Biology subjects

Gewurz, B. E.

Publications and source records attributed to Gewurz, B. E..

At least 19 recordsLinked to original sources

bioRxiv2026

The diverse functions of viral microproteins

A large fraction of the uncharacterized proteome consists of microproteins. Viruses, rich in microproteins with potent activities, provide a unique system for exploring their functions and evolution. Here, we systematically annotate viral microproteins through a phenotype-first approach: construction of a >50,000-element small ORF library spanning the microprotein-coding potential of human-infecting viruses, followed by broad screens that enrich candidates for high-resolution profiling by Perturb-seq, mass spectrometry, AI-guided structure prediction, and mechanistic studies. This atlas identified >2,000 active microproteins that modulate host pathways through diverse mechanisms, including molecular mimicry, hijacking of key regulators, and altering subcellular localization. We further show the oncogenic potential of BNLF2b, an uncharacterized Epstein-Barr virus microprotein linked to nasopharyngeal carcinoma. Three principles emerge: viral microproteins are diverse and often multifunctional, show widespread convergence toward shared functions, and serve as reservoirs of evolutionary innovation. These findings elucidate how viral microproteins occupy a large functional landscape, establish them as versatile tools for host manipulation, and provide a framework for functional annotation of uncharacterized proteins, contributing toward predictive systems virology.

systems biology↗

bioRxiv2026

Epstein-Barr virus transformation creates a methionine-dependent ferroptosis vulnerability in B cells

Epstein-Barr virus (EBV) causes over 200,000 cancers annually, including immunoblastic lymphomas in immunosuppressed hosts. Most transformed cells arrest, yet survive when deprived of the essential amino acid methionine. We instead find that EBV transformed lymphoblastoid cell lines (LCLs), which model the EBV latency III program-driven B-cell lymphoproliferative diseases of immunosuppressed hosts, rapidly die upon methionine restriction. Methionine restriction elevated LCL lipid reactive oxygen species and triggered ferroptosis. Whereas methionine restriction hypomethylates the EBV genome and triggers viral reactivation in latency I Burkitt cells by lowering the cellular methylation potential, the LCL latency III program instead redirected methionine toward redox defense, without altering the SAM/SAH ratio. Stable-isotope tracing revealed that latency III strongly induces transsulfuration, synthesizing cysteine de novo to support glutathione pools. The EBV oncoprotein LMP2A, which mimics B-cell receptor signaling, supported newly infected human B cell cystathionine-{beta}-synthase and cystathionine-{gamma}-lyase expression and methionine dependence, phenocopied by immunoglobulin crosslinking. In vivo, dietary methionine restriction impaired LCL xenograft outgrowth and depleted tumor cystine. Combined methioninase and cyst(e)inase administration blocked both cysteine sources, collapsed tumor glutathione levels, and triggered ferroptosis. Our results define methionine metabolism as a targetable ferroptosis vulnerability of EBV-transformed B cells. HighlightsO_LIMethionine restriction triggers EBV-transformed lymphoblastoid B cell ferroptosis C_LIO_LIEBV latency III induces transsulfuration to sustain LCL cysteine and glutathione C_LIO_LIMethioninase or dietary methionine restriction strongly impair LCL growth in vivo C_LIO_LIMethioninase plus cyst(e)inase collapses xenograft GSH levels and drives ferroptosis C_LI

biochemistry↗

bioRxiv2026

The HUSH Complex Dictates EBV-transformed B cell Sensitivity to NK Cell Surveillance Through Repression of NKG2A Ligand γ-Proto-cadherin

Natural Killer (NK) cells control Epstein-Barr virus (EBV), though how EBV+ B-cells escape NK surveillance to form tumors remains unknown. To gain insights, we performed a human genome-wide CRISPR-Cas9 screen in EBV-transformed lymphoblastoid cell lines (LCL). This revealed that the HUSH complex maintains LCL sensitivity to NK. HUSH knockout (KO) de-repressed LCL protocadherin gamma (PCDHG), typically expressed by neurons. PCDHG expression protected LCLs from NK and was necessary for HUSH KO-driven NK resistance. CRISPR analyses revealed NKG2A/CD94 as the PCDHG NK inhibitory counter-receptor, and NKG2A KO restored NK lysis of HUSH KO LCLs. HUSH perturbation impaired NK control of murine LCL xenografts in vivo. A subset of follicular lymphoma (FL) upregulate PCDHG, and PCDHG KO enhanced FL lysis by NK. Therefore, HUSH plays dual roles in foreign DNA surveillance and in support of NK, potentially as a neuronal dont kill me signal that can be exploited by transformed cells.

