Biology subjects

Hammerschmidt, W.

Publications and source records attributed to Hammerschmidt, W..

15 recordsLinked to original sources

bioRxiv2021

The EBNA2-EBF1 complex promotes oncogenic MYC expression levels and metabolic processes required for cell cycle progression of Epstein-Barr virus-infected B cells

Epstein-Barr virus (EBV) is a human tumor virus, which preferentially infects resting human B cells. Upon infection in vitro, EBV activates and immortalizes these cells. The viral latent protein EBV nuclear antigen (EBNA) 2 is essential for B cell activation and immortalization; it targets and binds the cellular and ubiquitously expressed DNA binding protein CBF1, thereby transactivating a plethora of viral and cellular genes. In addition, EBNA2 uses its N-terminal dimerization (END) domain to bind early B cell factor (EBF) 1, a pioneer transcription factor specifying the B cell lineage. We found that EBNA2 exploits EBF1 to support key metabolic processes and to foster cell cycle progression of infected B cells in their first cell cycles upon activation. An 1-helix within the END domain was found to promote EBF1 binding. EBV mutants lacking the 1-helix in EBNA2 can infect and activate B cells efficiently, but the activated cells fail to complete the early S phase of their initial cell cycle. Expression of MYC, target genes of MYC and E2F as well as multiple metabolic processes linked to cell cycle progression are impaired in EBV{Delta}1 infected B cells. Our findings indicate that EBF1 controls B cell activation via EBNA2 and, thus, has a critical role in regulating the cell cycle of EBV infected B cells. This is a function of EBF1 going beyond its well-known contribution to B cell lineage specification. Significance statementEpstein-Barr virus (EBV) infects primary B cells and establishes life-long latent infection in these cells. EBV nuclear antigen (EBNA) 2 drives early processes of B cell activation and cell cycle entry. The surface of the N-terminal dimerization domain of EBNA2 exposes a five amino acid -helix (1) that recruits EBF1 to activate MYC and downstream targets of both MYC and E2F to support critical metabolic processes in infected B cells and to drive them through S phase in the first cell cycle post-infection. Our study demonstrates how EBNA2 exploits EBF1, a key factor of B cell lineage specification to initiate proliferation and high-lights the 1-helix as a potential Achilles heel of the virus at the stage when latent infection is established.

molecular biology↗

bioRxiv2021

Systematic analysis of Epstein-Barr virus genes and their individual contribution to virus production and composition

Epstein-Barr virus (EBV), a member of the large herpesvirus family is a very complex human{gamma} -herpesvirus. Its complexity with respect to viral gene regulation, its preferred latent life style and technical difficulties are major obstacles, which hinder efficient virus generation in vitro to mass-produce or establish recombinant wild-type EBV or mutant derivatives. To explore conditions optimizing and improving virus production, we established and tested an EBV gene library with 77 expression plasmids and a set of designed shRNAs. With this tool set we investigated the contributions of individual viral genes in the context of virus synthesis and virion functions. Engineered virus stocks were systematically analyzed with respect to physical and bioparticle concentration, virus titer and virus uptake by primary human B cells, EBVs target cells in vivo. To quantitate virus uptake by these cells, we developed a novel {beta}-lactamase-based assay that can monitor fusion events of the viral envelope with membranes of recipient cells at the level of single cells. Based on our findings, EBV does not encode a dominant regulator that governs virus production, but our results identified several EBV genes such as BALF4, BVLF1 and BKRF4, encoding a viral glycoprotein, a transcriptional regulator of late viral genes, and a possible tegument protein, respectively, that improve virus production regarding virus yield, virus composition and quality, and virus uptake by primary human B cells. ImportanceFor more than 20 years HEK293 cells have been instrumental to produce virus stocks of recombinant EBV. The identification of this cell line as a source of infectious EB virions was unexpected because HEK293 cells are very distant from B cells and in particular plasma cells which produce EBV progeny in vivo. To our knowledge, no systematic analysis has addressed the fundamental question whether virus yield with respect to virus concentration, virion composition and functionality can be improved to produce recombinant EBV stocks in HEK293 cells. We tackled this question and analyzed 77 individual EBV genes for their possible contribution to virus yield. To analyze and compare different virus stocks we used established and developed novel assays to characterize important parameters of EB virions and their functions.

microbiology↗

bioRxiv2021

Inhibition of PLK1-dependent EBNA2 phosphorylation promotes lymphomagenesis in EBV-infected mice.

