Die Welt – ein (virtuelles?) Lebensdorf

Im letzten Schuljahr begann für die Schüler/innen der Klasse 2E des Gymnasiums Francesco Petrarca in Triest in ihrem Deutschunterricht ein neues Abenteuer: Sie bekamen die Möglichkeit, ein Theaterstück in der Fremdsprache zu entwickeln. Aus ihren eigenen Ideen beim Szenischen Improvisieren, angeleitet durch ihre Lehrerin und eine Theaterpädagogin aus Deutschland, entstanden einzelne Szenen und dann die Geschichte für ein ganzes Stück. Zum Proben, Organisieren von Auftritten und Theaterreisen agierten sie immer mutiger in der Fremdsprache und benutzten dafür die ihnen eigenen modernen Kommunikationsformen

Reference Genes for Expression Studies in Human CD8 + Naïve and Effector Memory T Cells under Resting and Activating Conditions

Abstract: Reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR) is widely used for mRNA quantification. To accurately measure changing gene transcript levels under different experimental conditions, the use of appropriate reference gene transcripts is instrumental. In T cell immunology, suitable reference genes have been reported for bulk CD4+ and CD8+ T cells. However, many CD4+ and CD8+ T cell subsets have been described in the past. Although they respond differently to given activation stimuli, proper validation of suitable reference genes in these subsets is lacking. In this study, we evaluated twelve commonly used reference gene products in human naïve (NV) and effector memory (EM) CD8+ T cells under non-activated and activated (2 h, 10 h and 20 h) conditions. We used five different statistical approaches for data analysis. Our results show that a number of widely used reference transcripts become differentially expressed under activating conditions. Using them as references markedly alters results as exemplified with IFNG mRNA expression. The only candidate reference gene products that remained stable during the activation process were 18S rRNA and SDHA mRNA, encouraging their usage as reference gene products for RT-qPCR experiments, when quantifying mRNA levels in human NV and EM CD8+ T cells

Memory CD8+ T Cells Balance Pro- and Anti-inflammatory Activity by Reprogramming Cellular Acetate Handling at Sites of Infection.

Serum acetate increases upon systemic infection. Acutely, assimilation of acetate expands the capacity of memory CD8+ T cells to produce IFN-γ. Whether acetate modulates memory CD8+ T cell metabolism and function during pathogen re-encounter remains unexplored. Here we show that at sites of infection, high acetate concentrations are being reached, yet memory CD8+ T cells shut down the acetate assimilating enzymes ACSS1 and ACSS2. Acetate, being thus largely excluded from incorporation into cellular metabolic pathways, now had different effects, namely (1) directly activating glutaminase, thereby augmenting glutaminolysis, cellular respiration, and survival, and (2) suppressing TCR-triggered calcium flux, and consequently cell activation and effector cell function. In vivo, high acetate abundance at sites of infection improved pathogen clearance while reducing immunopathology. This indicates that, during different stages of the immune response, the same metabolite-acetate-induces distinct immunometabolic programs within the same cell type

SDHA gain-of-function engages inflammatory mitochondrial retrograde signaling via KEAP1-Nrf2.

Whether screening the metabolic activity of immune cells facilitates discovery of molecular pathology remains unknown. Here we prospectively screened the extracellular acidification rate as a measure of glycolysis and the oxygen consumption rate as a measure of mitochondrial respiration in B cells from patients with primary antibody deficiency. The highest oxygen consumption rate values were detected in three study participants with persistent polyclonal B cell lymphocytosis (PPBL). Exome sequencing identified germline mutations in SDHA, which encodes succinate dehydrogenase subunit A, in all three patients with PPBL. SDHA gain-of-function led to an accumulation of fumarate in PPBL B cells, which engaged the KEAP1-Nrf2 system to drive the transcription of genes encoding inflammatory cytokines. In a single patient trial, blocking the activity of the cytokine interleukin-6 in vivo prevented systemic inflammation and ameliorated clinical disease. Overall, our study has identified pathological mitochondrial retrograde signaling as a disease modifier in primary antibody deficiency