immunology↗

bioRxiv2026

CRISPR Screens Reveal Epstein-Barr Virus-activated JunB as a Key Lymphoblastoid B cell Dependency Factor that Represses Cyclin Dependent Kinase Inhibitor P18INK4c

Epstein-Barr virus (EBV) persistently infects over 95% of adults worldwide and is associated with a range of cancers, including lymphomas and epithelial malignancies. Despite advances in understanding EBV biology, targeted therapies for EBV-associated cancers remain limited. To identify novel dependencies in EBV-infected cancers, we performed genome-wide CRISPR-Cas9 loss-of-function screens in EBV+ lymphoblastoid versus Burkitt lymphoma cells, which differ by EBV latency programs. JunB emerged as a critical LCL-selective host dependency factor. LCL JunB knockout significantly decreased proliferation, with reduced G2/M progression, but without inducing apoptosis. JunB was more highly expressed in B cells with the EBV latency III than latency I program and correlated with LMPist1 levels in newly infected B cells. LMP1 stimulated JunB expression in a manner dependent on its cytoplasmic tail TES1/CTAR1 region and on canonical NF-{kappa}B. EBV-activated JunB played an obligatory role in repression of the G1/S phase inhibitor CDKN2C/p18INK4c in LCLs but not Burkitt B cells. These findings establish an LMP1-JunB-p18INK4c axis as essential for EBV-driven lymphoblastoid B cell proliferation, suggest JunB-mediated cross-talk between Epstein-Barr nuclear antigens and LMP1, and highlight JunB as a potential therapeutic target for EBV-associated lymphoproliferative disorders.

microbiology↗

bioRxiv2026

A CRISPR-Cas9 screen Reveals STEEP1 as a Key Host Dependency Factor for Epstein-Barr Virus Latent Membrane Protein 1 Trafficking and Signaling

The Epstein-Barr virus (EBV) oncogene Latent membrane protein 1 (LMP1) is essential for B-cell transformation into continuously growing lymphoblastoid cell lines. LMP1 traffics to plasma membrane and intracellular signaling sites to mimic aspects of signaling by the B cell co-receptor CD40. LMP1 is expressed in many EBV-associated cancers, including post-transplant lymphoma, Hodgkin lymphoma, T/NK lymphoma and nasopharyngeal carcinoma, where it activates key growth and survival pathways. LMP1 signaling is also implicated in multiple sclerosis pathogenesis. To identify host dependency factors that support LMP1 trafficking and signaling, we performed a human genome-wide CRISPR-Cas9 screen in B cells. The screen identified both known and previously uncharacterized mediators of LMP1 signaling. The ER resident protein STEEP1, implicated in DNA sensor STING trafficking and signaling, was a top screen hit. Importantly, STEEP1 did not score in our prior B cell CRISPR screen for factors that support CD40 signaling, suggesting specificity. STEEP1 depletion strongly impaired LMP1 signaling, including activation of NF-kB and MAP kinase pathways. Mechanistically, STEEP1 associated with LMP1 in a manner dependent on the N-terminal cytoplasmic tail and supported LMP1 egress from the ER to signaling sites in both B and epithelial cells. Collectively, these findings reveal STEEP1 as a key host factor that supports trafficking of newly synthesized LMP1 molecules to intracellular signaling sites and highlights LMP1/STEEP1 interaction as a novel therapeutic target. ImportanceEpstein-Barr virus (EBV) infects most people worldwide. While infection is often benign, it causes infectious mononucleosis, is associated with a range of lymphomas, nasopharyngeal and gastric carcinoma and is a major trigger for autoimmune disease, including multiple sclerosis. The EBV encoded oncogene LMP1 is a key driver of EBV pathogenesis, and its signaling is necessary for viral immortalization of B lymphocytes into continuously growing lymphoblasts (LCLs). Here, we performed a CRISPR genetic screen to identify host factors that support continuous, ligand-independent signaling by LMP1. This analysis identified an ER-resident protein called STEEP1, previously implicated in support of trafficking of the DNA sensor STING, as a key LMP1 partner. We found that STEEP1 associates with LMP1 and supports LMP1 trafficking out of the endoplasmic reticulum to cellular signaling sites. As STEEP1 knockout impaired LMP1 function and LCL survival, our study identifies the STEEP1/LMP1 complex as a therapeutic target.