While Epstein-Barr virus (EBV) establishes a life-long latent infection in apparently healthy human immunocompetent hosts, immunodeficient individuals are at particular risk to develop lymphoproliferative B cell malignancies caused by EBV. A key EBV protein is the transcription factor EBV nuclear antigen 2 (EBNA2), which initiates B cell proliferation. Here, we combine biochemical, cellular and in vivo experiments demonstrating that the mitotic polo-like kinase 1 (PLK1) binds to EBNA2, phosphorylates its transactivation domain and thereby inhibits its biological activity. EBNA2 mutants that impair PLK1 binding or prevent EBNA2 phosphorylation are gain-of-function mutants. They have enhanced transactivation capacities, accelerate the proliferation of infected B cells and promote the development of monoclonal B cell lymphomas in infected mice. Thus, PLK1 coordinates the activity of EBNA2 to attenuate the risk of tumor incidences in favor of the establishment of latency in the infected but healthy host.

cancer biology↗

bioRxiv2020

Multiple viral microRNAs regulate interferon release and signaling early during infection with Epstein-Barr virus

Epstein-Barr virus (EBV), a human herpes virus, encodes 44 microRNAs (miRNAs), which regulate many genes with various functions in EBV-infected cells. Multiple target genes of the EBV miRNAs have been identified, some of which play important roles in adaptive antiviral immune responses. Using EBV mutant derivatives, we identified additional roles of viral miRNAs in governing versatile type I interferon (IFN) responses upon infection of human primary mature B cells. We also found that Epstein-Barr virus-encoded small RNAs (EBERs) and LF2, viral genes with previously reported functions in inducing or regulating IFN-I pathways, had negligible or even contrary effects on secreted IFN- in our model. Data mining and Ago PAR-CLIP experiments uncovered more than a dozen of previously uncharacterized, direct cellular targets of EBV miRNA associated with type I IFN pathways. We also identified indirect targets of EBV miRNAs in B cells, such as TRL7 and TLR9, in the pre-latent phase of infection. The presence of epigenetically naive, non-CpG methylated viral DNA was essential to induce IFN- secretion during EBV infection in a TLR9-dependent manner. In a newly established fusion assay, we verified that EBV virions enter a subset of plasmacytoid dendritic cells (pDCs) and determined that these infected pDCs are the primary producers of IFN- in EBV-infected peripheral blood mononuclear cells. Our findings document that many EBV-encoded miRNAs regulate type I IFN response in newly EBV infected primary human B cells in the pre-latent phase of infection and dampen the acute release of IFN- in pDCs upon their encounter with EBV. Author summaryAcute antiviral functions of all nucleated cells rely on type I interferon (IFN-I) pathways triggered upon viral infection. Host responses encompass the sensing of incoming viruses, the activation of specific transcription factors which induce transcription of IFN-I genes, the secretion of different IFN-I types and their recognition by the heterodimeric IFN-/{beta} receptor, the subsequent activation of JAK/STAT signaling pathways and, finally, the transcription of many IFN-stimulated genes (ISGs). In sum, these cellular functions establish a so-called antiviral state in infected and neighboring cells. To counteract these cellular defense mechanisms, viruses have evolved diverse strategies and encode gene products that target antiviral responses. Among such immune evasive factors are viral microRNAs (miRNAs) that can interfere with host gene expression. We discovered that multiple miRNAs encoded by Epstein-Barr virus (EBV) control over a dozen cellular genes that contribute to the anti-viral states of immune cells, specifically B cells and plasmacytoid dendritic cells (pDCs). We identified the viral DNA genome as the activator of IFN- and question the role of abundant EBV EBERs, that, contrary to previous reports, do not have an apparent inducing function in the IFN-I pathway early after infection.

microbiology↗

bioRxiv2020

Highly efficient CRISPR-Cas9-mediated gene knockout in primary human B cells for functional genetic studies of Epstein-Barr virus infection

