Sodium arsenite and arsenic trioxide differently affect the oxidative stress of lymphoblastoid cells: An intricate crosstalk between mitochondria, autophagy and cell death.

Although the toxicity of arsenic depends on its chemical forms, few studies have taken into account the ambiguous phenomenon that sodium arsenite (NaAsO2) acts as a potent carcinogen while arsenic trioxide (ATO, As2O3) serves as an effective therapeutic agent in lymphoma, suggesting that NaAsO2 and As2O3 may act via paradoxical ways to either promote or inhibit cancer pathogenesis. Here, we compared the cellular response of the two arsenical compounds, NaAsO2 and As2O3, on the Burkitt lymphoma cell model, the Epstein Barr Virus (EBV)-positive P3HR1 cells. Using flow cytometry and biochemistry analyses, we showed that a NaAsO2 treatment induces P3HR1 cell death, combined with drastic drops in ΔΨm, NAD(P)H and ATP levels. In contrast, As2O3-treated cells resist to cell death, with a moderate reduction of ΔΨm, NAD(P)H and ATP. While both compounds block cells in G2/M and affect their protein carbonylation and lipid peroxidation, As2O3 induces a milder increase in superoxide anions and H2O2 than NaAsO2, associated to a milder inhibition of antioxidant defenses. By electron microscopy, RT-qPCR and image cytometry analyses, we showed that As2O3-treated cells display an overall autophagic response, combined with mitophagy and an unfolded protein response, characteristics that were not observed following a NaAsO2 treatment. As previous works showed that As2O3 reactivates EBV in P3HR1 cells, we treated the EBV- Ramos-1 cells and showed that autophagy was not induced in these EBV- cells upon As2O3 treatment suggesting that the boost of autophagy observed in As2O3-treated P3HR1 cells could be due to the presence of EBV in these cells. Overall, our results suggest that As2O3 is an autophagic inducer which action is enhanced when EBV is present in the cells, in contrast to NaAsO2, which induces cell death. That's why As2O3 is combined with other chemicals, as all-trans retinoic acid, to better target cancer cells in therapeutic treatments.

Shikonin, an inhibitor of inflammasomes, inhibits Epstein-Barr virus reactivation.

Epstein-Barr virus (EBV) is a highly prevalent human herpesvirus that persists for life in more than 95% of the adult population. EBV usually establishes an asymptomatic life-long infection, but it is also associated with malignancies affecting B lymphocytes and epithelial cells mainly. The virus alternates between a latent phase and a lytic phase, both of which contribute to the initiation of the tumor process. So far, there is only a limited number of antiviral molecules against the lytic phase, most of them targeting viral replication. Recent studies provided evidence that EBV uses components of the NLRP3 inflammasome to enter the productive phase of its cycle following activation in response to various stimuli. In the present work, we demonstrate that shikonin, a natural molecule with low toxicity which is known to inhibit inflammasome, can efficiently repress EBV reactivation. Similar results were obtained with apigenin and OLT 1177, two other NLRP3 inflammasome inhibitors. It is shown herein that shikonin repressed the transcription of reactivation-induced NLRP3 thereby inhibiting inflammasome activation and EBV lytic phase induction.

Anti-Epstein-Barr Viral Agents from the Medicinal Herb-Derived Fungus Alternaria alstroemeriae Km2286.

Chromatographic separation on the liquid-state fermented products produced by the fungal strain Alternaria alstroemeriae Km2286 isolated from the littoral medicinal herb Atriplex maximowicziana Makino resulted in the isolation of compounds 1-9. Structures were determined by spectroscopic analysis as four undescribed perylenequinones, altertromins A-D (1-4), along with altertoxin IV (5), altertoxin VIII (6), stemphyperylenol (7), tenuazonic acid (8), and allo-tenuazonic acid (9). Compounds 1-6 exhibited antiviral activities against Epstein-Barr virus (EBV) with EC50 values ranging from 0.17 ± 0.07 to 3.13 ± 0.31 µM and selectivity indices higher than 10. In an anti-neuroinflammatory assay, compounds 1-4, 6, and 7 showed inhibitory activity of nitric oxide production in lipopolysaccharide-induced microglial BV-2 cells, with IC50 values ranging from 0.33 ± 0.04 to 4.08 ± 0.53 µM without significant cytotoxicity. This is the first report to describe perylenequinone-type compounds with potent anti-EBV and anti-neuroinflammatory activities.