microbiology↗

bioRxiv2026

Epstein-Barr Virus Latent Membrane Protein 1 Suppresses Ferroptosis via Pentose Phosphate Pathway and Glutathione Metabolism

Epstein-Barr virus (EBV) is associated with 200,000 cancers per year, including Burkitt lymphoma and post-transplant lymphomas. We previously reported that EBV latency oncogene programs dynamically remodel infected B cell metabolism and sensitivity to induction of ferroptosis, a programmed cell death pathway driven by lipid reactive oxygen species. However, much has remained unknown about how EBV remodels key redox defense pathways in support of infected B cell proliferation. Here, we identify EBV latent membrane protein 1 (LMP1), a key viral oncogene necessary for B cell immortalization and which mimics aspects of CD40 signaling, drives resistance to ferroptosis induction by erastin, a small molecule that blocks cystine uptake. LMP1 expression was sufficient to protect Burkitt cells from erastin ferroptosis induction. Mechanistically, signaling from the LMP1 TES2/CTAR2 region drove this phenotype, which was not shared by CD40 signaling, revealing that LMP1 evolved independent redox defense roles. Metabolomic analysis highlighted key LMP1 and TES2 signaling roles in support of antioxidant cysteine and glutathione levels. TES2 signaling supported cystine uptake, glutathione and NADPH pools in newly infected peripheral blood B cells. We identified PFKFB4, a host enzyme that shunts glucose into the pentose phosphate pathway to support NADPH production, as a major TES2 metabolic target. PFKFB4 knockdown increased EBV-transformed lymphoblastoid cell line lipid ROS levels, decreased glutathione and strongly sensitized them to ferroptosis induction by erastin treatment. PFKFB4 was also necessary for LMP1-mediated Burkitt B cell ferroptosis resistance. Collectively, these results identify PFKFB4 as a key host cell EBV metabolism remodeling target critical for infected B cell redox defense.

microbiology↗

bioRxiv2026

A tonsil organoid model reveals Epstein-Barr virus infected germinal center B cell states during primary infection

Epstein-Barr virus (EBV) colonizes secondary lymphoid tissues to establish persistent infection and is strongly associated with malignancy and autoimmunity. Our understanding of EBV infection biology is hindered by a lack of models that capture infected B cell activity in the lymphoid tissue microenvironment. We therefore developed an EBV human tonsil organoid model to evaluate key B cell states and antiviral responses, including after primary infection. EBV promoted B cell differentiation into germinal center (GC)-like phenotypes and transcriptomic analyses highlighted numerous B cell transcriptional programs unique to EBV-infected cells. B cell receptor repertoire analysis revealed that most EBV-infected B cells underwent class switching but only rarely participated in somatic hypermutation. CD4 T cells, highly activated by organoid infection, limited EBV+ B cell outgrowth. Our findings demonstrate human tonsil organoids as a physiologically relevant model to investigate key aspects of EBV immunity and pathogenesis.

immunology↗

bioRxiv2025

Epstein-Barr Virus Encoded lncRNAs Control the Viral Lytic Switch

Epstein-Barr virus (EBV) uses a biphasic lifecycle, switching between latent and lytic phases to persistently infect most adults. Latency is observed in most tumor cells of the 200,000 EBV-associated cancers/year. EBV reactivation is increasingly implicated in autoimmune diseases, including multiple sclerosis. However, mechanisms that regulate EBV reactivation have remained incompletely understood. Here, we leveraged multi-omic approaches to reveal the existence of pro-latency and a pro-lytic viral long noncoding RNAs (lncRNAs) that counter-regulate the lytic switch. Known reactivation triggers rapidly induced expression of the pro-lytic lncRNA, which encodes an RNA G-quadruplex that mediated its interaction with CTCF. The pro-lytic lncRNA occupies viral origin of lytic replication enhancers and promotes their looping to the immediate early lytic promoter to trigger reactivation. The pro-latency lncRNA duplexes with the pro-lytic RNA to impede its interactions with CTCF. These studies lay a foundation for therapeutic approaches to manipulate the EBV lytic switch.