BKV Agnoprotein Interacts with α-Soluble N-Ethylmaleimide-Sensitive Fusion Attachment Protein, and Negatively Influences Transport of VSVG-EGFP

Background: The human polyomavirus BK (BKV) infects humans worldwide and establishes a persistent infection in the kidney. The BK virus genome encodes three regulatory proteins, large and small tumor-antigen and the agnoprotein, as well as the capsid proteins VP1 to VP3. Agnoprotein is conserved among BKV, JC virus (JCV) and SV40, and agnoprotein-deficient mutants reveal reduced viral propagation. Studies with JCV and SV40 indicate that their agnoproteins may be involved in transcription, replication and/or nuclear and cellular release of the virus. However, the exact function(s) of agnoprotein of BK virus remains elusive. Principal Findings: As a strategy of exploring the functions of BKV agnoprotein, we decided to look for cellular interaction partners for the viral protein. Several partners were identified by yeast two-hybrid assay, among them a-SNAP which is involved in disassembly of vesicles during secretion. BKV agnoprotein and a-SNAP were found to partially co-localize in cells, and a complex consisting of agnoprotein and a-SNAP could be co-immunoprecipitated from cells ectopically expressing the proteins as well as from BKV-transfected cells. The N-terminal part of the agnoprotein was sufficient for the interaction with a-SNAP. Finally, we could show that BKV agnoprotein negatively interferes with secretion of VSVG-EGFP reporter suggesting that agnoprotein may modulate exocytosis. Conclusions: We have identified the first cellular interaction partner for BKV agnoprotein. The most N-terminal part of BKV agnoprotein is involved in the interaction with a-SNAP. Presence of BKV agnoprotein negatively interferes with secretion of VSVG-EGFP reporter

Processing of Genome 5′ Termini as a Strategy of Negative-Strand RNA Viruses to Avoid RIG-I-Dependent Interferon Induction

Innate immunity is critically dependent on the rapid production of interferon in response to intruding viruses. The intracellular pathogen recognition receptors RIG-I and MDA5 are essential for interferon induction by viral RNAs containing 5′ triphosphates or double-stranded structures, respectively. Viruses with a negative-stranded RNA genome are an important group of pathogens causing emerging and re-emerging diseases. We investigated the ability of genomic RNAs from substantial representatives of this virus group to induce interferon via RIG-I or MDA5. RNAs isolated from particles of Ebola virus, Nipah virus, Lassa virus, and Rift Valley fever virus strongly activated the interferon-beta promoter. Knockdown experiments demonstrated that interferon induction depended on RIG-I, but not MDA5, and phosphatase treatment revealed a requirement for the RNA 5′ triphosphate group. In contrast, genomic RNAs of Hantaan virus, Crimean-Congo hemorrhagic fever virus and Borna disease virus did not trigger interferon induction. Sensitivity of these RNAs to a 5′ monophosphate-specific exonuclease indicates that the RIG-I-activating 5′ triphosphate group was removed post-transcriptionally by a viral function. Consequently, RIG-I is unable to bind the RNAs of Hantaan virus, Crimean-Congo hemorrhagic fever virus and Borna disease virus. These results establish RIG-I as a major intracellular recognition receptor for the genome of most negative-strand RNA viruses and define the cleavage of triphosphates at the RNA 5′ end as a strategy of viruses to evade the innate immune response