Gene editing is now routine in all prokaryotic and metazoan cells but has not received much attention in immune cells when the CRISPR-Cas9 technology was introduced in the field of mammalian cell biology less than ten years ago. This versatile technology has been successfully adapted for gene modifications in human myeloid cells and T cells, among others, but applications to human primary B cells have been scarce and limited to activated B cells. This limitation has precluded conclusive studies into cell activation, differentiation or cell cycle control in this cell type. We report on highly efficient, simple and rapid genome engineering in primary resting human B cells using nucleofection of Cas9 ribonucleoprotein complexes. We provide proof-of-principle of gene editing in quiescent human B cells using two model genes: CD46 and CDKN2A. The latter encodes the cell cycle regulator p16INK4a which is an important target of Epstein-Barr virus (EBV). Infection of B cells carrying a knockout of CDKN2A with wildtype and EBNA3 oncoprotein mutant strains of EBV allowed us to conclude that EBNA3C controls CDKN2A, the only barrier to B cell proliferation in EBV infected cells. Together, this approach enables efficient targeting of specific gene loci in quiescent human B cells supporting basic research as well as immunotherapeutic strategies. Author summaryHuman hematopoietic stem cells and their derivatives of the myeloid and lymphoid lineages are important targets for gene correction or modifications using the CRISPR-Cas9 technology. Among others, this approach can support site-specific insertion of chimeric antigen receptors (CARs) or T cell receptors (TCRs) into primary T cells. Their subsequent adoptive transfer to patient donors is a promising immunotherapeutic concept that may control chronic infection or certain types of cancer. Human B cells have a similar potential but, in contrast to T cells, they are very sensitive, difficult to handle, and short-lived ex vivo precluding their genetic modification. Here, we provide means to manipulate primary human B cells genetically using in vitro assembled Cas9 ribonucleoprotein complexes and electroporation for their delivery. Our study demonstrates near-to-complete loss of a model target gene and provides examples to evaluate a cellular gene with a critical role during infection with Epstein-Barr virus (EBV).

immunology↗

bioRxiv2020

Human ORC/MCM density is low in active genes and correlates with replication time but does not solely define replication initiation zones

Eukaryotic DNA replication initiates during S phase from origins that have been licensed in the preceding G1 phase. Here, we compare ChIP-seq profiles of the licensing factors Orc2, Orc3, Mcm3, and Mcm7 with gene expression, replication timing and fork directionality profiles obtained by RNA-seq, Repli-seq and OK-seq. ORC and MCM are strongly and homogeneously depleted from transcribed genes, enriched at gene promoters, and more abundant in early-than in late-replicating domains. Surprisingly, after controlling these variables, no difference in ORC/MCM density is detected between initiation zones, termination zones, unidirectionally replicating and randomly replicating regions. Therefore, ORC/MCM density correlates with replication timing but does not solely regulate the probability of replication initiation. Interestingly, H4K20me3, a histone modification proposed to facilitate late origin licensing, was enriched in late replicating initiation zones and gene deserts of stochastic replication fork direction. We discuss potential mechanisms that specify when and where replication initiates in human cells.

cell biology↗

bioRxiv2020

Micro RNAs are minor constituents of extracellular vesicles and are hardly delivered to target cells

Mammalian cells release different types of vesicles, collectively termed extracellular vesicles (EVs). EVs contain cellular microRNAs (miRNAs) with an apparent potential to deliver their miRNA cargo to recipient cells to affect the stability of individual mRNAs and the cells transcriptome. The extent to which miRNAs are exported via the EV route and whether they contribute to cell-cell communication are controversial. To address these issues, we analyzed the capacity of EVs to deliver packaged miRNAs into target cells and to exert biological functions. We applied well-defined approaches to produce and characterize purified EVs with or without specific viral miRNAs. We found that only a small fraction of EVs carried miRNAs. EVs readily bound to different target cell types, but there was no EV-cell membrane fusion or delivery of cargo. Importantly, the functionality of cells exposed to miRNA-carrying EVs was not affected. These results suggest EV-borne miRNAs do not act as effectors and question their relevancy in paracrine cell-to-cell communication. AUTHOR SUMMARYThe majority of metazoan cells release vesicles of different types and origins, such as exosomes and microvesicles, now collectively termed extracellular vesicles (EVs). EVs have gained much attention because they contain microRNAs (miRNAs) and thus could regulate their specific mRNA targets in recipient or acceptor cells that take up EVs. Using a novel fusion assay with superior sensitivity and specificity, we revisited this claim but found no convincing evidence for an efficient functional uptake of EVs in many different cell lines and primary human blood cells. Even EVs engineered to fuse and deliver their miRNA cargo to recipient cells had no measurable effect on target mRNAs in very carefully controlled, quantitative experiments. Our negative results clearly indicate that EVs do not act as vehicles for miRNA-based cell-to-cell communication.