microbiology↗

bioRxiv2025

Arginine Metabolism Supports De Novo Pyrimidine Biosynthesis to Block DNA Damage and Maintain Epstein-Barr Virus Latency

Incompletely understood mechanisms serve to maintain Epstein-Barr virus (EBV) latency in most B-cell states, in which viral oncogene(s) are expressed but lytic antigens are repressed. Shortly after EBVs discovery and even before it was named, early pioneers Werne and Gertrude Henle identified that restriction of extracellular arginine de-represses EBV lytic antigens within Burkitt lymphoma tumor cells. However, for nearly 60 years, it has remained unknown how arginine metabolism supports EBV latency. To gain insights, we performed an amino acid restriction screen in Burkitt cell lines. This confirmed that arginine restriction was sufficient to trigger EBV reactivation in Burkitt B-cells and gastric carcinoma models. Arginine restriction strongly impaired de novo pyrimidine biosynthesis, and CRISPR or chemical genetic blockade of pyrimidine biosynthesis enzymes induced EBV immediate early and early lytic gene expression. However, arginine restriction blocked EBV lytic DNA replication and consequently also late gene expression, suggesting an abortive lytic cycle. Arginine restriction triggered DNA damage, which was an important driver of arginine restriction-driven EBV reactivation. Arginine restriction and DNA hypomethylation synergistically increased EBV reactivation. Together, our results highlight arginine and pyrimidine metabolism as potential targets for EBV lytic antigen induction therapy in B and epithelial cell contexts. ImportanceAltered metabolism is a hallmark of cancer, frequently increasing transformed cell dependence on extracellular amino acid supply. Despite current interest in EBV lytic antigen induction therapy, in which viral lytic reactivation sensitizes tumors to the highly cytotoxic effects of the antiviral ganciclovir, there has been no systemic study of extracellular amino acid that controls EBV latency. We identified that arginine uptake was important for the maintenance of EBV latency in both Burkitt lymphoma and gastric carcinoma contexts. Metabolic pathway analyses highlighted that arginine uptake and metabolism was required to supply pyrimidines. Disruption of arginine metabolism or de novo pyrimidine synthesis caused DNA damage. As arginine restriction was also found to cause Burkitt DNA hypermethylation, we provide evidence that the combination of arginine restriction and DNA hypomethylation by decitabine or by CRISPR approaches together induced EBV reactivation more highly than either alone, suggesting a therapeutic approach.

microbiology↗

bioRxiv2025

Epstein-Barr Virus Latent Membrane Protein 1 targets cIAP1, cIAP2 and TRAF2 for Proteasomal Degradation to Activate the Non-canonical NF-κB Pathway

The Epstein-Barr virus oncoprotein Latent Membrane Protein 1 (LMP1) is expressed in multiple malignancies and is critical for B-cell immortalization. LMP1 constitutively activates NF-{kappa}B signaling pathways, which are essential for EBV-mediated B cell transformation and for transformed B cell survival. Reverse genetic analysis revealed two LMP1 regions critical for primary human B cell immortalization, termed transformation effector site (TES) 1 and 2, which activate multiple host growth and survival pathways, in particular NF-{kappa}B. Of these, only TES1 signaling is required for B-cell transformation within the first several weeks of infection. TES1 signaling is also critical for EBV-transformed lymphoblastoid B-cell survival. However, precisely how TES1 initiates NF-{kappa}B signaling has remained incompletely understood. Here, we provide multiple lines of evidence that TES1 associates with cellular inhibitor of apoptosis protein 1 and 2 (cIAP1/2) in a tumor necrosis factor associated factor 3 (TRAF3) dependent manner. TES1 signaling drives cIAP1 autoubiquitination and targets TRAF2, cIAP1 and 2 for proteasomal degradation in a TRAF3 dependent manner. Overexpression of either cIAP1 or 2 impaired LMP1 TES1-mediated non-canonical NF-{kappa}B activation. Collectively, these studies suggest that LMP1 TES1 initiates non-canonical NF-{kappa}B signaling distinctly from CD40 and other host immunoreceptors, thereby highlighting a therapeutic target.

