Biology subjects

Luftig, M. A.

Publications and source records attributed to Luftig, M. A..

At least 19 recordsLinked to original sources

bioRxiv2025

Dissecting Epstein-Barr Virus Dependence Across Diverse Infected Cell Models

Epstein-Barr virus (EBV) is maintained clonally in a wide array of tumors and cells of lymphoproliferative disorders, yet the degree to which these infected cells continuously depend on EBV remains unresolved. To directly assess EBV dependence, we used two complementary Cas9-based viral eviction strategies: 1) targeting EBNA1 to promote episome dilution during cell division, and 2) targeting repetitive viral genomic regions to rapidly degrade EBV episomes. Using these approaches, we evaluated EBV-loss sensitivity across newly-derived, phenotypically diverse EBV-positive Burkitt lymphoma lines. We also evaluated cell lines from patients with EBV-associated T/NK and epithelial malignancies. We included the Burkitt Lymphoma cell line, Akata-GFP, as our EBV-loss-tolerant control and a lymphoblastoid cell line as our EBV-loss-sensitive control. We observed EBV-loss sensitivity in all the EBV-positive Burkitt Lymphoma cell lines and the chronic active EBV disease cell line. In contrast, the EBV-positive gastric adenocarcinoma lines were markedly less sensitive to EBV loss. EBV latency type did not predict EBV dependence, indicating that viral gene expression programs alone do not dictate reliance on the virus. These findings demonstrate that EBV dependence is prevalent across multiple EBV-associated disease models and is especially pronounced in early-state or newly derived lymphoid malignancies. AUTHOR SUMMARYEpstein-Barr virus (EBV) infects most people worldwide and is linked to many malignancies. Although EBV is present in cancer cells, it is unclear whether these cells require the virus to survive. To answer this question, we used CRISPR/Cas9-based methods to remove EBV from infected cells. Using these tools, we studied many different EBV-positive cell lines, including new Burkitt lymphoma cell lines. We found that most EBV-positive lymphoid cells required EBV for growth. In contrast, EBV-positive gastric cells were more tolerant of viral loss. These results show that many EBV-associated malignancies, especially early or newly established diseases, rely on EBV to survive.

microbiology↗

bioRxiv2025

HIV-associated non-Hodgkin lymphoma tumor-microenvironment axes differ by EBV status across cellular origins

Non-Hodgkin Lymphoma (NHL) is the main cancer-related mortality for people living with HIV (PLWH). NHL genetic and molecular classifications have been intensely studied and correlated with clinical outcomes, but critical unanswered questions relevant to malignancy persist. For example, tumors positive for Epstein-Barr virus (EBV) are aggressive and account for 30-50% of HIV-associated NHLs, yet insights on the nature of EBV in NHL pathogenesis or potential therapeutic vulnerabilities have been limited. Here, we examined HIV-associated NHLs stratified by histopathologic classification, cell of origin (COO), molecular subtype, and EBV status using genome-wide spatial transcriptomic analyses. Tumor tissues in EBV+ HIV-NHLs displayed enriched expression of mitochondrial respiration and purine metabolism signatures versus EBV- tumors. Immune infiltration of EBV+ tumors was limited, and immune and stromal regions of EBV+ HIV-NHLs displayed upregulation of the gene for the iron exporter ferroportin (SLC40A1), indicating immunosuppressive macrophage polarization. An immunosuppressive SPP1-CD44 axis and oncogenic GAS6-AXL interaction between tumor and stroma were specifically predicted for EBV+ HIV-NHLs with plasmablastic features. Tumor microenvironment (TME) similarities to secondary lymphoid tissues such as inverse CXCL12-CXCL13 gradients and evidence of matching between tumor B cell phenotypes and reticular cell signatures were also observed. Finally, we developed a simple yet flexible approach to quantify expression gradients and cell proximity to annotated regions. Thus, this study highlights potential avenues for NHL therapy tailored by EBV status and provides a unique resource to examine tumor-TME interactions in the HIV-immunocompromised context. Key PointsO_LISpatial transcriptomic landscapes resolve cell-of-origin- and EBV-associated HIV-NHL heterogeneity including immunosuppressive myeloid responses to EBV+ tumors C_LIO_LISpatial gradients including CXCL12 and CXCL13 indicate that tumor-stromal interactions mimic lymphoid tissue organization and depend on B cell development stage C_LIO_LIDevelopment of a direction-agnostic method to calculate spatial expression gradients relative to annotated tissue and cell types of interest C_LIO_LIPredicted pharmacologic vulnerabilities in clinical samples are replicated in vitro and confirmed by small molecule screening C_LI

cancer biology↗

bioRxiv2025

Single-Cell Profiling of HDAC Inhibitor-Induced EBV Lytic Heterogeneity Defines Abortive and Refractory States in B Lymphoblasts