cell biology↗

bioRxiv2019

Active transcription regulates ORC/MCM distribution whereas replication timing correlates with ORC density in human cells

Eukaryotic replication initiates during S phase from origins that have been licensed in the preceding G1 phase. Here, we compare ChIP-seq profiles of the licensing factors Orc2, Orc3, Mcm3, and Mcm7 with replication initiation events obtained by Okazaki fragment sequencing. We demonstrate that MCM is displaced from early replicating, actively transcribed gene bodies, while ORC is mainly enriched at active TSS. Late replicating, H4K20me3 containing initiation zones display enhanced ORC and MCM levels. Furthermore, we find early RTDs being primarily enriched in ORC, compared to MCM, indicating that ORC levels are involved in organizing the temporal order of DNA replication. The organizational connection between active transcription and replication competence directly links changes in the transcriptional program to flexible replication patterns, which ensures the cells flexibility to respond to environmental cues.

cell biology↗

bioRxiv2019

KLF4 acts as a tumor suppressor in human B-cells and patients’ B-cell leukemias growing in mice

Clinic-close methods are not available that prioritize and validate potential therapeutic targets in individual tumors from the vast bulk of descriptive expression data. We developed a novel technique to express transgenes in established patient-derived xenograft (PDX) models in vivo to fill this gap. With this technique at hand, we analyzed the role of transcription factor Kruppel-like factor 4 (KLF4) in B-cell acute lymphoblastic leukemia (B-ALL) PDX models at different disease stages. In competitive pre-clinical in vivo trials, we found that re-expression of wild type KLF4 reduced leukemia load in PDX models of B-ALL, with strongest effects after conventional chemotherapy at minimal residual disease (MRD). A non-functional KLF4 mutant had no effect in this model. Re-expressing KLF4 sensitized tumor cells in the PDX model towards systemic chemotherapy in vivo. Of major translational relevance, Azacitidine upregulated KLF4 levels in the PDX model and a KLF4 knockout reduced Azacitidine-induced cell death, suggesting that Azacitidine can regulate KLF4 re-expression. These results support applying Azacitidine in patients with B-ALL to regulated KLF4 as a therapeutic option. Taken together, our novel technique allows studying the function of dysregulated genes in a highly clinic-related, translational context and testing clinically applicable drugs in a relevant pre-clinical model.

cancer biology↗

bioRxiv2019

The first days in the life of naïve human B-lymphocytes infected with Epstein-Barr virus