The three-dimensional structure of Epstein-Barr virus genome varies by latency type and is regulated by PARP1 enzymatic activity.

Epstein-Barr virus (EBV) persists in human B-cells by maintaining its chromatinized episomes within the nucleus. We have previously shown that cellular factor Poly [ADP-ribose] polymerase 1 (PARP1) binds the EBV genome, stabilizes CTCF binding at specific loci, and that PARP1 enzymatic activity correlates with maintaining a transcriptionally active latency program. To better understand PARP1's role in regulating EBV latency, here we functionally characterize the effect of PARP enzymatic inhibition on episomal structure through in situ HiC mapping, generating a complete 3D structure of the EBV genome. We also map intragenomic contact changes after PARP inhibition to global binding of chromatin looping factors CTCF and cohesin across the EBV genome. We find that PARP inhibition leads to fewer total unique intragenomic interactions within the EBV episome, yet new chromatin loops distinct from the untreated episome are also formed. This study also illustrates that PARP inhibition alters gene expression at the regions where chromatin looping is most effected. We observe that PARP1 inhibition does not alter cohesin binding sites but does increase its frequency of binding at those sites. Taken together, these findings demonstrate that PARP has an essential role in regulating global EBV chromatin structure and latent gene expression.

Prognostic Role of the Expression of Latent-Membrane Protein 1 of Epstein-Barr Virus in Classical Hodgkin Lymphoma.

The prognostic impact of the presence of Epstein-Barr virus (EBV) in classical Hodgkin lymphoma (cHL) is controversial. Previous studies reported heterogeneous results, rendering difficult the clinical validation of EBV as a prognostic biomarker in this lymphoma. The objective of this study was to evaluate the survival impact of the expression of EBV Latent-Membrane Protein 1 (EBV-LMP1) in tumoral Hodgkin-Reed-Sternberg (HRS) cells of primary diagnostic samples of cHL. Formalin-Fixed Paraffin-Embedded (FFPE) lymph node samples from 88 patients with cHL were analyzed. Patients were treated with the standard first-line chemotherapy (CT) with Adriamycin, Bleomycin, Vinblastine and Dacarbazine (ABVD) followed by radiotherapy. The Kaplan-Meier method and the Cox proportional hazards model were used for carrying out the survival analysis. In order to investigate whether the influence of EBV was age-dependent, analyses were performed both for patients of all ages and for age-stratified subgroups. In bivariate analysis, the expression of EBV was associated with older age (p = 0.011), mixed cellularity subtype cHL (p < 0.001) and high risk International Prognostic Score (IPS) (p = 0.023). Overall survival (OS) and progression-free survival (PFS) were associated with the presence of bulky disease (p = 0.009) and advanced disease at diagnosis (p = 0.016). EBV-positive cases did not present a significantly lower OS and PFS in comparison with EBV-negative cases, for all ages and when stratifying for age. When adjusted for covariates, absence of bulky disease at diagnosis (HR: 0.102, 95% CI: 0.02-0.48, p = 0.004) and limited disease stages (I-II) (HR: 0.074, 95% CI: 0.01-0.47, p = 0.006) were associated with a significant better OS. For PFS, limited-disease stages also retained prognostic impact in the multivariate Cox regression (HR: 0.145, 95% CI: 0.04-0.57, p = 0.006). These results are of importance as the early identification of prognostic biomarkers in cHL is critical for guiding and personalizing therapeutic decisions. The prognostic role of EBV in cHL could be modulated by the type of CT protocol employed and interact with the rest of presenting features.