microbiology↗

bioRxiv2025

Systematic investigation of Epstein-Barr virus transcriptional regulator interactions with the human genome

We systematically investigate interactions between Epstein-Barr virus (EBV) transcriptional regulators (vTRs) and the human genome. Starting with 16 known and candidate vTRs, we identify nine whose introduction into human cells results in substantial alterations to host gene expression. Genome-scale determination of vTR genomic binding events and alterations to chromatin accessibility reveals a detailed map of EBVs functional interactions with the human genome, including >100,000 vTR binding events impacting almost a quarter of all human genes. BMRF1 emerges as a potent regulator, impacting >7,000 genes and altering >37,000 chromatin regions. Our results provide new evidence that EBV RTA interacts with and stabilizes the binding of human RBPJ. Network analysis reveals that many human genes are targeted by multiple EBV vTRs, highlighting the vast coordinated impact of EBV on human gene expression. This study provides a valuable, extensive resource for examining EBV-induced alterations to human gene regulation, with data available on multiple platforms.

genomics↗

bioRxiv2025

Insights into the Absence of Lymphoma Despite Fulminant Epstein-Barr Virus Infection in Patients with XIAP Deficiency

X-linked Lymphoproliferative Syndromes (XLP), which arise from mutations in the SH2D1A or XIAP genes, are characterized by the inability to control Epstein-Barr Virus (EBV) infection. While primary EBV infection triggers severe diseases in each, lymphomas occur at high rates with XLP-1 but not with XLP-2. Why XLP-2 patients are apparently protected from EBV-driven lymphomagenesis, in contrast to all other described congenital conditions that result in heightened susceptibility to EBV, remains a key open question. To gain insights, we cross-compared newly EBV infected versus immune stimulated B-cells from XLP-2 patients or upon XIAP CRISPR knockout, relative to healthy controls. XIAP perturbation impeded outgrowth of newly EBV-infected primary human B-cells, though had no impact on proliferation of B-cells stimulated by CD40 ligand and interleukin-21 or upon established EBV-immortalized lymphoblastoid cell lines (LCLs). B-cells from XLP-2 patients or in which XIAP was depleted by CRISPR editing exhibited a markedly lower EBV transformation efficiency than healthy control B-cells. Mechanistically, nascent EBV infection activated p53-mediated apoptosis signaling, whose effects on transforming B-cell death were counteracted by XIAP. In the absence of XIAP, EBV infection triggered high rates of apoptosis, not seen with CD40L/IL-21 stimulation. Moreover, inflammatory cytokines are present at high levels in XLP-2 patient serum with fulminant EBV infection, which heightened apoptosis induction in newly EBV-infected cells. These findings highlight the crucial role of XIAP in supporting early stages of EBV-driven B-cell immortalization and provide insights into the absence of EBV+ lymphoma in XLP-2 patients. Key pointsO_LIXIAP loss-of-function markedly impairs EBV+ B-cells outgrowth over the first week post-infection, particularly in the presence of IFN-{gamma}. C_LIO_LIXIAP mutation impedes EBV-driven B-cell transformation by potentiating p53-driven caspase activation and apoptosis. C_LI

microbiology↗

bioRxiv2024

Epstein-Barr Virus Latent Membrane Protein 1 Subverts IMPDH pathways to drive B-cell oncometabolism

