Modulation des 7SK small nuclear Ribonukleoproteinkomplexes durch das Typ-IV-Effektorprotein AnkG von Coxiella burnetii

The obligate intracellular, Gram-negative bacterium Coxiella burnetii is the causative agent of Q fever. Humans are usually infected by inhalation of contaminated aerosols of animal products. Interestingly, 1-10 bacteria can be sufficient to establish an infection. Q fever occurs in an acute and a chronic form. The chronic form can lead to a lethal endocarditis. Treatment of the chronic form needs doxycycline and chloroquine for up to 18 months. This long-term treatment and the occurrence of the post Q-fever fatigue syndrome, which cannot be treated efficiently, show the urgency of further investigating these bacteria to develop new possibilities of treatment. For successful infection, intracellular survival, and replication C. burnetii utilizes more than 150 effector proteins, which are translocated from bacterial cytoplasm to host cell cytoplasm by a type-4-secretion system. The function of the majority of these effectors is unknown. However, for the effector proteins CaeA, CaeB, and AnkG an anti-apoptotic activity has been described. Amongst them AnkG is the best characterized. Preceding to this work it has been known that the amino acids 1-69 are necessary and sufficient for the anti-apoptotic activity. Furthermore, a nuclear localization for AnkG is essential for its anti-apoptotic effect. This localization is reached by interaction with the host cell proteins p32 and Importin-α1. However, the underlying molecular mechanism was unknown and should be analyzed in the scope of this study. It could be shown that the amino acids 1-28 are necessary and sufficient for the anti- apoptotic activity of AnkG. Besides, the binding of AnkG to the nuclear host cell proteins DDX21, MTA2, CDC5L, hnRNPM, and TSR1 could be confirmed. These proteins are involved in RNA metabolism (e.g. splicing) and regulatory functions in cell survival. Localization analysis by ectopic co-expression of AnkG and the binding partner showed co- localization of AnkG with DDX21, MTA2, and CDC5L within the nucleus. Furthermore, an influence on the localization of MTA2 and CDC5L by AnkG could be shown. In both cases the localization of host cell proteins were altered from a homogenous distribution in the nucleus to a concentrated localization in substructures in the nucleus. For MTA2 and CDC5L it has been shown that localization can be critical for molecular function. CDC5L in turn influences the localization of AnkG by replacing it in substructures in the nucleus. Additionally, AnkG influences the localization of DDX21 after apoptosis induction. AnkG inhibits the migration of DDX21 from the nucleoli to the nucleoplasm leading to an anti- apoptotic phenotype. The interaction between DDX21 and AnkG has been characterized. It could been shown that DDX21 binds with the amino acids 1-28 of AnkG, the anti-apoptotic part. The amino acid 27is critical for this interaction. After mutating this arginine to serine the interaction is disrupted, what could indicate in interaction by salt bridges. Hitherto, it has been known that DDX21 interacts with the 7SK small nuclear ribonucleoprotein complex (7SK snRNP). Therefore, interaction between AnkG and the 7SK snRNP has been analyzed. It could be shown that AnkG also interacts with LARP7, a scaffolding protein of the 7SK snRNP. Additionally, AnkG interacts with the 7SK RNA. This RNA is responsible for the release of the positive transcription elongation factor b (P-TEFb), which can lead to anti-apoptotic transcription activity. Further analysis showed that ectopic expression of AnkG facilitates the release of CDK9, the catalytic subunit of P-TEFb, from the 7SK snRNP. Hence, it was supposed that AnkG might influence host cell transcription. RNA sequencing showed that expression of AnkG leads to upregulation of anti-apoptotic genes while pro-apoptotic genes are downregulated. Furthermore, the data showed an influence of AnkG on genes involved in the cytoskeleton and trafficking as well as encoding transcription factors or are involved in signaling. It could be shown that the mutant AnkG1-28 R27S, which does not bind DDX21, also lose the capability of binding to LARP7 and the 7SK RNA. Since this mutant is not anti-apoptotic anymore, it is obvious that interaction of AnkG with DDX21 and the 7SK snRNP is essential for its anti-apoptotic activity. Using CDK9 inhibitors showed also that depletion of CDK9 leads to a loss of the anti-apoptotic effect of AnkG. In summary, the data showed an interaction of AnkG with DDX21 and the 7SK snRNP and facilitates the release of P-TEFb and CDK9, respectively. This mechanism is critical for the anti-apoptotic activity of AnkG since the depletion of CDK9 leads to a loss of the anti- apoptotic effect. Furthermore, the role of AnkG during infection was analyzed to evaluate a possible importance of the anti-apoptotic activity for the infection. Therefore, an AnkG deletion mutant (∆ankG) was used. In the scope of that it could been shown that ∆ankG has similar infection rates compared to the wild type. However, ∆ankG shows a defect in establishing and maturating of the Coxiella-containing vacuole (CCV). The CCVs of ∆ankG are smaller and there are more vacuoles per cell in comparison to the wild type. The intracellular replication of ∆ankG is impaired as well. However, this defect was independent of the anti-apoptotic activity of