25-Hydroxycholesterol Inhibits Kaposi's Sarcoma Herpesvirus and Epstein-Barr Virus Infections and Activates Inflammatory Cytokine Responses.

Oncogenic gammaherpesviruses express viral products during latent and lytic infection that block the innate immune response. Previously, we found that Kaposi's sarcoma herpesvirus (KSHV/human herpesvirus-8) viral microRNAs (miRNAs) downregulate cholesterol biogenesis, and we hypothesized that this prevents the production of 25-hydroxycholesterol (25HC), a cholesterol derivative. 25HC blocks KSHV de novo infection of primary endothelial cells at a postentry step and decreases viral gene expression of LANA (latency-associated nuclear antigen) and RTA. Herein we expanded on this observation by determining transcriptomic changes associated with 25HC treatment of primary endothelial cells using RNA sequencing (RNA-Seq). We found that 25HC treatment inhibited KSHV gene expression and induced interferon-stimulated genes (ISGs) and several inflammatory cytokines (interleukin 8 [IL-8], IL-1α). Some 25HC-induced genes were partially responsible for the broadly antiviral effect of 25HC against several viruses. Additionally, we found that 25HC inhibited infection of primary B cells by a related oncogenic virus, Epstein-Barr virus (EBV/human herpesvirus-4) by suppressing key viral genes such as LMP-1 and inducing apoptosis. RNA-Seq analysis revealed that IL-1 and IL-8 pathways were induced by 25HC in both primary endothelial cells and B cells. We also found that the gene encoding cholesterol 25-hydroxylase (CH25H), which converts cholesterol to 25HC, can be induced by type I interferon (IFN) in human B cell-enriched peripheral blood mononuclear cells (PBMCs). We propose a model wherein viral miRNAs target the cholesterol pathway to prevent 25HC production and subsequent induction of antiviral ISGs. Together, these results answer some important questions about a widely acting antiviral (25HC), with implications for multiple viral and bacterial infections. IMPORTANCE A cholesterol derivative, 25-hydroxycholesterol (25HC), has been demonstrated to inhibit infections from widely different bacteria and viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, its mechanism of activity is still not fully understood. In this work, we look at gene expression changes in the host and virus after 25HC treatment to find clues about its antiviral activity. We likewise demonstrate that 25HC is also antiviral against EBV, a common cancer-causing virus. We compared our results with previous data from antiviral screening assays and found the same pathways resulting in antiviral activity. Together, these results bring us closer to understanding how a modified form of cholesterol works against several viruses.

Association of Infectious Mononucleosis in Childhood and Adolescence With Risk for a Subsequent Multiple Sclerosis Diagnosis Among Siblings.