Epstein-Barr virus (EBV) is associated with multiple types of cancers, many of which express the key viral oncoprotein Latent Membrane Protein 1 (LMP1). LMP1 is the only EBV-encoded protein whose expression is sufficient to transform both epithelial and B-cells. Although metabolism reprogramming is a cancer hallmark, much remains to be learned about how LMP1 alters lymphocyte oncometabolism. To gain insights into key B-cell metabolic pathways subverted by LMP1, we performed systematic metabolomic analyses on B cells with conditional LMP1 expression. This approach highlighted that LMP highly induces de novo purine biosynthesis, with xanthosine-5-P (XMP) as one of the most highly LMP1-upregulated metabolites. Consequently, IMPDH inhibition by mycophenolic acid (MPA) triggered apoptosis of LMP1-expressing EBV-transformed lymphoblastoid cell lines (LCL), a key model for EBV-driven immunoblastic lymphomas. Whereas MPA instead caused growth arrest of Burkitt lymphoma cells with the EBV latency I program, conditional LMP1 expression triggered their apoptosis. Although both IMPDH isozymes are expressed in LCLs, only IMPDH2 was critical for LCL survival, whereas both contributed to proliferation of Burkitt cells with the EBV latency I program. Both LMP1 C-terminal cytoplasmic tail domains critical for primary human B-cell transformation were important for XMP production, and each contributed to LMP1-driven Burkitt cell sensitivity to MPA. MPA also de-repressed EBV lytic antigens including LMP1 in latency I Burkitt cells, highlighting crosstalk between the purine biosynthesis pathway and the EBV epigenome. These results suggest novel oncometabolism-based therapeutic approaches to LMP1-driven lymphomas. IMPORTANCEAltered metabolism is a hallmark of cancer, yet much remains to be learned about how EBV rewires host cell metabolism to support multiple malignancies. While the oncogene LMP1 is the only EBV-encoded gene that is sufficient to transform murine B-cells and rodent fibroblasts, knowledge has remained incomplete about how LMP1 alters host cell oncometabolism to aberrantly drive infected B-cell growth and survival. Likewise, it has remained unknown whether LMP1 expression creates metabolic vulnerabilities that can be targeted by small molecule approaches to trigger EBV-transformed B-cell programmed cell death. We therefore used metabolomic profiling to define how LMP1 signaling remodels the B-cell metabolome. We found that LMP1 upregulated purine nucleotide biosynthesis, likely to meet increased demand. Consequently, LMP1 expression sensitized Burkitt B-cells to growth arrest upon inosine monophosphate dehydrogenase blockade. Thus, while LMP1 itself may not be a therapeutic target, its signaling induces dependence on downstream druggable host cell nucleotide metabolism enzymes, suggesting rational therapeutic approaches.

microbiology↗

bioRxiv2024

The DNA loop release factor WAPL suppresses Epstein-Barr virus latent membrane protein expression to maintain the highly restricted latency I program

Epstein-Barr virus (EBV) uses latency programs to colonize the memory B-cell reservoir, and each program is associated with human malignancies. However, knowledge remains incomplete of epigenetic mechanisms that maintain the highly restricted latency I program, present in memory and Burkitt lymphoma cells, in which EBNA1 is the only EBV-encoded protein expressed. Given increasing appreciation that higher order chromatin architecture is an important determinant of viral and host gene expression, we investigated roles of Wings Apart-Like Protein Homolog (WAPL), a host factor that unloads cohesins to control DNA loop size and that was discovered as an EBNA2-associated protein. WAPL knockout (KO) in Burkitt cells de-repressed LMP1 and LMP2A expression but not other EBV oncogenes to yield a viral program reminiscent of EBV latency II, which is rarely observed in B-cells. WAPL KO also increased LMP1/2A levels in latency III lymphoblastoid cells. WAPL KO altered EBV genome architecture, triggering formation of DNA loops between the LMP promoter region and the EBV origins of lytic replication (oriLyt). Hi-C analysis further demonstrated that WAPL KO reprograms EBV genomic DNA looping. LMP1 and LMP2A de-repression correlated with decreased histone repressive marks at their promoters. We propose that EBV coopts WAPL to negatively regulate latent membrane protein expression to maintain Burkitt latency I. Author SummaryEBV is a highly prevalent herpesvirus etiologically linked to multiple lymphomas, gastric and nasopharyngeal carcinomas, and multiple sclerosis. EBV persists in the human host in B-cells that express a series of latency programs, each of which is observed in a distinct type of human lymphoma. The most restricted form of EBV latency, called latency I, is observed in memory cells and in most Burkitt lymphomas. In this state, EBNA1 is the only EBV-encoded protein expressed to facilitate infected cell immunoevasion. However, epigenetic mechanisms that repress expression of the other eight EBV-encoded latency proteins remain to be fully elucidated. We hypothesized that the host factor WAPL might have a role in restriction of EBV genes, as it is a major regulator of long-range DNA interactions by negatively regulating cohesin proteins that stabilize DNA loops, and WAPL was found in a yeast 2-hybrid screen for EBNA2-interacting host factors. Using CRISPR together with Hi-ChIP and Hi-C DNA architecture analyses, we uncovered WAPL roles in suppressing expression of LMP1 and LMP2A, which mimic signaling by CD40 and B-cell immunoglobulin receptors, respectively. These proteins are expressed together with EBNA1 in the latency II program. We demonstrate that WAPL KO changes EBV genomic architecture, including allowing the formation of DNA loops between the oriLyt enhancers and the LMP promoter regions. Collectively, our study suggests that WAPL reinforces Burkitt latency I by preventing the formation of DNA loops that may instead support the latency II program.

