Podcasts about ebna1

TWiV explains genetic changes in nOPV2 strains that paralyzed children, outbreak of raccoon distemper in Toronto, EcoHealth Alliance gets its NIH grant back, and breakage at chromosomal fragile sites caused by the EBNA1 protein of Epstein-Barr virus as a mechanism for carcinogenesis. Hosts: Vincent Racaniello, Rich Condit, Kathy Spindler, and Angela Mingarelli Subscribe (free): Apple Podcasts, Google Podcasts, RSS, email Become a patron of TWiV! Links for this episode Register for ASV 2023 Research assistant position at FDA (pdf) MicrobeTV Discord Server nOPV2 after two years (Polioeradication) Raccoon distemper in Toronto (CityNews Everywhere) NIH reinstates EcoHealth grant (Nature) Chromosomal breakage by EBNA1 (Nature) Agent of chromosomal instability (Nature) Timestamps by Jolene. Thanks! Weekly Picks Angela – Night Science Kathy – Kermit, Jim Henson, Rainbow Connection Rich – Alexander Skarsgård Explains the Answer to Everything Vincent – Beyond the Noise with Dr. Paul Offit Intro music is by Ronald Jenkees Send your virology questions and comments to twiv@microbe.tv

Orc6 is a crucial component of the replication initiation machinery in eukaryotes. Its study helps us to better understand one of the most basic cell functions: DNA replication. The first step in replication initiation is the assembly and DNA binding of the origin recognition complex (ORC). Although members of ORC are highly conserved and well studied, Orc6 evolved faster than the other members of the complex, has a different structure and is less well understood than Orc1-5. The low number of studies on Orc6 in human cells and the low predictability of results obtained from model systems necessitates an in depth analysis of HsOrc6. The aim of this study is to better understand the role of Orc6 in the human cell by exploring the mechanism of Orc6-Orc1-5 interaction and Orc6-DNA binding, studying the function of Orc6 in live cells, and mapping its interaction network to find new pathways and functions of the protein. The study concludes that Orc6 interacts via its C terminus with Orc1-5 in vitro. It might also have a second pre-RC interaction domain, probably close to the nuclear localization signal. Orc6 is not required for Orc1-5-DNA binding, but is able to enhance the process in vitro. It is also able to bind DNA in the absence of Orc1-5 with high affinity. In vivo experiments present evidence on the ability of Orc6 to recruit the replication machinery to itself on DNA. Although this ability is less efficient than that of EBNA1, a viral factor, it is sufficiently strong to support autonomous replication of plasmids in human cell lines for at least four weeks. Truncated variants of the protein containing at least one of the predicted Orc1-5 interaction sites are sufficient to allow replication. This finding can be used to design autonomous-replicating plasmids for use in gene therapy. Mapping the interaction network of Orc6 leads to the conclusion that HsOrc6 is only weakly attached to pre-RC factors, and is likely to have an important role in mitosis and possibly be involved in other pathways.