Epstein-Barr virus (EBV) is associated with multiple malignancies including Burkitt lymphoma (BL), Hodgkins lymphomas, nasopharyngeal carcinomas (NPC), and gastric cancers. Canonically, EBV positive tumors display latent gene expression programs that are difficult to target pharmacologically. To overcome this hurdle, lytic reactivation therapies have been developed based on HDAC inhibition with limited mechanistic studies. We therefore characterized the impact of pan-HDAC inhibitor, panobinostat, and class I HDAC inhibitor, nanatinostat, on the growth, survival, and lytic reactivation of four EBV-positive cell lines: P3HR1-ZHT BL, Jijoye BL, IBL-1 immunoblastic lymphoma, and de novo infection derived lymphoblastoid cell lines (LCL). All lines were sensitive, enabling us to define ranges of sensitivity within which to use single cell approaches to assess early EBV lytic gene expression, cell cycle state, and apoptosis. We observed that each EBV-positive model of malignancy responded uniquely to the same HDAC inhibitors and that lytic reactivation was successful in only a small percentage of the cell population. To elucidate the potential role of host factors in preventing successful lytic reactivation, we performed single-cell RNA sequencing on the P3HR1-ZHT BL line treated with the HDAC inhibitor panobinostat. We observed that abortive lytic cells, or cells that do not successfully progress through the lytic cycle, upregulated genes downstream of NF-{kappa}B activity. Additionally, genes involved in immune signaling including the CD137/CD137L signaling axis, were upregulated in abortive lytic cells. These data have important implications for how we approach oncolytic therapies for EBV-associated malignancies. AUTHOR SUMMARYEpstein-Barr virus (EBV) is an extremely prevalent human herpesvirus that is associated with a variety of cancers and autoimmune diseases. EBV establishes latent infection in the host and, under various circumstances, can reactivate the lytic cycle to produce more infectious particles. In the context of EBV-associated malignancies, the virus is most often maintained in a latent state, which makes it difficult to target with pharmaceuticals. To develop more viral targeted strategies, kick and kill regiments have been investigated. This therapy involves reactivating the virus with an HDAC inhibitor followed by treatment with an antiviral drug. It is well established that reactivating EBV with pharmaceuticals is often inefficient and leads to heterogeneous responses, including an abortive lytic trajectory. To better characterize the overall effect of two classes of HDAC inhibitors in various models of EBV-associated malignancies, we utilized single-cell techniques to capture various responses to stimuli. Consistent with prior studies, HDAC inhibition led to both successful and abortive lytic populations. Single-cell RNA sequencing provided evidence of upregulated immune signaling pathways in this abortive lytic population. This study provides in depth characterization of lytic reactivation with a biologically relevant stimulus.

microbiology↗

bioRxiv2024

Sp140L Is a Novel Herpesvirus Restriction Factor

Herpesviruses, including Epstein-Barr Virus (EBV) - a human oncogenic viruses and essential trigger of multiple sclerosis, must bypass host DNA sensing mechanisms to establish lifelong, latent infection. Therefore, herpesviruses encode viral proteins to disrupt key host factors involved in DNA sensing and viral restriction. The first viral latency protein expressed, EBNA-LP, is essential for transformation of naive B cells and establishment of viral gene expression, yet its role in evading host defenses remains unclear. Using single-cell RNA sequencing of EBNA-LP-Knockout (LPKO)- infected B cells, we reveal an antiviral response landscape implicating the speckled proteins as key cellular restriction factors countered by EBNA-LP. Specifically, loss of SP100 or the primate-specific SP140L reverses the restriction of LPKO, suppresses a subset of canonically interferon-stimulated genes, and restores transcription of essential latent viral genes and cellular proliferation. Notably, we also identify Sp140L as a restriction target of the herpesvirus saimiri ORF3 protein, implying a role for Sp140L in immunity to other diverse DNA viruses. This study reveals Sp140L as a restriction factor that we propose links sensing and transcriptional suppression of viral DNA to an IFN-independent innate immune response, likely relevant to all nuclear DNA viruses. Significance StatementHerpesviruses, including the oncogenic Epstein-Barr virus (EBV), are restricted by DNA sensing during initial infection and therefore encode viral proteins to antagonize key restriction factors. We found that the EBV latency protein EBNA-LP, disrupts the speckled proteins Sp100 and Sp140L - an evolutionarily recent protein with unknown function, which we find promotes an anti-viral state that suppresses cellular proliferation, characterized by high induction of cellular anti-viral genes and suppressed transcription of essential viral latency genes. Sp140L also restricts the herpesvirus saimiri, which we find antagonizes Sp140L through the viral protein ORF3. Our study therefore identifies Sp140L as a novel restriction factor of diverse herpesviruses, and likely all DNA viruses, during a critical stage of initial viral infection.