Characterization of the viral gene products p10 and P of the Borna disease virus

Das Borna Disease Virus (BDV, Bornavirus) besitzt ein einzelsträngiges RNA-Genom negativer Polarität und ist innerhalb der Ordnung Mononegavirales der Prototyp einer eigenen Virusfamilie, die der Bornaviridae. Eine außergewöhnliche Eigenschaft des Virus ist seine nukleäre Transkription und Replikation, eine weitere besteht in seiner Fähigkeit, als neurotropes Virus sowohl in vivo als auch in vitro persistente Infektionen zu etablieren. Die zugrunde liegenden Mechanismen sowohl der Replikation als auch der Persistenz sind derzeit noch unzureichend verstanden, auch deshalb, weil das Virus noch relativ „jung“ ist: Erste komplette Sequenzen des RNA-Genoms wurden 1994 publiziert und erst vor einigen Monaten gelang die Generierung rekombinanter Viren auf der Basis klonierter cDNA. Im Mittelpunkt dieser Arbeit standen das p10 Protein und das Phosphoprotein (P), die von der gemeinsamen Transkriptionseinheit II in überlappenden Leserahmen kodiert werden. Als im Kern der Wirtszelle replizierendes Virus ist das Bornavirus auf zelluläre Importmechanismen angewiesen, um den Kernimport aller an der Replikation beteiligten viralen Proteine zu gewährleisten. Das p10 Protein ist ein negativer Regulator der viralen RNA-abhängigen RNA-Polymerase (L). In vitro Importexperimente zeigten, dass p10 über den klassischen Importin alpha/beta abhängigen Kernimportweg in den Nukleus transportiert wird. Dies war unerwartet, da p10 kein vorhersagbares klassisches Kernlokalisierungssignal (NLS) besitzt und weist darauf hin, dass der zelluläre Importapparat offensichtlich flexibler ist als allgemein angenommen. Die ersten 20 N-terminalen AS vermitteln sowohl Kernimport als auch die Bindung an den Importrezeptor Importin alpha. Durch Di-Alanin-Austauschmutagenese wurden die für diesen Transportprozess essentiellen AS identifiziert und die Bedeutung hydrophober und polarer AS-Reste demonstriert. Die Fähigkeit des Bornavirus, persistente Infektionen zu etablieren, wirft die Frage auf, wie das Virus die zellulären antiviralen Abwehrmechanismen, insbesondere das Typ I Interferon (IFN)-System, unterwandert. Das virale P Protein wurde in dieser Arbeit als potenter Antagonist der IFN-Induktion charakterisiert. Es verhindert die Phosphorylierung des zentralen Transkriptionsfaktors IRF3 durch die zelluläre Kinase TBK1 und somit dessen Aktivierung. Der Befund, dass P mit TBK1 Komplexe bildet und zudem auch als Substrat für die zelluläre Kinase fungiert, erlaubt es, erstmalig einen Mechanismus zu postulieren, in dem ein virales Protein (BDV-P) als putatives TBK1-Pseudosubstrat die IRF3-Aktivierung kompetitiv hemmt.The Borna Disease Virus (BDV) harbors a single stranded RNA genome of negative polarity. Within the order of Mononegavirales it is the prototype of a new virus family named Bornaviridae. Unique features of this neurotrope virus are its nuclear transcription and replication as well as its ability to establish persistent infections both in vivo and in vitro. The underlying mechanisms of BDV replication and persistence are currently not well understood amongst others due to the fact that BDV is quite a young virus: First complete sequences of the RNA genome have been published in 1994. Only a few months ago the generation of a recombinant Bornavirus from cloned cDNA has been accomplished. The work presented here focused on the viral p10 protein and the phosphoprotein P that are both encoded by two overlapping reading frames of the transcription unit II. Nuclear replication of the Bornavirus relies on cellular import mechanisms to allow for nuclear import of viral proteins involved in viral replication. The p10 protein has been described as a negative regulator of the viral RNA dependent RNA polymerase (L). In vitro import experiments revealed that p10 translocates into the nucleus via the classical importin alpha/beta; dependent pathway. This was unexpected since p10 does not contain a predictable classical nuclear localization signal (NLS) suggesting that the cellular import machinery is more flexible than generally believed. The first 20 amino acids mediate nuclear import and binding to the import receptor importin alpha. Analysis of di-alanine-exchange mutants identified essential amino acids and furthermore revealed the impact of hydrophobic and polar side chains in receptor binding and nuclear import. The ability of the Bornavirus to establish persistent infections rises the question of how the virus circumvents cellular antiviral defense mechanisms, in particular the type I interferon system. This work characterizes the viral P protein as a potent antagonist of IFN beta induction. It prevents the activation of the central transcription factor IRF3 by interfering with the cellular kinase TBK1. The finding that P forms complexes with TBK1 and moreover serves as a kinase substrate allows to postulate a mechanism for the first time, in which a viral protein (BDV-P) acts as a putative TBK1 pseudo-substrate and thereby competitively inhibits IRF3 activation