Cellular gene regulation depends on fundamental epigenetic mechanisms, but epigenetic modifications also govern the regulation of the life cycle of Epstein-Barr virus (EBV). Promoter usage during latency depends on DNA methylation of the viral genome and CpG-methylation of certain promoters with meZREs is an indispensable prerequisite to switch from the latent to the lytic phase. In my thesis, I wanted to assess the underlying epigenetic principles of EBV’s gene regulation during the establishment of latency and upon lytic reactivation. My results suggested a new classification of viral promoters and phases of gene regulation that depend on the epigenetic state of the viral chromatin. According to this new model, EBV’s infectious cycle consists of an initial abortive lytic, a latent, and a productive lytic phase, and the viral promoters can be classified into default-on, poised-on, and poised-off promoters. Default-on promoters are immediately active upon infection of primary B cells leading to the so-called abortive lytic phase of an EBV infection. Default-on promoters encounter the cell in an environment that supports binding of the basal transcription machinery to activate viral gene transcription. Default-on promoters include the promoters of BALF1, BHRF1, BZLF1, BRLF1, BNLF2a, BCRF1, and Wp. The protein products are indispensible for the permanent establishment of EBV’s genome in latently infected cells supporting growth transformation, immune evasion, and anti-apoptotic cellular pathways. Default-on promoters are epigenetically silenced upon occupancy of EBV’s DNA with nucleosomes very early after infection and a switch to poised-on promoters is initiated. Poised-on promoters embody an epigenetically active state upon infection, but their activation requires an additional, virus-encoded factor to allow initiation of transcription. Wp-induced expression of EBNA1 promotes the switch to the poised-on promoter Cp to sustain long-term EBNA expression. Other poised-on genes including the viral structural proteins are not provided with their cofactor initially. During latency, these promoters are repressed through compaction of chromatin by high nucleosome occupancy, trimethylation of H3K27, and a stable transmission of repressive modifications by Polycomb-mediated long-term silencing. The establishment of a defined DNA methylation pattern on EBV’s DNA further represses poised-on promoters. DNA methylation is a prerequisite for the activation of a third promoter class, termed default-off promoters. Default-off promoters are bound and transactivated by BZLF1 in a methylation-dependent manner. Upon infection of primary B cells, EBV’s DNA is completely unmethylated, impeding an early expression of default-off genes. Only two to three weeks post infection the viral genome has acquired a proper epigenetic configuration that supports transcription of default-off genes. Binding of BZLF1 alone does not suffice to recruit the cellular transcription machinery including RNA polymerase II, but the chromatin requires remodeling, including a loss of nucleosomes and repressive modifications at default-off promoter sites. Default-off genes encode the viral lytic DNA replication machinery. The newly synthesized DNA templates lack epigenetic modifications because lytic DNA amplification is uncoupled from cellular DNA replication, eliminating the epigenetic maintenance mechanisms during the synthesis of viral progeny. As a consequence, default-on promoters and silenced poised-on promoters, which rely on unmethylated, epigenetically naïve templates, become also activated in the onset of the lytic phase. Silenced poised-on promoters require additionally a viral cofactor for their activation. This so-far unknown factor is probably provided upon lytic DNA amplification allowing the transcription of genes encoding for structural proteins that are necessary for the packaging of viral progeny. The released EBV progeny is epigenetically unmodified and ready to infect other cells. In essence, the regulation of EBV’s life cycle by epigenetic mechanisms is a paradigm for viral coevolution with its host. Repressive epigenetic modifications are common cellular defense mechanism to fight invading pathogens. EBV has hijacked this system for the regulation of promoter usage during its own life cycle, which has become a key principle of EBV’s success in infecting and persisting in its host.

EBV ist ätiologisch eng mit verschiedenen malignen Erkrankungen des Menschen verbunden Die meisten Erkenntnisse über die Funktionen viraler Proteine, die z. B. bei der B-Zell-Trans-formation eine Rolle spielen, stammen aus Zellkulturexperimenten, denen allerdings die Komponenten und die Komplexität eines lebenden Organismus fehlen, weswegen ein Tiermodell wünschenswert ist. Eine Möglichkeit nähere Informationen über die Machbarkeit eines Tiermodells zu bekommen, führt über die genetische Manipulation von embryonalen Stammzellen (ES-Zellen) der Maus. Dazu wurde die genetische Information des Epstein-Barr Virus direkt in EBNA1-positive ES-Zellen der Maus einbracht und die Aufrechterhaltung des Gesamtgenoms als extrachromosomales Plasmid nachgewiesen werden. Die ES-Zellen wurden dann in vitro zu B-Zellen differenziert, um den transformierenden Phänotyp dieses Virus in murinen B-Zellen zu analysieren. Sowohl in den ES-Zellen als auch in den in vitro differenzierten B-Zellen wurde eine Expression der Gene LMP1 und LMP2A gefunden, nicht aber eine Expression des Gens EBNA2. Dieses Expressionsmuster ist charakteristisch für die Latenz II des Virus. Die viralen EBNA-Promotoren waren in beiden Zellarten aktiv, aber eine genaue Analyse ergab Hinweise auf Probleme bei der Transkription bzw. bei der mRNA-Prozessierung. Dies ist vermutlich der Grund für das Fehlen einer EBNA2-Genexpression.