microbiology↗

bioRxiv2024

Human cytomegalovirus extensively re-organizes the human genome, diminishing TEAD1 transcription factor activity

Human cytomegalovirus (HCMV) infects up to 80% of the worlds population. Here, we show that HCMV infection leads to widespread changes in human chromatin accessibility and chromatin looping, with hundreds of thousands of genomic regions affected 48 hours after infection. Integrative analyses reveal HCMV-induced perturbation of Hippo signaling through drastic reduction of TEAD1 transcription factor activity. We confirm extensive concordant loss of TEAD1 binding, active H3K27ac histone marks, and chromatin looping interactions upon infection. Our data position TEAD1 at the top of a hierarchy involving multiple altered important developmental pathways. HCMV infection reduces TEAD1 activity through four distinct mechanisms: closing of TEAD1-bound chromatin, reduction of YAP1 and phosphorylated YAP1 levels, reduction of TEAD1 transcript and protein levels, and alteration of TEAD1 exon-6 usage. Altered TEAD1-based mechanisms are highly enriched at genetic risk loci associated with eye and ear development, providing mechanistic insight into HCMVs established roles in these processes.

genomics↗

bioRxiv2024

Epstein-Barr Virus Orchestrates Spatial Reorganization and Immunomodulation within the Classic Hodgkin Lymphoma Tumor Microenvironment

Classic Hodgkin Lymphoma (cHL) is a tumor composed of rare malignant Hodgkin and Reed-Sternberg (HRS) cells nested within a T-cell rich inflammatory immune infiltrate. cHL is associated with Epstein-Barr Virus (EBV) in 25% of cases. The specific contributions of EBV to the pathogenesis of cHL remain largely unknown, in part due to technical barriers in dissecting the tumor microenvironment (TME) in high detail. Herein, we applied multiplexed ion beam imaging (MIBI) spatial pro-teomics on 6 EBV-positive and 14 EBV-negative cHL samples. We identify key TME features that distinguish between EBV-positive and EBV-negative cHL, including the relative predominance of memory CD8 T cells and increased T-cell dysfunction as a function of spatial proximity to HRS cells. Building upon a larger multi-institutional cohort of 22 EBV-positive and 24 EBV-negative cHL samples, we orthogonally validated our findings through a spatial multi-omics approach, coupling whole transcriptome capture with antibody-defined cell types for tu-mor and T-cell populations within the cHL TME. We delineate contrasting transcriptomic immunological signatures between EBV-positive and EBV-negative cases that differently impact HRS cell proliferation, tumor-immune interactions, and mecha-nisms of T-cell dysregulation and dysfunction. Our multi-modal framework enabled a comprehensive dissection of EBV-linked reorganization and immune evasion within the cHL TME, and highlighted the need to elucidate the cellular and molecular fac-tors of virus-associated tumors, with potential for targeted therapeutic strategies.

cancer biology↗

bioRxiv2024

Epstein-Barr Virus BALF0/1 Subverts the Caveolin and ERAD Pathways to Target B-cell Receptor Complexes for Degradation

