The Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNALP) is essential for the immortalization of naive B lymphocytes (NBLs). However, the mechanisms remain elusive. To understand EBNALP's role in B-cell transformation, we compare NBLs infected with wild-type EBV and an EBNALP-null mutant EBV using multi-omics techniques. EBNALP inactivation alters enhancer-promoter interactions, resulting in decreased CCND2 and increased CASP1 and BCL2L11 expression. Mechanistically, EBNALP interacts with and colocalizes with the looping factor YY1. Depletion of EBNALP reduces YY1 DNA-binding and enhancer-promoter interactions, similar to effects observed with YY1 depletion. Furthermore, EBNALP colocalizes with DPF2, a protein that binds to H3K14ac and H4K16ac. CRISPR depletion of DPF2 reduces both EBNALP and YY1 DNA binding, suggesting that the DPF2/EBNALP complex may tether YY1 to DNA to increase enhancer-promoter interactions. EBNALP inactivation also increases enhancer-promoter interactions at the CASP1 and BCL2L11 loci, along with elevated DPF2 and YY1 binding and DNA accessibility. Our data suggest that EBNALP regulates YY1 to rewire the host genome, which might facilitate naive B-cell transformation.

Induction of cuproptosis in tumor cells is an emerging direction for cancer drug development. Plumbagin (PLB), a natural biological molecule, has anticancer activities, partially via copper-dependent mechanisms. But it remains unclear if PLB can induce cuproptosis in hepatocellular carcinoma (HCC). In this study, PLB showed HCC-suppressive activities and caused representative molecular phenotypes of cuproptosis, whereas tetrathiomolybdate, an inhibitor of cuproptosis, could alleviate these effects the most. The mRNA and protein expression levels of the primary hepatic copper exporter, ATPase copper transporting beta (ATP7B), decreased in PLB-treated HCC cells, which might cause the accumulation of intracellular copper and trigger cuproptosis. An upstream ATP7B-regulatory microRNA, microRNA-302a-3p (miR-302a-3p), was identified by quantification and validated by the overexpression/inhibition experiment and luciferase reporter assay. Moreover, PLB was found to reduce the protein level of DNA-methyltransferase 1 (DNMT1), thereby enhancing the promoter hypomethylation and the expression of miR-302a-3p. Gene manipulation experiments further demonstrated that ATP7B, miR-302a-3p, and DNMT1 mediated PLB-induced cuproptosis. Preliminary clinical analyses showed that low ATP7B expression levels were associated with better prognosis, supporting the importance of ATP7B-lowering therapeutic strategies in HCC. Together, our results indicate that PLB triggers HCC cuproptosis via the DNMT1/miR-302a-3p/ATP7B axis, providing a potential therapeutic strategy for HCC.

This study characterizes the alterations in peripheral blood lymphocyte subsets and cytokine levels in patients with respiratory syncytial virus (RSV) infection and evaluates their clinical relevance. We collected clinical data from 215 RSV-positive inpatients. Patients were stratified into distinct groups according to different criteria; within-group comparisons were performed. In the RSV-infected group, absolute counts of all peripheral blood lymphocyte subsets were significantly lower than in controls and showed a negative correlation with disease severity. Conversely, all measured cytokines were markedly elevated in the infected group and positively correlated with the severity of RSV infection. Within the infected group, elderly patients (≥65 years) showed significantly different lymphocyte-subset counts and cytokine profiles compared with non-elderly patients. Similarly, individuals with high-risk diseases exhibited significant differences in these parameters relative to those without such diseases. RSV infection induces abnormalities in peripheral blood lymphocyte subsets and cytokine levels. The magnitude of these immune changes is linked to disease severity, patient age, and selected comorbidities, suggesting their potential utility as adjunct biomarkers for clinical assessment.