Das Epstein-Barr Virus (EBV) infiziert primäre humane B-Zellen und kann deren unbegrenzte Proliferation induzieren. Dieser Prozess der Wachstumstransformation von B–Zellen ist ein Modellsystem, das die pathogenen Mechanismen bei der Tumorentstehung widerspiegelt. Das Epstein-Barr Virus nukleäre Antigen 1 (EBNA1) wurde als essentiell für den Prozess der Wachstumstransformation primärer humaner B-Lymphozyten beschrieben, weil es an der latenten Replikation über den viralen Replikations-Ursprung oriP, der extrachromosomalen Erhaltung des Virus-Episoms und der transkriptionellen Trans-aktivierung der latenten Gene beteiligt ist (Rickinson und Kieff, 2001). Dieses Postulat wurde nie experimentell untersucht, da die genetische Analyse mit den bisherigen Methoden nicht möglich war. Das Maxi-EBV-System macht das Genom von EBV einer genetischen Manipulation zugänglich und erlaubt auch die Herstellung von Viren, denen essentielle Gene fehlen (Delecluse et al., 1998). Ein Ziel meiner Doktorarbeit war die Herstellung und Analyse eines EBNA1-negativen Virus. Entgegen der Lehrmeinung war es mit EBNA1-negativem Maxi-EBV möglich, wachstums-transformierte Zellklone nach Infektion von primären humanen B-Lymphozyten zu etablieren. Das virale Genom war in sämtlichen erhaltenen lymphoblastoiden Zelllinien so integriert, dass alle untersuchten latenten EBV-Proteine exprimiert wurden. Meine Ergebnisse zeigen eindeutig, dass EBNA1 prinzipiell für die Wachstumstransformation entbehrlich ist. Mit EBNA1-positiven Viren werden die primären B-Zellen jedoch mindestens um den Faktor 10.000 besser wachstumstransformiert. Da EBNA1 den episomalen Status des Virusgenoms vermittelt, scheint die Etablierung des EBV-Genoms in infizierten Zellen der limitierende Schritt zu sein. Auch in vivo im SCID-Maus-Modell erwies sich EBNA1 als entbehrlich für die Tumorbildung, womit es nicht als essentielles Onkogen von EBV betrachtet werden kann. Ein weiterer im Rahmen dieser Doktorarbeit untersuchter Aspekt war die Frage, ob EBNA1 für die extrachromosomale Erhaltung und Replikation des EBV-Episoms durch heterologe Genprodukte ersetzt werden kann. Zu diesem Zweck wurden Fusionsproteine aus der DNA-Bindedomäne von EBNA1 mit den zellulären Proteinen Histon H1 bzw. HMG-I (Mitglied der hoch mobilen Protein-Gruppe) hergestellt. Ich konnte zeigen, dass HMG-I:EBNA1- und H1:EBNA1-Fusionsproteine in der Lage sind, kleine oriP-enthaltende Plasmide und Maxi-EBVs episomal zu erhalten und die zelluläre Replikations-Maschinerie zu rekrutieren. Zusätzlich dazu unterstützen die Fusionsproteine im EBNA1-negativen Maxi-EBV die Produktion infektiöser Viren. Für ein konditional regulierbares Vektorsystem wurden Fusionsproteine aus der EBNA1-Transaktivierungsdomäne und der DNA-Bindedomäne des Tet-Repressors (TetR) hergestellt. Diese Proteine sollten mit Tet-Operator-Sequenzen (TetO, TetR-Bindemotiv) interagieren, die multimerisiert auf oriP-basierte Vektoren kloniert wurden. Dadurch sollte die Erhaltung der oriP-basierten Vektoren in der Zelle konditional regulierbar gestaltet werden. Es gelang in dieser Doktorarbeit zum ersten Mal ein System zu etablieren, mit dem Plasmide episomal erhalten werden und bei Zugabe von Doxyzyklin konditional regulierbar verloren gehen. Dieses erstmals realisierte konditional regulierbare Vektorsystem schafft neue Wege, die virale und zelluläre Replikation genauer zu untersuchen. Außerdem öffnen sich Möglichkeiten für eine sicherere Gentherapie, da die viralen Anteile auf ein Minimum reduziert werden können. Mit einem solchen System könnten EBV-Genvektoren in B-Zellen eingeführt werden und nach Expression des auf dem Vektor kodierten, therapeutischen Gens könnte die Genfähre durch Tetrazyklin-Applikation wieder aus dem Patienten entfernt werden.