microbiology↗

bioRxiv2024

Human genetic variation reveals FCRL3 is a lymphocyte receptor for Yersinia pestis

Yersinia pestis is the gram-negative bacterium responsible for plague, one of the deadliest and most feared diseases in human history. This bacterium is known to infect phagocytic cells, such as dendritic cells and macrophages, but interactions with non-phagocytic cells of the adaptive immune system are frequently overlooked despite the importance they likely hold for human infection. To discover human genetic determinants of Y. pestis infection, we utilized nearly a thousand genetically diverse lymphoblastoid cell lines in a cellular genome-wide association study method called Hi-HOST (High-throughput Human in-vitrO Susceptibility Testing). We identified a nonsynonymous SNP, rs2282284, in Fc receptor like 3 (FCRL3) associated with bacterial invasion of host cells (p=9x10-8). FCRL3 belongs to the immunoglobulin superfamily and is primarily expressed in lymphocytes. rs2282284 is within a tyrosine-based signaling motif, causing an asparagine-to-serine mutation (N721S) in the most common FCRL3 isoform. Overexpression of FCRL3 facilitated attachment and invasion of non-opsonized Y. pestis. Additionally, FCRL3 colocalized with Y. pestis at sites of cellular attachment, suggesting FCRL3 is a receptor for Y. pestis. These properties were variably conserved across the FCRL family, revealing molecular requirements of attachment and invasion, including an Ig-like C2 domain and a SYK interaction motif. Direct binding was confirmed with purified FCRL5 extracellular domain. Following attachment, invasion of Y. pestis was dependent on SYK and decreased with the N721S mutation. Unexpectedly, this same variant is associated with risk of chronic hepatitis C virus infection in BioBank Japan. Thus, Y. pestis hijacks FCRL proteins, possibly taking advantage of an immune receptor to create a lymphocyte niche during infection.

genetics↗

bioRxiv2024

Fatty acid desaturases link cell metabolism pathways to promote proliferation of Epstein-Barr virus-infected B cells

Epstein-Barr virus (EBV) is a gamma herpesvirus that infects up to 95% of the human population by adulthood, typically remaining latent in the host memory B cell pool. In immunocompromised individuals, EBV can drive the transformation and rapid proliferation of infected B cells, ultimately resulting in neoplasia. The same transformation process can be induced in vitro, with EBV-infected peripheral blood mononuclear cells (PBMCs) forming immortalized lymphoblastoid cell lines (LCLs) within weeks. In this study, we found that the fatty acid desaturases stearoyl-CoA desaturase 1 (SCD1) and fatty acid desaturase 2 (FADS2) are upregulated by EBV, and crucial for EBV-induced B cell proliferation. Additionally, we found that pharmacological and genetic inhibition of both SCD1 and FADS2 results in a significantly greater reduction in proliferation and cell cycle arrest, compared to perturbing either enzyme individually. Additionally, we found that inhibiting either SCD1 or FADS2 alone hypersensitizes LCLs to palmitate-induced apoptosis, recapitulating studies in other tissue types. Further lipidomic and metabolic analysis of dual SCD1/FADS2-inhibited LCLs revealed an increase in free unsaturated fatty acids, a reduction of oxidative phosphorylation, and a reduction of glycolysis, thereby linking the activity of SCD1 and FADS2 to overall growth-promoting metabolism. Lastly, we show that SCD1 and FADS2 are important in the growth of clinically derived EBV+ immunoblastic lymphoma cells. Collectively, these data demonstrate a previously uncharacterized role of lipid desaturation in EBV+ transformed B cell proliferation, revealing a metabolic pathway that can be targeted in future anti-lymphoma therapies.