HCC is a globally high-incidence malignant tumour, and its pathogenesis is still unclear. Recently, STRN3 has been found to be elevated in various tumours, but its expression and biological functions in HCC have not been studied. In the study, clinical correlation analysis was performed on 371 liver cancer patients from TCGA database and liver cancer tissues and normal tissues from the GEO database. qRT-PCR and western blotting were used to detect relevant proteins in cells, and CCK8 and colony formation experiments were performed to analyse cell proliferation ability. Transwell and wound healing experiments were performed to detect cell invasion ability, and flow cytometry was used to detect cell apoptosis. Single-cell sequencing data and multiple immunofluorescence were analysed for the expression abundance and distribution of certain proteins. Immunohistochemistry was used to assess the expression of STRN3 in patients' tumour and adjacent non-cancerous tissues. The results indicated STRN3 was highly expressed in liver tumour tissues and was closely associated with poor prognosis. Knockdown of STRN3 could significantly inhibit cell proliferation and migration ability. At the same time, we found that STRN3 could inhibit the Hippo pathway and promote the entry of YAP protein into the nucleus. Our study first found that STRN3 could promote tumour growth by inhibiting the Hippo pathway. The study of STRN3 can promote the understanding and treatment of the occurrence and development of HCC.

The human genome is organized in an extremely complexed yet ordered way within the nucleus. Genome organization plays a critical role in the regulation of gene expression. Viruses manipulate the host machinery to influence host genome organization to favor their survival and promote disease development. Epstein-Barr virus (EBV) is a common human virus, whose infection is associated with various diseases, including infectious mononucleosis, cancer, and autoimmune disorders. This review summarizes our current knowledge of how EBV uses different strategies to control the cellular 3D genome organization to affect cell gene expression to transform normal cells into lymphoblasts.

Background and objectivesTo investigate the application of intelligent puncture blood collection robots in anticoagulated blood specimens, the satisfaction of subjects with the two blood collection methods, and the feasibility of intelligent blood collection devices to replace manual blood collection methods in clinical work.Materials and methodsA total of 154 volunteers from Zhongshan Hospital Fudan University were recruited to compare the test results of anticoagulant blood samples between blood collection robot and manual blood collection, a questionnaire was used to inquire about the volunteers' feelings about the two blood collection methods; the blood collection data of 6,255 patients willing to use the robot for blood collection were collected to analyze the success rate of blood collection.ResultsThe blood collection robot is superior to manual specimen collection in terms of volume and pain of specimen collection, and the puncture success rate is 94.3%. The anticoagulated blood specimens collected by the robot had 11 indexes statistically different from the results of manual blood collection, but the differences did not affect the clinical diagnosis and prognosis.ConclusionThe intelligent robotic blood collection is less painful and has better acceptance by patients, which can be used for clinical anticoagulated blood specimen collection.

Mutational detail in EBV (S3) and host samples(S4). Full legend is provided in Supplementary methods.

Table S5J-part3, expression values (transcripts per million; T.P.M.) of all genes (rows) in each sample (columns).

We probed the lifecycle of EBV on a cell-by-cell basis using single cell RNA sequencing (scRNA-seq) data from nine publicly available lymphoblastoid cell lines (LCL). While the majority of LCLs comprised cells containing EBV in the latent phase, two other clusters of cells were clearly evident and were distinguished by distinct expression of host and viral genes. Notably, both were high expressors of EBV LMP1 / BNLF2 and BZLF1 compared to another cluster that expressed neither gene. The two novel clusters differed from each other in their expression of EBV lytic genes, including glycoprotein gene GP350 . The first cluster, comprising GP350 − LMP1 hi cells, expressed high levels of HIF1A and was transcriptionally regulated by HIF1-α. Treatment of LCLs with Pevonedistat, a drug that enhances HIF1-α signaling, markedly induced this cluster. The second cluster, containing GP350 + LMP1 hi cells, expressed EBV lytic genes. Host genes that are controlled by super-enhancers (SEs), such as transcription factors MYC and IRF4 , had the lowest expression in this cluster. Functionally, the expression of genes regulated by MYC and IRF4 in GP350 + LMP1 hi cells were lower compared to other cells. Indeed, induction of EBV lytic reactivation in EBV + AKATA reduced the expression of these SE-regulated genes. Furthermore, CRISPR-mediated perturbation of the MYC or IRF4 SEs in LCLs induced the lytic EBV gene expression, suggesting that host SEs and/or SE target genes are required for maintenance of EBV latency. Collectively, our study revealed EBV associated heterogeneity among LCLs that may have functional consequence on host and viral biology. Importance Epstein-Barr virus (EBV) establishes a life-long latency program within host cells. As such, EBV immortalized lymphoblastoid cells (LCLs) often carry the latent EBV genome and only a small percentage of LCLs containing lytic EBV. However, the cellular programs that distinguish latent from lytic cells and the heterogeneity of cells in latent or lytic phases remains poorly explored. To explore these unknowns, we reanalyzed publicly available single cell RNA-seq data from nine LCLs. This approach permitted the simultaneous study of cells in both latent and lytic phases. We identified three cell populations with distinct lytic/latent activity and further characterized the transcriptomes of these cells. We also identified a new role of super-enhancers in regulating EBV lytic replication. Collectively, our studies revealed EBV associated heterogeneity among LCLs that contribute to EBV life cycle and biology.