Importance: Epstein-Barr virus and its acute manifestation, infectious mononucleosis (IM), are associated with an increased risk of multiple sclerosis (MS). Whether this association is confounded by susceptibility to infection is still debated. Objective: To assess whether hospital-diagnosed IM during childhood, adolescence, or young adulthood is associated with subsequent MS diagnosis independent of shared familial factors. Design, Setting, and Participants: This population-based cohort study used the Swedish Total Population Register to identify individuals born in Sweden from January 1, 1958, to December 31, 1994. Participants aged 20 years were followed up from January 1, 1978, to December 31, 2018, with a median follow-up of 15.38 (IQR, 8.68-23.55; range, 0.01-40.96) years. Data were analyzed from October 2020 to July 2021. Exposure: Hospital-diagnosed IM before 25 years of age. Main Outcomes and Measures: Diagnoses of MS from 20 years of age were identified. Risk of an MS diagnosis associated with IM in childhood (birth to 10 years of age), adolescence (11-19 years of age), and early adulthood (20-24 years of age [time-dependent variable]) were estimated using conventional and stratified (to address familial environmental or genetic confounding) Cox proportional hazards regression. Results: Of the 2 492 980 individuals (1 312 119 men [52.63%] and 1 180 861 women [47.37%]) included, 5867 (0.24%) had an MS diagnosis from 20 years of age (median age, 31.50 [IQR, 26.78-37.54] years). Infectious mononucleosis in childhood (hazard ratio [HR], 1.98; 95% CI, 1.21-3.23) and adolescence (HR, 3.00; 95% CI, 2.48-3.63) was associated with an increased risk of an MS diagnosis that remained significant after controlling for shared familial factors in stratified Cox proportional hazards regression (HRs, 2.87 [95% CI, 1.44-5.74] and 3.19 [95% CI, 2.29-4.46], respectively). Infectious mononucleosis in early adulthood was also associated with risk of a subsequent MS diagnosis (HR, 1.89; 95% CI, 1.18-3.05), but this risk was attenuated and was not significant after controlling for shared familial factors (HR, 1.51; 95% CI, 0.82-2.76). Conclusions and Relevance: These findings suggest that IM in childhood and particularly adolescence is a risk factor associated with a diagnosis of MS, independent of shared familial factors.

Targeting cancer cell plasticity by HDAC inhibition to reverse EBV-induced dedifferentiation in nasopharyngeal carcinoma.

Application of differentiation therapy targeting cellular plasticity for the treatment of solid malignancies has been lagging. Nasopharyngeal carcinoma (NPC) is a distinctive cancer with poor differentiation and high prevalence of Epstein-Barr virus (EBV) infection. Here, we show that the expression of EBV latent protein LMP1 induces dedifferentiated and stem-like status with high plasticity through the transcriptional inhibition of CEBPA. Mechanistically, LMP1 upregulates STAT5A and recruits HDAC1/2 to the CEBPA locus to reduce its histone acetylation. HDAC inhibition restored CEBPA expression, reversing cellular dedifferentiation and stem-like status in mouse xenograft models. These findings provide a novel mechanistic epigenetic-based insight into virus-induced cellular plasticity and propose a promising concept of differentiation therapy in solid tumor by using HDAC inhibitors to target cellular plasticity.

Stress-Induced Epstein-Barr Virus Reactivation.

Epstein-Barr virus (EBV) is typically found in a latent, asymptomatic state in immunocompetent individuals. Perturbations of the host immune system can stimulate viral reactivation. Furthermore, there are a myriad of EBV-associated illnesses including various cancers, post-transplant lymphoproliferative disease, and autoimmune conditions. A thorough understanding of this virus, and the interplay between stress and the immune system, is essential to establish effective treatment. This review will provide a summary of the interaction between both psychological and cellular stressors resulting in EBV reactivation. It will examine mechanisms by which EBV establishes and maintains latency and will conclude with a brief overview of treatments targeting EBV.

Curcumol inhibits EBV-positive Nasopharyngeal carcinoma migration and invasion by targeting nucleolin.

Metastasis is a major cause of recurrence and death in patients with EBV-positive Nasopharyngeal carcinoma (NPC). Previous reports documented that curcumol has both anti-cancer and anti-viral effects, but there is little literature systematically addressing the mechanism of curcumol in EBV-positive tumors. Previously we found that nucelolin (NCL) is a target protein of curcumol in CNE2 cells, an EBV-negative NPC, and in this experiment, we reported a critical role for NCL in promoting migration and invasion of C666-1 cells, an EBV-positive NPC, and found that the expression of NCL determined the level of curcumol's efficacy. Mechanistically, NCL interacted with Epstein-Barr Virus Nuclear Antigen 1 (EBNA1) to activate VEGFA/VEGFR1/PI3K/AKT signaling pathway, which in turn promoted NPC cell invasion and metastasis. Moreover, further study showed that the differential expression of NCL and curcumol intervention only had a regulatory effect on the nuclear accumulation of VEGFR1, which strengthened the anti-cancer effect of curcumol mediated through NCL. Our findings indicated that curcumol exerted anti EBV-positive NPC invasion and metastasis by downregulating EBNA1 and inhibiting VEGFA/VEGFR1/PI3K/AKT signaling by targeting NCL, which provides a novel pharmacological basis for curcumol's clinical use in treating patients with EBV-positive NPC.