microbiology↗

bioRxiv2024

Epstein-Barr virus reactivation induces divergent abortive, reprogrammed, and host shutoff states by lytic progression

Viral infection leads to heterogeneous cellular outcomes ranging from refractory to abortive and fully productive states. Single cell transcriptomics enables a high resolution view of these distinct post-infection states. Here, we have interrogated the host-pathogen dynamics following reactivation of Epstein-Barr virus (EBV). While benign in most people, EBV is responsible for infectious mononucleosis, up to 2% of human cancers, and is a trigger for the development of multiple sclerosis. Following latency establishment in B cells, EBV reactivates and is shed in saliva to enable infection of new hosts. Beyond its importance for transmission, the lytic cycle is also implicated in EBV-associated oncogenesis. Conversely, induction of lytic reactivation in latent EBV-positive tumors presents a novel therapeutic opportunity. Therefore, defining the dynamics and heterogeneity of EBV lytic reactivation is a high priority to better understand pathogenesis and therapeutic potential. In this study, we applied single-cell techniques to analyze diverse fate trajectories during lytic reactivation in two B cell models. Consistent with prior work, we find that cell cycle and MYC expression correlate with cells refractory to lytic reactivation. We further found that lytic induction yields a continuum from abortive to complete reactivation. Abortive lytic cells upregulate NF{kappa}B and IRF3 pathway target genes, while cells that proceed through the full lytic cycle exhibit unexpected expression of genes associated with cellular reprogramming. Distinct subpopulations of lytic cells further displayed variable profiles for transcripts known to escape virus-mediated host shutoff. These data reveal previously unknown and promiscuous outcomes of lytic reactivation with broad implications for viral replication and EBV-associated oncogenesis. AUTHOR SUMMARY / SIGNIFICANCEViral infections profoundly alter host cell biological programming in ways that potentiate disease. Epstein-Barr virus (EBV) is a particularly prevalent human pathogen associated with diverse cancers and several autoimmune disorders. EBV predominantly establishes latent infection in B cells and can promote B cell malignancies through functions of well-characterized latent oncoproteins. Aspects of the viral lytic cycle also clearly contribute to EBV-associated diseases, although pathologic roles of lytic reactivation are incompletely understood. Here we use single-cell techniques to examine cellular responses to EBV lytic reactivation in multiple B cell models. Consistent with prior studies, reactivation from latency is incomplete (abortive) in some cells and successful in others. Abortive and full lytic trajectories exhibit distinct biological responses that each may promote pathogenesis and reinforce bimodal latent-lytic control. Intriguingly, a portion on cells that proceed through the lytic cycle exhibits unexpected and striking expression of genes associated with cellular reprogramming, pluripotency, and self-renewal. Collectively, this study provides a valuable resource to understand diverse host-virus dynamics and fates during viral reactivation and identifies multiple modes of EBV lytic pathogenesis to investigate in future research.

microbiology↗

bioRxiv2024

Incorporation of Epstein-Barr viral variation implicates significance of LMP1 in survival prediction and prognostic subgrouping in Burkitt lymphoma

While Epstein-Barr virus (EBV) plays a role in Burkitt lymphoma (BL) tumorigenesis, it is unclear if EBV genetic variation impacts clinical outcomes. From 130 publicly available whole-genome tumor sequences of EBV-positive BL patients, we used least absolute shrinkage and selection operator (LASSO) regression and Bayesian variable selection models within a Cox proportional hazards framework to select the top EBV variants, putative driver genes, and clinical features associated with patient survival time. These features were incorporated into survival prediction and prognostic subgrouping models. Our model yielded 22 EBV variants including seven in LMP1 as most associated with patient survival time. Using the top EBV variants, driver genes, and clinical features, we defined three prognostic subgroups that demonstrated differential survival rates, laying the foundation for incorporating EBV variants such as those in LMP1 as predictive biomarker candidates in future studies.

genomics↗

bioRxiv2024

Epstein-Barr virus protein EBNA-LP engages YY1 through leucine-rich motifs to promote naïve B cell transformation