Shown are information on host mutational pattern and host response networks to EBV infection. These are additional supporting information for Figure 5-6.

Background Nasopharyngeal carcinoma (NPC) is a malignant head and neck cancer with a high incidence in Southern China and Southeast Asia. Patients with remote metastasis and recurrent NPC have poor prognosis. Thus, a better understanding of NPC pathogenesis may identify novel therapies to address the unmet clinical needs.Methods H3K27ac ChIP-seq and HiChIP was applied to understand the enhancer landscapes and the chromosome interactions. Whole genome sequencing was conducted to analyze the relationship between genomic variations and epigenetic dysregulation. CRISPRi and JQ1 treatment were used to evaluate the transcriptional regulation of SOX2 SEs. Colony formation assay, survival analysis and in vivo subcutaneous patient-derived xenograft assays were applied to explore the function and clinical relevance of SOX2 in NPC.Findings We globally mapped the enhancer landscapes and generated NPC enhancer connectomes, linking NPC specific enhancers and SEs. We found five overlapped genes, including SOX2, among super-enhancer regulated genes, survival related genes and NPC essential genes. The mRNA expression of SOX2 was repressed when applying CRISPRi targeting different SOX2 SEs or JQ1 treatment. Next, we identified a genetic variation (Chr3:181422197, G > A) in SOX2 SE which is correlated with higher expression of SOX2 and poor survival. In addition, SOX2 was highly expressed in NPC and is correlated with short survival in patients with NPC. Knock-down of SOX2 suppressed tumor growth in vitro and in vivo. Interpretation Our study demonstrated the super-enhancer landscape with chromosome interactions and identified super-enhancer driven SOX2 promotes tumorigenesis, suggesting that SOX2 is a potential therapeutic target for patients with NPC.

Shown are information related to EBV integration, EBV gene manipulation, and EBV mutational patterns. These are additional supporting information for Figure 1-4.

A, detail information on all 1342 cancer samples used in this study. B, parts per million (P.P.M.) values for each cancer along with the number of reads that mapped to the EBV genome and the library size after removing low-quality reads and PCR duplicates. C, 56 EBV-integrated loci in the human genome. For each integration (rows), shown are the integration location, the nearest gene and its hg19 coordinates, the number of samples with the integration in each tissue type, and the coordinates of overlapping SEs from genesets shown in Fig. 1E. The total number of EBV+ samples analyzed in each tumor type is indicated in the header row. D, for each sample (rows), shown are the EBV integration location. E, expression (transcripts per million; T.P.M.) of all EBV genes (rows) in each sample (columns).

list of DEGs (fold change >1.5, FDR<0.05) between EBV+ and EBV- samples in each cancer (A-G). For each gene, shown are the mean expression value across EBV- and EBV+ cancer samples. The log-fold change, p-value and FDR values were obtained using differential expression analysis in edgeR. H, genes involved in Type I and Type II interferon activity (see methods). I, interferon activity of EBV+ tumor types. Shown in columns are cancer types, classification into IFN+ or IFN- groups for EBV+ samples or control group for EBV- samples, EBV status, Type I and Type II interferon signature score, and z-scores of genes related to Type I and Type II interferon signatures, for each sample (rows). J, expression values (transcripts per million; T.P.M.) of all genes (rows) in each sample (columns).