Simiao Qingwen Baidu decoction inhibits Epstein-Barr virus-induced B lymphoproliferative disease and lytic viral replication.

CONTEXT: Simiao Qingwen Baidu decoction (SQBD), a traditional Chinese medicine prescription, can ameliorate Epstein-Barr virus (EBV) induced disease. However, its mechanism still remains unknown. OBJECTIVE: To detect the mechanism of SQBD in EBV-induced B lymphoproliferative disease in vitro. MATERIALS AND METHODS: Sprague-Dawley (SD) rats (n = 20) were given SQBD (10 mL/kg) by gavage once a day for 7 d. SQBD-containing serum was obtained from abdominal aortic blood of rats, and diluted with medium to obtain 5%, 10% or 20%-medicated serum. SD rats (n = 10) were given normal saline, and normal serum was collected as a control. EBV-transformed B cells (CGM1) were cultured in medium containing 5%, 10% or 20%-medicated serum. CGM1 cells were treated with normal serum as a control. Cell viability and apoptosis were examined. The expression and activity of proteins were assessed. RESULTS: We found that IC50 (83 ± 26.07%, 24 h; 69.88 ± 4.69%, 48 h) of 10% medicated serum was higher than that of 5% (25.47 ± 6.98%, 24 h; 21.62 ± 7.30%, 48 h) and 20%-medicated serum (51 ± 7.25%, 24 h; 56.03 ± 2.56%, 48 h). Moreover, SQBD promoted apoptosis of CGM1 cells by regulating EBV latency proteins expression. SQBD inhibited EBV-induced lytic viral replication. CONCLUSIONS: Our data confirmed that SQBD inhibits EBV-induced B lymphoproliferative disease and lytic viral replication. This work provides a theoretical basis for the mechanism of SQBD in EBV-induced B lymphoproliferative disease, and SQBD may be an effectively therapeutic drug for EBV-induced B lymphoproliferative disease.

Rationally repurposed nitroxoline inhibits preclinical models of Epstein-Barr virus-associated lymphoproliferation.

Repurposing of currently used drugs for new indications benefits from known experience with those agents. Rational repurposing can be achieved when newly uncovered molecular activities are leveraged against diseases that utilize those mechanisms. Nitroxoline is an antibiotic with metal-chelating activity used to treat urinary tract infections. This small molecule also inhibits the function of bromodomain and extraterminal (BET) proteins that regulate oncogene expression in cancer. Lymphoproliferation driven by the Epstein-Barr virus (EBV) depends on these same proteins. We therefore tested the efficacy of nitroxoline against cell culture and small animal models of EBV-associated lymphoproliferation. Nitroxoline indeed reduces cell and tumor growth. Nitroxoline also acts faster than the prototype BET inhibitor JQ1. We suggest that this rational repurposing may hold translational promise.

Arsenicals, the Integrated Stress Response, and Epstein-Barr Virus Lytic Gene Expression.

Following our observation that clofoctol led to Epstein-Barr virus (EBV) lytic gene expression upon activation of the integrated stress response (ISR), we decided to investigate the impact of As2O3 on viral lytic gene expression. As2O3 has also been reported to activate the ISR pathway by its activation of the heme-regulated inhibitor (HRI). Our investigations show that As2O3 treatment leads to eIF2α phosphorylation, upregulation of ATF4 and TRB3 expression, and an increase of EBV Zta gene expression in lymphoid tumor cell lines as well as in naturally infected epithelial cancer cell lines. However, late lytic gene expression and virion production were blocked after arsenic treatment. In comparison, a small molecule HRI activator also led to increased Zta expression but did not block late lytic gene expression, suggesting that As2O3 effects on EBV gene expression are also mediated through other pathways.