Epstein-Barr Virus (EBV) is associated with numerous cancers including B cell lymphomas. In vitro, EBV transforms primary B cells into immortalized Lymphoblastoid Cell Lines (LCLs) which serves as a model to study the role of viral proteins in EBV malignancies. EBV induced cellular transformation is driven by viral proteins including EBV-Nuclear Antigens (EBNAs). EBNA-LP is important for the transformation of naive but not memory B cells. While EBNA-LP was thought to promote gene activation by EBNA2, EBNA-LP Knock Out (LPKO) virus-infected cells express EBNA2-activated genes efficiently. Therefore, a gap in knowledge exists as to what roles EBNA-LP plays in naive B cell transformation. We developed a trans-complementation assay wherein transfection with wild-type EBNA-LP rescues the transformation of peripheral blood- and cord blood-derived naive B cells by LPKO virus. Despite EBNA-LP phosphorylation sites being important in EBNA2 co-activation; neither phospho-mutant nor phospho-mimetic EBNA-LP was defective in rescuing naive B cell outgrowth. However, we identified conserved leucine-rich motifs in EBNA-LP that were required for transformation of adult naive and cord blood B cells. Because cellular PPAR-{gamma} coactivator (PGC) proteins use leucine-rich motifs to engage transcription factors including YY1, a key regulator of DNA looping and metabolism, we examined the role of EBNA-LP in engaging cellular transcription factors. We found a significant overlap between EBNA-LP and YY1 in ChIP-Seq data and confirmed their biochemical association in LCLs by endogenous co-immunoprecipitation. Moreover, we found that the EBNA-LP leucine-rich motifs were required for YY1 interaction in LCLs. Finally, we used Cas9 to knockout YY1 in primary total B cells and naive B cells prior to EBV infection and found YY1 to be essential for EBV-mediated transformation. We propose that EBNA-LP engages YY1 through conserved leucine-rich motifs to promote EBV transformation of naive B cells. Author SummaryEpstein-Barr Virus (EBV) is associated with various B cell lymphomas, particularly in immunosuppressed individuals. In the absence of a functional immune system, viral latency proteins, including EBV Nuclear Antigens (EBNAs) act as oncoproteins to promote tumorigenesis. EBNA-LP is one of the first viral proteins produced after infection and is important for the transformation of naive B cells. However, the roles of EBNA-LP during infection are largely undefined. In this study, developed an assay in which the role of wild type and mutant EBNA-LP could be investigated in context of primary naive B cells infected with an EBNA-LP Knock Out virus. Using this assay, we identified highly conserved leucine-rich motifs within EBNA-LP that are important for transformation of EBV-infected naive B cells. These conserved motifs associate with the cellular transcription factor YY1, an important transcriptional regulator in B cell development and in many cancers, that we now show is essential for outgrowth of EBV infected B cells. Our study provides further insights into the mechanisms by which EBV transforms naive B cells.

microbiology↗

bioRxiv2022

Fate-resolved gene regulatory signatures of individual B lymphocytes in the early stages of Epstein-Barr Virus infection

Epstein-Barr Virus (EBV) infection of B lymphocytes elicits diverse host responses via complex, well-adapted transcriptional control dynamics. Consequently, this host-pathogen interaction provides a powerful system to explore fundamental cellular processes that contribute to consensus fate decisions including cell cycle arrest, apoptosis, proliferation, and differentiation. Here we capture these responses and fates with matched single-cell transcriptomics and chromatin accessibility, from which we construct a genome-wide multistate model of early infection dynamics. Notably, our model captures a previously uncharacterized EBV+ analog of a multipotent activated precursor state that can yield early memory B cells. We also find that a marked global reduction in host chromatin accessibility occurs during the first stages of infection in subpopulations of EBV+ cells that display senescent and pre-apoptotic hallmarks induced by innate antiviral sensing and proliferation-linked DNA damage. However, cells in proliferative infection trajectories exhibit greater accessibility at select host sites linked to B cell activation and survival genes as well as key regions within the viral genome. To further investigate such loci, we implement a bioinformatic workflow (crisp-ATAC) to identify phenotype-resolved regulatory signatures. This customizable method applies user-specified logical criteria to produce genome-wide single-cell ATAC-and ChIP-seq range intersections that are used as inputs for cis-linkage prediction and ontology tools. The resulting tri-modal data yield exquisitely detailed hierarchical perspectives of the transforming regulatory landscape during critical stages of an oncogenic viral infection that simulates antigen-induced B cell activation and differentiation. We anticipate these resources will guide investigations of gene regulatory modules controlling EBV-host dynamics, B cell effector fates, and lymphomagenesis. To demonstrate the utility of this resource, this work concludes with the discovery of EBV infection dynamics in FCRL4+ / TBX21+ Tissue-Like Memory B cells, an unconventional subset with notable associations to numerous immune disorders.