Detailed information on different computational analysis performed in this study. This file additionally includes supplementary table legends and Table S2.

Table S5J-part2, expression values (transcripts per million; T.P.M.) of all genes (rows) in each sample (columns).

Epstein-Barr virus (EBV) immortalization of resting B lymphocytes (RBLs) to lymphoblastoid cell lines (LCLs) models human DNA tumor virus oncogenesis. RBL and LCL chromatin interaction maps are compared to identify the spatial and temporal genome architectural changes during EBV B cell transformation. EBV induces global genome reorganization where contact domains frequently merge or subdivide during transformation. Repressed B compartments in RBLs frequently switch to active A compartments in LCLs. LCLs gain 40% new contact domain boundaries. Newly gained LCL boundaries have strong CTCF binding at their borders while in RBLs, the same sites have much less CTCF binding. Some LCL CTCF sites also have EBV nuclear antigen (EBNA) leader protein EBNALP binding. LCLs have more local interactions than RBLs at LCL dependency factors and super-enhancer targets. RNA Pol II HiChIP and FISH of RBL and LCL further validate the Hi-C results. EBNA3A inactivation globally alters LCL genome interactions. EBNA3A inactivation reduces CTCF and RAD21 DNA binding. EBNA3C inactivation rewires the looping at the CDKN2A/B and AICDA loci. Disruption of a CTCF site at AICDA locus increases AICDA expression. These data suggest that EBV controls lymphocyte growth by globally reorganizing host genome architecture to facilitate the expression of key oncogenes.

Epstein-Barr virus (EBV) persists in human cells as episomes. EBV episomes are chromatinized and their 3D conformation varies greatly in cells expressing different latency genes. We used HiChIP, an assay which combines genome-wide chromatin conformation capture followed by deep sequencing (Hi-C) and chromatin immunoprecipitation (ChIP), to interrogate the EBV episome 3D conformation in different cancer cell lines. In an EBV-transformed lymphoblastoid cell line (LCL) GM12878 expressing type III EBV latency genes, abundant genomic interactions were identified by H3K27ac HiChIP. A strong enhancer was located near the BILF2 gene and looped to multiple genes around BALFs loci. Perturbation of the BILF2 enhancer by CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) altered the expression of BILF2 enhancer-linked genes, including BARF0 and BALF2, suggesting that this enhancer regulates the expression of linked genes. H3K27ac ChIP followed by deep sequencing (ChIP-seq) identified several strong EBV enhancers in T/NK (natural killer) lymphoma cells that express type II EBV latency genes. Extensive intragenomic interactions were also found which linked enhancers to target genes. A strong enhancer at BILF2 also looped to the BALF loci. CRISPRi also validated the functional connection between BILF2 enhancer and BARE1 gene. In contrast, H3K27ac HiChIP found significantly fewer intragenomic interactions in type I EBV latency gene-expressing primary effusion lymphoma (PEL) cell lines. These data provided new insight into the regulation of EBV latency gene expression in different EBV-associated tumors. IMPORTANCE EBV is the first human DNA tumor virus identified, discovered over 50 years ago. EBV causes similar to 200,000 cases of various cancers each year. EBV-encoded oncogenes, noncoding RNAs, and microRNAs (miRNAs) can promote cell growth and survival and suppress senescence. Regulation of EBV gene expression is very complex. The viral C promoter regulates the expression of all EBV nuclear antigens (EBNAs), some of which are very far away from the C promoter. Another way by which the virus activates remote gene expression is through DNA looping. In this study, we describe the viral genome looping patterns in various EBV-associated cancer cell lines and identify important EBV enhancers in these cells. This study also identified novel opportunities to perturb and eventually control EBV gene expression in these cancer cells.

[This corrects the article DOI: 10.1371/journal.ppat.1007416.].