Epstein-Barr virus (EBV) establishes persistent infection, causes infectious mononucleosis, is a major trigger for multiple sclerosis and contributes to multiple cancers. Yet, knowledge remains incomplete about how the virus remodels host B cells to support lytic replication. We previously identified that EBV lytic replication results in selective depletion of plasma membrane B-cell receptor (BCR) complexes, comprised of immunoglobulin and the CD79A and CD79B signaling chains. Here, we used proteomic and biochemical approaches to identify that the EBV early lytic protein BALF0/1 is responsible for EBV lytic cycle BCR degradation. Mechanistically, an immunoglobulin heavy chain cytoplasmic tail KVK motif was required for ubiquitin-mediated BCR degradation, while CD79A and CD79B were dispensable. BALF0/1 subverted caveolin-mediated endocytosis to internalize plasma membrane BCR complexes and to deliver them to the endoplasmic reticulum. BALF0/1 stimulated immunoglobulin heavy chain cytoplasmic tail ubiquitination, which together with the ATPase valosin-containing protein/p97 drove ER-associated degradation of BCR complexes by cytoplasmic proteasomes. BALF0/1 knockout reduced the viral load of secreted EBV particles from B-cells that expressed a monoclonal antibody against EBV glycoprotein 350 and increased viral particle immunoglobulin incorporation. Consistent with downmodulation of plasma membrane BCR, BALF0/1 overexpression reduced viability of a diffuse large B-cell lymphoma cell line dependent upon BCR signaling. Collectively, our results suggest that EBV BALF0/1 downmodulates immunoglobulin upon lytic reactivation to block BCR signaling and support virion release. SIGNIFICANCEEBV uses a biphasic lifecycle, in which it switches between a latent state that facilitates immune evasion and a lytic state, where virion are secreted. However, when EBV infects a B-cell that makes antibody against a virion protein, EBV must have a strategy to escape becoming trapped, since maturing virion and antibody each traffic through the secretory pathway. We identified that an EBV-encoded protein expressed, BALF0/1, associates with and targets immunoglobulin complexes for degradation. Intriguingly, BALF0/1 subverts the caveolin-1 and ERAD pathways to route antibody from the plasma membrane to cytoplasmic proteasomes for degradation. We present evidence that this enhances EBV secretion from cells that produce antibody against a viral glycoprotein, which could otherwise trap virus.

microbiology↗

bioRxiv2023

Characterization of Target Gene Regulation by the Two Epstein-Barr Virus Oncogene LMP1 Domains Essential for B-cell Transformation

The Epstein-Barr virus (EBV) oncogene latent membrane protein 1 (LMP1) mimics CD40 signaling and is expressed by multiple malignancies. Two LMP1 C-terminal cytoplasmic tail regions, termed transformation essential sites (TES) 1 and 2, are critical for EBV transformation of B lymphocytes into immortalized lymphoblastoid cell lines (LCL). However, TES1 versus TES2 B-cell target genes have remained incompletely characterized, and whether both are required for LCL survival has remained unknown. To define LCL LMP1 target genes, we profiled transcriptome-wide effects of acute LMP1 CRISPR knockout (KO) prior to cell death. To then characterize specific LCL TES1 and TES2 roles, we conditionally expressed wildtype, TES1 null, TES2 null or double TES1/TES2 null LMP1 alleles upon endogenous LMP1 KO. Unexpectedly, TES1 but not TES2 signaling was critical for LCL survival. The LCL dependency factor cFLIP, which plays obligatory roles in blockade of LCL apoptosis, was highly downmodulated by loss of TES1 signaling. To further characterize TES1 vs TES2 roles, we conditionally expressed wildtype, TES1 and/or TES2 null LMP1 alleles in two Burkitt models. Systematic RNAseq analyses revealed gene clusters that responded more strongly to TES1 versus TES2, that respond strongly to both or that are oppositely regulated. Robust TES1 effects on cFLIP induction were again noted. TES1 and 2 effects on expression of additional LCL dependency factors, including BATF and IRF4, and on EBV super-enhancers were identified. Collectively, these studies suggest a model by which LMP1 TES1 and TES2 jointly remodel the B-cell transcriptome and highlight TES1 as a key therapeutic target. ImportanceEpstein-Barr virus (EBV) causes multiple human cancers, including B-cell lymphomas. In cell culture, EBV converts healthy human B-cells into immortalized ones that grow continuously, which model post-transplant lymphomas. Constitutive signaling from two cytoplasmic tail domains of the EBV oncogene Latent Membrane Protein 1 (LMP1) is required for this transformation, yet there has not been systematic analysis of their host gene targets. We identified that only signaling from the membrane proximal domain is required for survival of these EBV-immortalized cells and that its loss triggers apoptosis. We identified key LMP1 target genes, whose abundance changed significantly with loss of LMP1 signals, or that were instead upregulated in response to switching on signaling by one or both LMP1 domains in an EBV-uninfected human B-cell model. These included major anti-apoptotic factors necessary for EBV-infected B-cell survival. Bioinformatics analyses identified clusters of B-cell genes that respond differently to signaling by either or both domains.

cancer biology↗