immunology↗

bioRxiv2021

Comparing instance segmentation methods for analyzing clonal growth of single cells in microfluidic chips

Appropriately tailored segmentation techniques can extract detailed quantitative information from biological image datasets to characterize and better understand sample distributions. Practically, high-resolution characterization of biological samples such as cell populations can provide insights into the sources of variance in biomarker expression, drug resistance, and other phenotypic aspects, but it is still unclear what is the best method for extracting this information from large image-based datasets. We present a software pipeline and comparison of multiple image segmentation methods to extract single-cell morphological and fluorescence quantitation from time lapse images of clonal growth rates using a recently reported microfluidic system. The inputs in all pipelines consist of thousands of unprocessed images and the outputs are the detection of cell counts, chamber identifiers, and individual morphological properties of each clone over time detected through multi-channel fluorescence and bright field imaging. Our conclusion is that unsupervised learning methods for cell segmentation substantially outperform supervised statistical methods with respect to accuracy and have key advantages including individual cell instance detection and flexibility through model training. We expect this system and software to have broad utility for researchers interested in high-throughput single-cell biology.

bioengineering↗

bioRxiv2020

Massively parallel quantification of phenotypic heterogeneity in single cell drug responses

Single cell analysis tools have made significant advances in characterizing genomic heterogeneity, however tools for measuring phenotypic heterogeneity have lagged due to the increased difficulty of handling live biology. Here, we report a single cell phenotyping tool capable of measuring image-based clonal properties at scales approaching 100,000 clones per experiment. These advances are achieved by exploiting a novel flow regime in ladder microfluidic networks that, under appropriate conditions, yield a mathematically perfect cell trap. Machine learning and computer vision tools are used to control the imaging hardware and analyze the cellular phenotypic parameters within these images. Using this platform, we quantified the responses of tens of thousands of single cell-derived acute myeloid leukemia (AML) clones to targeted therapy, identifying rare resistance and morphological phenotypes at frequencies down to 0.05%. This approach can be extended to higher-level cellular architectures such as cell pairs and organoids and on-chip live-cell fluorescence assays.

bioengineering↗

bioRxiv2020

Monocarboxylate transporter antagonism reveals metabolic vulnerabilities of viral-driven lymphomas

Epstein-Barr Virus (EBV) is a ubiquitous herpesvirus that typically causes asymptomatic infection but can promote B lymphoid tumors in the immune-suppressed. In vitro, EBV infection of primary B cells stimulates glycolysis during immortalization into lymphoblastoid cell lines (LCLs). Lactate export during glycolysis is crucial for continued proliferation of many cancer cells-part of a phenomenon known as the "Warburg effect," and is mediated by the monocarboxylate transporters 1 and 4 (MCT1 and MCT4). However, the role of MCT1/4 has yet to be studied in EBV-associated malignancies which display Warburg-like metabolism in vitro. Here, we show that EBV infection of B lymphocytes directly promotes temporal induction of MCT1 and MCT4 through the viral proteins EBNA2 and LMP1 respectively, with MCT1 being induced early after infection and MCT4 late. Remarkably, singular MCT1 inhibition early, and dual MCT1/4 inhibition in LCLs using a novel MCT4-selective inhibitor led to growth arrest and lactate buildup. Metabolic profiling in LCLs revealed significatly reduced oxygen consumption rates (OCR) and NAD+/NADH ratios, contrary to prevous observations of increased OCR and unaltered NAD+/NADH ratios in MCT1/MCT4-inhibited cancer cells. Furthermore, U-13C6 glucose labeling of MCT1/4-inhibited LCLs also revealed increased labeling of glutathione in the presence of elevated ROS and depleted glutathione pools, as well as increased labeling of de novo pyrimidine biosynthetic intermediates, suggesting broad effects on LCL metabolism. These vulnerabilities sensitized LCLs as well as EBV+, and the related gammaherpesvirus KSHV+ lymphoma cell lines to killing by metformin and phenformin, pointing at a novel therapeutic approach for viral lymphomas.