Epstein-Barr virus (EBV) infects and activates resting human B-lymphocytes, reprograms them, induces their proliferation, and establishes a latent infection in them. In established EBV-infected cell lines many viral latent genes are expressed. Their roles in supporting the continuous proliferation of EBV-infected B cells in vitro are known, but their functions in the early, pre-latent phase of infection have not been investigated systematically. In studies during the first eight days of infection using derivatives of EBV with mutations in single genes of EBVs we found only EBNA2 to be essential for activating naive human B-lymphocytes, inducing their growth in cell volume, driving them into rapid cell divisions, and preventing cell death in a subset of infected cells. EBNA-LP, LMP2A and the viral microRNAs have supportive, auxiliary functions, but mutants of LMP1, EBNA3A, EBNA3C, and the noncoding EBER RNAs had no discernable phenotype compared with wild-type EBV. B cells infected with a double mutant of EBNA3A and 3C had an unexpected proliferative advantage and did not regulate the DNA damage response (DDR) of the infected host cell in the pre-latent phase. Even EBNA1 which has very critical long-term functions in maintaining and replicating the viral genomic DNA in established cell lines, was dispensable for the early activation of infected cells. Our findings document that the virus dose is a critical parameter and indicate that EBNA2 governs the infected cells initially and implements a strictly controlled temporal program independent of other viral latent genes. It thus appears that EBNA2 is sufficient to control all requirements for clonal cellular expansion and to reprogram human B-lymphocytes from energetically quiescent to activated cells.\n\nAuthor summaryThe preferred target of Epstein-Barr virus (EBV) are human resting B-lymphocytes. We found that their infection induces a well-coordinated, time-driven program that starts with a substantial increase in cell volume followed by cellular DNA synthesis after three days and subsequent rapid rounds of cell divisions on the next day accompanied by some DNA replication stress (DRS). Two to three days later the cells decelerate and turn into stably proliferating lymphoblast cell lines. With the aid of 16 different recombinant EBV strains we investigated the individual contributions of EBVs multiple latent genes during early B-cell infection and found that many do not exert a detectable phenotype or contribute little to EBVs pre-latent phase. The exception is EBNA2 that is essential in governing all aspects of B-cell reprogramming. EBV relies on EBNA2 to turn the infected B-lymphocytes into proliferating lymphoblasts preparing the infected host cell for the ensuing stable, latent phase of viral infection. In the early steps of B-cell reprogramming viral latent genes other than EBNA2 are dispensable but some, EBNA-LP for example, support the viral program and presumably stabilize the infected cells once viral latency is established.

microbiology↗

bioRxiv2019

Epstein-Barr virus inactivates the transcriptome and disrupts the chromatin architecture of its host cell in the first phase of lytic reactivation

Epstein-Barr virus (EBV), a herpes virus also termed HHV 4 and the first identified human tumor virus, establishes a stable long-term latent infection in human B cells, its preferred host. Upon induction of EBVs lytic phase the latently infected cells turn into a virus factory, a process, that is governed by EBV. In the lytic, productive phase all herpesviruses ensure the efficient induction of all lytic viral genes to produce progeny, but certain of these genes also repress the ensuing antiviral responses of the virally infected host cells, regulate their apoptotic death or control the cellular transcriptome. We now find that EBV causes previously unknown massive and global alterations in the chromatin of its host cell upon induction of the viral lytic phase and prior to the onset of viral DNA replication. The viral initiator protein of the lytic cycle, BZLF1, binds to >105 binding sites with different sequence motifs in cellular chromatin and in a concentration dependent manner. Concomitant with DNA binding, silent chromatin opens locally as shown by ATAC-seq experiments, while previously wide-open cellular chromatin becomes inaccessible on a global scale within hours. While viral transcripts increase drastically, the induction of the lytic phase results in a massive reduction of cellular transcripts and a loss of chromatin-chromatin interactions of cellular promoters with their distal regulatory elements as shown in Capture-C experiments. Our data document that EBVs lytic cycle induces discrete early processes that disrupt the architecture of host cellular chromatin and repress the cellular epigenome and transcriptome likely supporting the efficient de novo synthesis of this herpesvirus.

cell biology↗

bioRxiv2018

Epstein-Barr virus reprograms human B-lymphocytes immediately in the pre-latent phase of infection

Epstein-Barr virus (EBV) is a human tumor virus and a model of herpesviral latency. The virus efficiently infects resting human B-lymphocytes and induces their continuous proliferation in vitro, which mimics certain aspects of EBVs oncogenic potential in vivo. This seminal finding was made 50 years ago, but how EBV activates primary human B-lymphocytes and how lymphoblastoid cell lines (LCLs) evolve from the EBV-infected lymphocytes is uncertain. We conducted a systematic time-resolved longitudinal study of cellular functions and transcriptional profiles of newly infected naive primary B-lymphocytes. EBV reprograms these human cells comprehensively and globally. Rapid and extensive transcriptional changes occur within 24 hours of infection and precede any metabolic and phenotypic changes. Within the next 48 hours, the virus activates the cells, changes their phenotypes with respect to cell size, RNA and protein content and induces metabolic pathways to cope with the increased demand for energy, supporting an efficient cell cycle entry on day three post infection. The transcriptional program that EBV initiates consists of three waves of clearly discernable clusters of cellular genes that peak on day one, two, or three and regulate RNA synthesis, metabolic pathways and cell division, respectively. Upon the onset of cell doublings on day four the cellular transcriptome appears to be completely reprogrammed to support the activated and proliferating cell, but three additional clusters of EBV regulated genes adjust the infected immune cells to fine-tune cell signaling, migration, and immune response pathways, eventually. Our study reveals that more than 98 % of the 13,000 expressed genes in B-lymphocytes are regulated upon infection demonstrating that EBV governs the entire biology of its target cell.