The time-dependent effects of early-onset Epstein-Barr viremia on adult acute leukemia patients following allo-HSCT with ATG-containing MAC regimen.

Epstein-Barr virus (EBV) viremia is a common complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). The purpose of this study was to evaluate the impacts of early-onset EBV viremia in acute leukemia (AL) patients who underwent allo-HSCT with anti-thymocyte globulin (ATG)-containing myeloablative conditioning (MAC) regimen. Two hundred and ninety-six patients were included between January 2013 and December 2015. In 126 patients (42.6%) who developed early-onset EBV viremia, with a median time of 48 (range 18~99) days after allo-HSCT. The cumulative incidence of EBV viremia at 30 and 90 days after allo-HSCT were 4.1 and 39.9%, respectively. Prognostic analysis showed that the adjusted overall survival in early-EBVpos group was significantly lower than early-EBVneg group within the first 26.7 months after allo-HSCT [hazard ratio (HR), 1.63, P = 0.012], but significantly higher than those afterward (after 26.7 months: HR 0.11, P = 0.035); for the adjusted event-free survival, early-EBVpos group was significantly inferior in early-EBVpos group within the first 10.8 months after transplantation (HR: 1.55, P = 0.042), and this adverse effect was not detected any more after 10.8 months (HR: 0.58, P = 0.107). Compared with early-EBVneg group after adjusting by aGVHD and CMV viremia, HR for death from transplant-related mortality was 2.78-fold higher in patients with early-EBV viremia in piecewise constant Cox analysis (P = 0.006), and this adverse effect was not detected any more after the cut-point time (HR: 0.67, P = 0.361). No differences in terms of relapse and relapse mortality were observed between early-EBVpos and early-EBVneg group (P > 0.05). In conclusion, the impacts on transplant outcomes of early-EBV viremia were time-dependent, which may help to optimize management strategies for early-EBV viremia after allo-HSCT, especially in AL patients with ATG-containing MAC regimen.

Prazoles Targeting Tsg101 Inhibit Release of Epstein-Barr Virus following Reactivation from Latency.

Epstein-Barr virus (EBV) is a ubiquitous herpesvirus responsible for several diseases, including cancers of lymphoid and epithelial cells. EBV cancers typically exhibit viral latency; however, the production and release of EBV through its lytic phase are essential for cancer development. Antiviral agents that specifically target EBV production do not currently exist. Previously, we reported that the proton pump inhibitor tenatoprazole, which blocks the interaction of ubiquitin with the ESCRT-1 factor Tsg101, inhibits production of several enveloped viruses, including EBV. Here, we show that three structurally distinct prazoles impair mature particle formation postreactivation and identify the impact on stages of replication. The prazoles did not impair expression of lytic genes representative of the different kinetic classes but interfered with capsid maturation in the nucleus as well as virion transport from the nucleus. Replacement of endogenous Tsg101 with a mutant Tsg101 refractory to prazole-mediated inhibition rescued EBV release. These findings directly implicate Tsg101 in EBV nuclear egress and identify prazoles as potential therapeutic candidates for conditions that rely on EBV replication, such as chronic active EBV infection and posttransplant lymphoproliferative disorders. IMPORTANCE Production of virions is necessary for the ubiquitous Epstein-Barr virus (EBV) to persist in humans and can set the stage for development of EBV cancers in at-risk individuals. In our attempts to identify inhibitors of the EBV lytic phase, we previously found that a prazole proton pump inhibitor, known to block the interaction of ubiquitin with the ESCRT-1 factor Tsg101, blocks production of EBV. We now find that three structurally distinct prazoles impair maturation of EBV capsids and virion transport from the nucleus and, by interfering with Tsg101, prevent EBV release from lytically active cells. Our findings not only implicate Tsg101 in EBV production but also identify widely used prazoles as candidates to prevent development of posttransplant EBV lymphomas.