cancer biology↗

bioRxiv2020

Epstein-Barr virus promotes survival through germinal center light zone chromatin architecture

BFL-1 is an understudied anti-apoptotic protein upregulated in cancer and Epstein-Barr virus (EBV)-immortalized lymphoblastoid cell lines (LCLs). We have previously shown that BFL-1 is regulated through viral EBNA3A-mediated alterations in B-cell chromatin conformation (Price et al., 2017). Here, we extend those findings to define cis- and trans-acting factors that regulate BFL-1 in LCLs and reliance on BFL-1 for survival from extrinsic apoptosis. Beyond LCLs, BFL-1 is expressed in B cells maturing in the germinal center (GC). We therefore characterized the gene expression profiles and chromatin landscape of maturing human tonsillar B-cell subsets. While chromatin accessibility at the BFL-1 locus increases as naive B cells enter the GC reaction, BFL-1 expression increases during the transition from dark zone to light zone (LZ) correlating with association between enhancer regions and the transcriptional start site. The relationship between LCLs and LZ B cells suggests that EBV phenocopies GC biology to enhance their survival in establishing latent infection.

immunology↗

bioRxiv2020

Single-cell characterization of transcriptomic heterogeneity in lymphoblastoid cell lines

Lymphoblastoid Cell Lines (LCLs) are generated by transforming primary B cells with Epstein-Barr Virus (EBV) and are used extensively as model systems in viral oncology, immunology, and human genetics research. In this study, we characterized single-cell transcriptomic profiles of five LCLs and present a simple discrete-time simulation to explore the influence of stochasticity on LCL clonal evolution. Single-cell RNA sequencing revealed substantial phenotypic heterogeneity within and across LCLs with respect to immunoglobulin isotype; virus-modulated host pathways involved in survival, proliferation, and differentiation; viral replication state; and oxidative stress. This heterogeneity is likely attributable to intrinsic variance in primary B cells and host-pathogen dynamics. Stochastic simulations demonstrate that initial primary cell heterogeneity, random sampling, time in culture, and even mild differences in phenotype-specific fitness can contribute substantially to dynamic diversity in populations of nominally clonal cells.

immunology↗

bioRxiv2019

Epstein Barr virus genomes reveal population structure and type 1 association with endemic Burkitt lymphoma

Endemic Burkitt lymphoma (eBL), the most prevalent pediatric cancer in sub-Saharan Africa, is associated with malaria and Epstein Barr virus (EBV). In order to better understand the role of EBV in eBL, we improved viral DNA enrichment methods and generated a total of 98 new EBV genomes from both eBL cases (N=58) and healthy controls (N=40) residing in the same geographic region in Kenya. Comparing cases and controls, we found that EBV type 1 was significantly associated with eBL with 74.5% of patients (41/55) versus 47.5% of healthy children (19/40) carrying type 1 (OR=3.24, 95% CI=1.36 - 7.71, P=0.007). Controlling for EBV type, we also performed a genome-wide association study identifying 6 nonsynonymous variants in the genes EBNA1, EBNA2, BcLF1, and BARF1 that were enriched in eBL patients. Additionally, we observed that viruses isolated from plasma of eBL patients were identical to their tumor counterpart consistent with circulating viral DNA originating from the tumor. We also detected three intertypic recombinants carrying type 1 EBNA2 and type 2 EBNA3 regions as well as one novel genome with a 20 kb deletion resulting in the loss of multiple lytic and virion genes. Comparing EBV types, genes show differential variation rates as type 1 appears to be more divergent. Besides, type 2 demonstrates novel substructures. Overall, our findings address the complexities of EBV population structure and provide new insight into viral variation, which has the potential to influence eBL oncogenesis.\n\nKey PointsO_LIEBV type 1 is more prevalent in eBL patients compared to the geographically matched healthy control group.\nC_LIO_LIGenome-wide association analysis between cases and controls identifies 6 eBL-associated nonsynonymous variants in EBNA1, EBNA2, BcLF1, and BARF1 genes.\nC_LIO_LIAnalysis of population structure reveals that EBV type 2 exists as two genomic sub groups.\nC_LI