microbiology↗

bioRxiv2018

Money or smiles: Independent ERP effects of associated monetary reward and happy faces

In comparison to neutral faces, facial expressions of emotion are known to gain attentional prioritization, mainly demonstrated by means of event-related potentials (ERPs). Recent evidence indicated that such a preferential processing can also be elicited by neutral faces when associated with increased motivational salience via reward. It remains, however, an open question whether impacts of inherent emotional salience and associated motivational salience might be integrated. To this aim, expressions and outcomes were orthogonally combined. Participants (N=42) learned to explicitly categorize happy and neutral faces as either reward- or zero-outcome-related via an associative learning paradigm. ERP components (P1, N170, EPN, and LPC) were measured throughout the experiment, and separately analyzed before (learning phase) and after (consolidation phase) reaching a pre-defined learning criterion. Happy facial expressions boosted early processing stages, as reflected in enhanced amplitudes of the N170 and EPN, both during learning and consolidation. In contrast, effects of monetary reward became evident only after successful learning and in form of enlarged amplitudes of the LPC, a component linked to higher-order evaluations. Interactions between expressions and associated outcome were absent in all ERP components of interest. The present study provides novel evidence that acquired salience impacts stimulus processing but independent of the effects driven by happy facial expressions.

neuroscience↗

bioRxiv2018

BZLF1 interacts with the chromatin remodeler INO80 promoting escape from latent infections with Epstein-Barr virus

A hallmark of Epstein-Barr virus (EBV) infections is its latent phase, when all viral lytic genes are repressed. Repression results from a high nucleosome occupancy and epigenetic silencing by cellular factors such as the Polycomb repressive complex 2 (PRC2) and DNA methyltransferases that respectively introduce repressive histone marks and DNA methylation. The viral transcription factor BZLF1 acts as molecular switch to induce the transition from the latent to the lytic or productive phase of EBVs life cycle. It is unknown how BZLF1 can bind to the epigenetically silenced viral DNA and whether it directly reactivates the viral genome through chromatin remodeling. We addressed these fundamental questions and found that BZLF1 binds to nucleosomal DNA motifs both in vivo and in vitro, a property characteristic of bona fide pioneer factors. BZLF1 co-precipitates with cellular chromatin remodeler ATPases, and the knock-down of one of them, INO80, impaired lytic reactivation and virus synthesis. We conclude that BZLF1 reactivates the EBV genome by directly binding to silenced chromatin and recruiting cellular chromatin remodeling enzymes, which implement a permissive state for viral transcription. BZLF1 shares this mode of action with a limited number of cellular pioneer factors, which are instrumental in transcriptional activation, differentiation, and reprogramming in all eukaryotic cells.

microbiology↗

bioRxiv2017

Implicit reward associations impact face processing: Time-resolved evidence from event-related brain potentials and pupil dilations

The present study aimed at investigating whether associated motivational salience causes preferential processing of inherently neutral faces similar to emotional expressions by means of event-related brain potentials (ERPs) and changes of the pupil size. To this aim, neutral faces were implicitly associated with monetary outcome, while participants (N = 44) performed a subliminal face-matching task that ensured performance around chance level and thus an equal proportion of gain, loss, and zero outcomes. Motivational context strongly impacted processing of all - even task-irrelevant - stimuli prior to the target face, indicated by enhanced amplitudes of subsequent ERP components and increased pupil size. In a separate test session, previously associated faces as well as novel faces with emotional expressions were presented within the same task but without motivational context and performance feedback. Most importantly, previously gain-associated faces amplified the LPC, although the individually contingent face-outcome assignments were not made explicit during the learning session. Emotional expressions impacted the N170 and EPN components. Modulations of the pupil size were absent in both motivationally-associated and emotional conditions. Our findings demonstrate that neural representations of neutral stimuli can acquire increased salience via implicit learning, with an advantage for gain over loss associations.

neuroscience↗