review what is known about the establishment and regulation of latency in EBV infected B-cells, the role of EBV-specific T-cells in lymphoma, and strategies to convert latency I tumors to latency II/III.

Strategies to target the central nervous system HIV reservoir.

PURPOSE OF THE REVIEW: The central nervous system (CNS) is an hotspot for HIV persistence and may be a major obstacle to overcome for curative strategies. The peculiar anatomical, tissular and cellular characteristics of the HIV reservoir in the CNS may need to be specifically addressed to achieve a long-term HIV control without ART. In this review, we will discuss the critical challenges that currently explored curative strategies may face in crossing the blood-brain barrier (BBB), targeting latent HIV in brain-resident myeloid reservoirs, and eliminating the virus without eliciting dangerous neurological adverse events. RECENT FINDINGS: Latency reversing agents (LRA), broadly neutralizing monoclonal antibodies (bNabs), chimeric antigen receptor (CAR) T-cells, and adeno-associated virus 9-vectored gene-therapies cross the BBB with varying efficiency. Although brain penetration is poor for bNAbs, viral vectors for in vivo gene-editing, certain LRAs, and CAR T-cells may reach the cerebral compartment more efficiently. All these approaches, however, may encounter difficulties in eliminating HIV-infected perivascular macrophages and microglia. Safety, including local neurological adverse effects, may also be a concern, especially if high doses are required to achieve optimal brain penetration and efficient brain cell targeting. SUMMARY: Targeting the CNS remains a potential problem for the currently investigated HIV curing strategies. In vivo evidence on CNS effectiveness is limited for most of the investigated strategies, and additional studies should be focused on evaluating the interplay between the cerebral HIV reservoir and treatment aiming to achieve an ART-free cure.

Epstein-Barr virus latency patterns in polymorphic lymphoproliferative disorders and lymphomas in immunodeficiency settings: Diagnostic implications.

Epstein-Barr virus (EBV) is responsible for many B cell lymphoproliferative disorders (LPD) spanning subclinical infection to immunodeficiency-related neoplasms. EBV establishes a latent infection in the host B cell as defined histologically by the expression of EBV latent membrane proteins and nuclear antigens. Herein, we characterize the latency patterns of immunodeficiency-related neoplasms including post-transplant lymphoproliferative disorders (PTLD) and therapy-related LPD (formerly iatrogenic) with latent membrane protein-1 (LMP-1) and EBV nuclear antigen-2 (EBNA-2) immunohistochemistry. The latency pattern was correlated with immunodeficiency and dysregulation (IDD) status and time from transplant procedure. 38 cases of EBV+ PTLD in comparison to 27 cases of classic Hodgkin lymphoma (CHL) and diffuse large B cell lymphoma (DLBCL) arising in either the therapy-related immunodeficiency setting (n = 12) or without an identified immunodeficiency (n = 15) were evaluated for EBV-encoded small RNAs by in situ hybridization (EBER-ISH) and for LMP-1 and EBNA-2 by immunohistochemistry. A full spectrum of EBV latency patterns was observed across PTLD in contrast to CHL and DLBCL arising in the therapy-related immunodeficiency setting. Polymorphic-PTLD (12 of 16 cases, 75 %) and DLBCL-PTLD (9 of 11 cases, 82 %) showed the greatest proportion of cases with latency III pattern. Whereas, EBV+ CHL in an immunocompetent patient showed exclusively latency II pattern (13 of 13 cases, 100 %). The majority of EBV+ PTLD occurred by three years of transplant procedure date and were enriched for latency III pattern (21 of 22 cases, 95 %). Immunohistochemical identification of EBV latency by LMP-1 and EBNA-2 can help classify PTLD in comparison to other EBV+ B cell LPD and lymphomas arising in therapy-related immunodeficiency and non-immunodeficiency settings.

Dynamic bidirectional regulation of NLRC3 and gammaherpesviruses during viral latency in B lymphocytes.

While most NOD-like receptors (NLRs) are predominately expressed by innate immune cells, NLRC3, an inhibitory NLR of immune signaling, exhibits the highest expression in lymphocytes. The role of NLRC3 or any NLRs in B lymphocytes is completely unknown. Gammaherpesviruses, including human Epstein-Barr virus (EBV) and murine gammaherpesvirus 68 (MHV-68), establish latent infection in B lymphocytes, which requires elevated NF-κB. This study shows that during latent EBV infection of human B cells, viral-encoded latent membrane protein 1 (LMP1) decreases NLRC3 transcript. LMP1-induced-NF-κB activation suppresses the promoter activity of NLRC3 via p65 binding to the promoter. Conversely, NLRC3 inhibits NF-κB activation by promoting the degradation of LMP1 in a proteasome-dependent manner. In vivo, MHV-68 infection reduces Nlrc3 transcripts in splenocytes, and Nlrc3-deficient mice show greater viral latency than controls. These results reveal a bidirectional regulatory circuit in B lymphocytes, where viral latent protein LMP1 reduces NLRC3 expression, while NLRC3 disrupts gammaherpesvirus latency, which is an important step for tumorigenesis.

Enhancer-promoter activation by the Kaposi sarcoma-associated herpesvirus episome maintenance protein LANA.

Higher-order genome structure influences the transcriptional regulation of cellular genes through the juxtaposition of regulatory elements, such as enhancers, close to promoters of target genes. While enhancer activation has emerged as an important facet of Kaposi sarcoma-associated herpesvirus (KSHV) biology, the mechanisms controlling enhancer-target gene expression remain obscure. Here, we discover that the KSHV genome tethering protein latency-associated nuclear antigen (LANA) potentiates enhancer-target gene expression in primary effusion lymphoma (PEL), a highly aggressive B cell lymphoma causally associated with KSHV. Genome-wide analyses demonstrate increased levels of enhancer RNA transcription as well as activating chromatin marks at LANA-bound enhancers. 3D genome conformation analyses identified genes critical for latency and tumorigenesis as targets of LANA-occupied enhancers, and LANA depletion results in their downregulation. These findings reveal a mechanism in enhancer-gene coordination and describe a role through which the main KSHV tethering protein regulates essential gene expression in PEL.

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.