Rituximab-containing immuno-chemotherapy regimens are effective for the elimination of EBV for EBV-HLH with only and mainly B lymphocytes of EBV infection.

Patients with Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH) have a poor prognosis. This study investigated the efficacy of rituximab-containing immuno-chemotherapy regimens for EBV-HLH. In this study, 15 patients were treated with rituximab-containing regimens. The treatment efficacy and adverse events were evaluated. In 10 patients, EBV DNA became negative after the first course of treatment. The lymphocyte types infected by EBV in the 10 patients were only infected with B cells and mainly infected with B cells. In the other 5 patients, the EBV DNA of peripheral blood mononuclear cells (PBMC) before and after treatment with the regimens had no statistical difference (P = 0.111). In addition, in these 5 patients, EBV mainly infected T and NK cells. Among the 5 patients without a significant decline in EBV DNA of PBMC, 2 patients received allogeneic hematopoietic stem cell transplantation and turned negative for EBV DNA. This study suggests that rituximab-containing regimens are effective therapy for EBV-HLH with only and mainly B lymphocytes infected by EBV, especially for eliminating EBV.

EBV-PTLD in a patient after haploidentical stem-cell transplantation with post-transplant cyclophosphamide.

Here, we describe the case of a male patient with Epstein-Barr virus post-transplantation lymphoproliferative disorder (EBV-PTLD), which developed 18 months after a haploidentical hematopoietic stem cell transplantation (haplo-HSCT) and the administration of post-transplant cyclophosphamide (PTCy). Of note, no anti-thymoglobulin was used in the entire clinical course. Prior to the onset of EBV-PTLD, the patient had pulmonary chronic graft-versus-host disease and was treated with prednisolone and tacrolimus. After stopping immunosuppressive therapy, he was diagnosed with EBV-positive infectious mononucleosis PTLD, and EBV-associated hemophagocytic syndrome; therefore, dexamethasone and rituximab monotherapies were administered. After four courses of rituximab, EBV-DNA was no longer detected in the peripheral blood, and the patient's laboratory data improved. Overall, this study highlights the need to predict the risk factors associated with the development of EBV-PTLD in transplanted patients after haplo-HSCT with PTCy.

Sildenafil prevents HDACi-induced Epstein-Barr virus reactivation through the PKG pathway in NK/T cell lymphoma; potential implications for HDACi-mediated fatal complications.

Romidepsin, a histone deacetylase (HDAC) inhibitor, has been approved for the treatment of relapsed and refractory peripheral T-cell lymphoma. However the use of romidepsin reportedly causes potent EBV (Epstein-Barr virus) reactivation leading to severe adverse events in patients with natural killer (NK)/T-cell lymphoma (NKTL). As inhibition of EBV lytic cycle reactivation may help prevent romidepsin-induced adverse events in NKTL, we herein set out to identify a safe and effective drug for inhibiting EBV reactivation and examine its mechanism of inhibition. EBV reactivation was evaluated by qRT-PCR of BZLF1 and BRLF1 mRNA expression, qPCR of EBV DNA, and immunoblotting of viral EA-D protein. High-throughput screening of FDA-approved drugs was performed to identify safe and effective molecules and test their effect on romidepsin-induced EBV reactivation in the EBV-positive NKTL cell lines, SNK6 and NK92MI. We found that phosphodiesterase 5 (PDE5) inhibitors, including sildenafil (Viagra; Pfizer), appeared to be nontoxic and effective inhibitors of romidepsin-induced EBV reactivation. Clinical relevance was investigated by qPCR of EBV in two primary effusion samples of NKTL patients. We also investigated the molecular consequences downstream of sildenafil-induced PDE5 inhibition in NKTL cells. A negative correlation was established between the cGMP/PKG pathway and EBV reactivation in NKTL cells. On a molecular level, PDE5 inhibition downregulates BZLF1 and BRLF1 through cGMP/PKG signaling-induced ZNF overexpression. Co-treatment with romidepsin and sildenafil (inhibiting HDAC and PDE5, respectively) showed a synergistic inhibitory effect on NKTL cells, highlighting PDE5 as an attractive target for future therapy in NKTL.