genomics↗

bioRxiv2019

Identification of Host Biomarkers of EBV Latency IIb and Latency III

Deciphering the molecular pathogenesis of virally induced cancers is challenging due, in part, to the heterogeneity of both viral and host gene expression. Epstein-Barr Virus (EBV) is a ubiquitous herpesvirus prevalent in B-cell lymphomas of the immune suppressed. EBV infection of primary human B cells leads to their immortalization into lymphoblastoid cell lines (LCLs) serving as a model of these lymphomas. In previous studies, our lab has described a temporal model for immortalization with an initial phase characterized by expression of the Epstein-Barr Nuclear Antigens (EBNAs), high c-Myc activity, and hyper-proliferation in the absence of the Latent Membrane Proteins (LMPs), called latency IIb. This is followed by the long-term outgrowth of LCLs expressing the EBNAs along with the LMPs, particularly the NF{kappa}B-activating LMP1, defining latency III. LCLs, however, express a broad distribution of LMP1 such that a subset of these cells expresses LMP1 at levels seen in latency IIb, making it difficult to distinguish these two latency states. In this study, we performed mRNA-Seq on early EBV-infected latency IIb cells and latency III LCLs sorted by NF{kappa}B activity. We found that latency IIb transcriptomes clustered independently from latency III independent of NF{kappa}B. We identified and validated mRNAs defining these latency states. Indeed, we were able to distinguish latency IIb cells from LCLs expressing low levels of LMP1 using multiplex RNA-FISH targeting EBV EBNA2, LMP1, and human CCR7. This study defines latency IIb as a bona fide latency state independent from latency III and identifies biomarkers for understanding EBV-associated tumor heterogeneity.\n\nIMPORTANCEEBV is a ubiquitous pathogen with >95% of adults harboring a life-long latent infection in memory B cells. In immunocompromised individuals, latent EBV infection can result in lymphoma. The established expression profile of these lymphomas is latency III, which includes expression of all latency genes. However, single cell analysis of EBV latent gene expression in these lymphomas suggests heterogeneity where most cells express the transcription factor, EBNA2, and only a fraction express the membrane protein LMP1. Our work describes an early phase after infection where the EBNAs are expressed without LMP1, called latency IIb. However, LMP1 levels within latency III vary widely making these states hard to discriminate. This may have important implications for therapeutic responses. It is crucial to distinguish these states to understand the molecular pathogenesis of these lymphomas. Ultimately, better tools to understand the heterogeneity of these cancers will support more efficacious therapies in the future.

cancer biology↗

bioRxiv2017

The Epstein-Barr Virus miR-BHRF1 microRNAs Regulate Viral Gene Expression in cis

The Epstein-Barr virus (EBV) miR-BHRF1 microRNA (miRNA) cluster has been shown to facilitate B-cell transformation and promote the rapid growth of the resultant lymphoblastoid cell lines (LCLs). However, we find that expression of physiological levels of the miR-BHRF1 miRNAs in LCLs transformed with a miR-BHRF1 null mutant ({triangleup}123) fails to increase their growth rate. We demonstrate that the pri-miR-BHRF1-2 and 1-3 stem-loops are present in the 3UTR of transcripts encoding EBNA-LP and that excision of pre-miR-BHRF1-2 and 1-3 by Drosha destabilizes these mRNAs and reduces expression of the encoded protein. Therefore, mutational inactivation of primiR-BHRF1-2 and 1-3 in the {triangleup}123 mutant upregulates the expression of not only EBNA-LP but also EBNA-LP-regulated mRNAs and proteins, including LMP1. We hypothesize that this overexpression causes the reduced transformation capacity of the {triangleup}123 EBV mutant. Thus, in addition to regulating cellular mRNAs in trans, miR-BHRF1-2 and 1-3 also regulate EBNA-LP mRNA expression in cis.\n\nHighlightsO_LIThe EBV miR-BHRF1 microRNAs do not up upregulate B cell growth in trans.\nC_LIO_LIEBNA-LP expression is downregulated by pri-miR-BHRF1-2 and 1-3 acting in cis.\nC_LIO_LILoss of miR-BHRF1-2 and 1-3 causes EBNA-LP overexpression and inhibits B cell growth.\nC_LIO_LINovel alternative splicing of EBV Cp/Wp transcripts was identified.\nC_LI

microbiology↗