EBV is a γ-herpes virus which is able to infect human resting B-cells and to transform them into permanently growing lymphoblastoid cell lines (LCLs). EBNA2 (Epstein-Barr virus nuclear antigen 2) is one of the first viral proteins expressed after in vitro infection and interacts with different cellular proteins like RBP-Jκ and PU.1. The EBNA2 protein acts as a transcriptional activator of the viral Latent Membrane Proteins 1 and 2 (LMP1 and LMP2) and the viral nuclear genes EBNA1, EBNA3A, -3B, -3C, EBNA-LP. Additionally EBNA2 is also able to transactivate cellular genes like CD21, CD23 or c-myc. To study the different EBNA2 target genes and the function of EBNA2 a LCL was established (ER/EB2-5 cells, Kempkes et al., 1995) harboring an estrogen-inducible EBNA2. In the presence of estrogen the ER/EBNA2 fusion protein (estrogen receptor binding domain) is located in the nucleus were EBNA2 can transactivate its target genes, whereas in the absence of estrogen the ER/EBNA2 fusion protein is kept in the cytoplasm and therefore inactive. The cells proliferate in the presence of estrogen and they arrest in the absence resulting in a phenotype similar to resting B-lymphocytes. By using the ER/EB2-5 cell line I could clearly show that the cell surface molecule CD83, belonging to the immunoglobuline superfamily (Zhou et al., 1992), is upregulated after the activation of EBNA2. By using a derivative ER/EB2-5 cell line that constitutively expressed LMP1 I could show that CD83 is still expressed even in the absence of functional EBNA2 suggesting that LMP1, the viral target gene of EBNA2, is responsible for the induction of CD83. Therefore I analysed the activation of the CD83 promoter by LMP1. LMP1 is a transmembrane protein with a short intracellular N-terminus, 6 hydrophobic transmembrane domains and a long intracellular C-terminus, containing C-terminal activator regions CTAR1, 2 and 3. The different CTAR regions are responsible for activating genes via NF-κB, ATF, AP1 and STAT signaling pathways. For the activation of its target genes LMP1 uses the same signaling molecules (TRAF, TRADD) as family members of the TNF-R family (CD40, TNF-R1, TNF-R2). The CD83 promoter was activated by LMP1 as shown by promoter luciferase reporter assays in 293-T cells. The induction was not observed in the absence of a NF-κB binding site in a CD83 promoter mutant. Furthermore LMP1 mutants which are mutated in the binding regions for TRAF2 (CTAR1) or TRADD (CTAR2) are not able to transactivate the CD83 promoter. By co-transfection of LMP1 and dominant/negative IκB the CD83 promoter could not be activated because of inactivation of NF-κB. These experiments clearly demonstrate that the CD83 promoter is transactivated by LMP1 via NF-κB. Additionally to the regulation of CD83 I was also interested in the functional role of CD83. Until now only little is known about the function of CD83. CD83 seems to have a specific role in the decision to single positive CD4+ T-cells in the thymus (Fujimoto et al., 2002). I have tested a possible co-stimulatory function of CD83 to CD4+ T-cells by retroviral expression of CD83 in non-professional antigen presenting cells (RCC). Indeed CD83 expression increased the CD4+ response in comparison to CD80 or GFP retroviral infected RCC cells. In mixed lymphocyte reactions this co-stimulatory effect could not be clearly demonstrated although a soluble CD83-Ig showed a small inhibitory influence. The identification of a CD83 ligand molecule could give new insights into the function of CD83. Therefore a CD83-Ig fusion protein as well as a CD83-tetramer construct were generated and used to screen for a potential ligand of CD83. First results showed that the CD83-Ig fusion protein and the CD83-tetramer construct bound to CD4+ and to CD8+ T-cells of isolated PBMCs as well as to activated T-cells in a culture of mixed T-cell populations.