Effects of chloroquine or hydroxychloroquine treatment on non-SARS-CoV2 viral infections: A systematic review of clinical studies.

Chloroquine (CQ) and hydroxychloroquine (HCQ) have been used as antiviral agents for the treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) infection. We performed a systematic review to examine whether prior clinical studies that compared the effects of CQ and HCQ to a control for the treatment of non-SARS-CoV2 infection supported the use of these agents in the present SARS-CoV2 outbreak. PubMed, EMBASE, Scopus and Web of Science (PROSPERO CRD42020183429) were searched from inception through 2 April 2020 without language restrictions. Of 1766 retrieved reports, 18 studies met our inclusion criteria, including 17 prospective controlled studies and one retrospective study. CQ or HCQ were compared to control for the treatment of infectious mononucleosis (EBV, n = 4), warts (human papillomavirus, n = 2), chronic HIV infection (n = 6), acute chikungunya infection (n = 1), acute dengue virus infection (n = 2), chronic HCV (n = 2), and as preventive measures for influenza infection (n = 1). Survival was not evaluated in any study. For HIV, the virus that was most investigated, while two early studies suggested HCQ reduced viral levels, four subsequent ones did not, and in two of these CQ or HCQ increased viral levels and reduced CD4 counts. Overall, three studies concluded CQ or HCQ were effective; four concluded further research was needed to assess the treatments' effectiveness; and 11 concluded that treatment was ineffective or potentially harmful. Prior controlled clinical trials with CQ and HCQ for non-SARS-CoV2 viral infections do not support these agents' use for the SARS-CoV2 outbreak.

()-Epigallocatechin-3-Gallate Inhibits EBV Lytic Replication via Targeting LMP1-Mediated MAPK Signal Axes.

EpsteinBarr virus (EBV)-encoded latent membrane protein 1 (LMP1) plays an important oncogenic role in the viral latent infection. Recently, increasing evidence indicates that the high expression of LMP1 during EBV lytic cycle is related to the viral lytic replication. However, the mechanism by which LMP1 regulates EBV lytic replication remains unclear. ()-Epigallocatechin-3-gallate (EGCG) prevents carcinogenesis by directly targeting numerous membrane proteins and effectively inhibits EBV lytic cascade. Here, we demonstrated that LMP1 promotes EBV lytic replication through the downstream signal molecules MAPKs, including ERKs, p38, and JNKs. LMP1 induces the phosphorylation of p53 through MAPKs to enhance the ability of wild-type p53 (wt-p53) to activate expression of BZLF1 gene, while the JNKs/c-Jun signal axis appears to be involved in EBV lytic replication induced by LMP1 in p53 mutant manner. We provided the first evidence that EGCG directly targets the viral membrane LMP1 (K d=0.36 M, n=1) using fluorescence quenching, isothermal titration calorimetry (ITC) assay, and CNBR-activated Sepharose 4B pull-down affinity chromatography. Furthermore, we revealed that EGCG inhibits EBV lytic replication via suppressing LMP1 and thus blocking the downstream MAPKs/wt-p53 signal axis in AGS-EBV cells and JNKs/c-Jun signal axis in p53 mutant B95.8 cells. Our study, for the first time, reports the binding and inhibitory efficacy of EGCG to the LMP1, which is a key oncoprotein encoded by EBV. These findings suggest the novel function of LMP1 in the regulation of EBV lytic cycle and reveal the new role of EGCG in EBV-associated malignancies through suppressing viral reactivation.