AnkG since infection of cells with inhibited intrinsic apoptosis pathways does not rescue the wild type phenotype. In summary, these data show a critical role of AnkG for intracellular replication and CCV maturation. Besides, it could be shown that the host cell protein MTA2 binds the anti-apoptotic part of AnkG. MTA2 is known to facilitate the deacetylation of the transcription factor p53 leading to an inhibition of pro-apoptotic activity. The lysine 120 of p53 is critical for its activation by acetylation. Preliminary mass spectrometry analysis showed an effect of AnkG on p53 acetylation. Expression of AnkG might lead to a deacetylation of lysine 120 of p53 and therefore to inactivation. Furthermore, interaction between AnkG and RNAs, especially non-coding RNAs could be confirmed. However, the consequences of these interactions are unknown and have to be further investigated. In summary, it could been shown that the anti-apoptotic mechanism of AnkG functions via interaction with DDX21 and the 7SK snRNP. Additionally, AnkG is involved in intracellular replication and the maturation of the CCV.Das obligat intrazelluläre, Gram-negative Bakterium Coxiella burnetii ist der Erreger des Q- Fiebers. Eine humane C. burnetii Infektion tritt üblicherweise durch Inhalation von kontaminierten Aerosolen tierischer Produkte auf. Hierbei können 1-10 Bakterien ausreichen, um eine Infektion zu etablieren. Für Q-Fieber gibt es eine akute und eine chronische Form, wobei letztere zu einer letalen Endokarditis führen kann. Die Behandlung der chronischen Form erfordert die Gabe von Doxycyclin und Chloroquin über einen Zeitraum von 18 Monaten. Diese lange Therapiedauer sowie das Auftreten eines Post Q-Fieber fatigue Syndroms, für das es keine gezielte Therapie gibt, machen die weitere Erforschung des Bakteriums notwendig, um verbesserte Therapien zu entwickeln. Für die Etablierung der Infektion und die intrazelluläre Replikation ist das Typ-4- Sekretionssystem essentiell, durch das C. burnetii mehr als 150 Effektorproteine vom bakteriellen Zytoplasma in das Wirtszellzytoplasma transloziert. Von den meisten dieser Effektorproteine ist die Funktion unbekannt. Doch für einige wurde eine Funktion beschrieben. So konnte festgestellt werden, dass die Effektorproteine CaeA, CaeB und AnkG anti-apoptotisch auf die Wirtszelle wirken und somit dazu beitragen das intrazelluläre Überleben der Bakterien zu fördern. Hierbei ist AnkG das am besten charakterisierte anti- apoptotische Effektorprotein. Vor Anfertigung dieser Arbeit war bekannt, dass die Aminosäuren 1-69 von AnkG ausreichend und notwendig für die anti-apoptotische Aktivität sind. Zudem ist eine nukleäre Lokalisation von AnkG kritisch für die anti-apoptotische Wirkung. Diese Lokalisation wird durch die Interaktion mit den Wirtszellproteinen p32 und Importin-α1 erreicht. Der molekulare Mechanismus der anti-apoptotischen Aktivität von AnkG war unbekannt und sollte im Rahmen dieser Arbeit analysiert werden. So konnte festgestellt werden, dass die Aminosäuren 1-28 notwendig und ausreichend für die anti-apoptotische Aktivität von AnkG sind. Zudem wurden die Bindung der nukleären Wirtszellproteine DDX21, MTA2, CDC5L, hnRNPM und TSR1 an AnkG verifiziert. Diese Proteine sind alle in den RNA-Metabolismus (z.B. Splicing) oder die Regulation der Zellvitalität involviert. Lokalisationsanalysen bei ektopischer Ko-Expression der Bindepartner mit AnkG ergaben eine Ko-Lokalisation von AnkG mit DDX21, MTA2 und CDC5L im Nukleus. Zudem konnte gezeigt werden, dass AnkG die Lokalisation von MTA2 und CDC5L beeinflusst. In beiden Fällen liegen die Proteine ohne Expression von AnkG homogen im Nukleus verteilt vor und werden bei Ko-Expression mit AnkG in Substrukturen im Nukleus konzentriert. CDC5L hat zudem einen Einfluss auf die Lokalisation von AnkG und verdrängt dieses aus Substrukturen im Nukleus. Sowohl für MTA2 als auch CDC5L wurde beschrieben, dass die Lokalisation wichtig für ihre regulatorischen Funktionen auf die Zellvitalität ist. Die Lokalisation von DDX21 wird ebenfalls durch AnkG beeinflusst, allerdings nur nach Induktion der intrinsischen Apopotose. Hier inhibiert AnkG die Migration von DDX21 aus den Nukleoli in das Nukleoplasma und inhibiert somit die Apoptose. Zunächst wurde die Interaktion von DDX21 und AnkG weiter charakterisiert. Es konnte festgestellt werden, dass DDX21 mit den Aminosäuren 1-28 von AnkG, dem anti-apoptotischen Bereich, interagiert. Hierbei ist das Arginin an Position 27 kritisch. Wird dieses durch ein Serin ersetzt, findet keine Bindung zwischen AnkG und DDX21 mehr statt, was darauf hindeuten könnte, dass die Bindung über eine Salzbrücke erfolgt. Da für DDX21 bekannt ist, dass es mit dem 7SK small nuclear Ribonukleoproteinkomplex (7SK snRNP) interagiert, wurde eine Bindung von AnkG an diesen Komplex untersucht. Es konnte festgestellt werden, dass AnkG mit LARP7, einem Gerüstprotein des 7SK snRNP, interagiert. Zusätzlich wurde gezeigt, dass AnkG auch mit der 7SK RNA interagiert. Diese bildet das Rückgrat des Komplexes und ist für die Freisetzung des positiven Transkriptionselongationsfaktors b (P-TEFb) von dem Komplex verantwortlich. Dies kann zu anti-apoptotischer Transkriptionsaktivität führen. Weitere Analysen zeigten, dass bei ektopischer Expression von AnkG eine vermehrte Freisetzung von CDK9, der katalytischen Untereinheit von P-TEFb, stattfindet. Daraus folgte die Hypothese, dass AnkG hierüber Einfluss auf die Transkription der Wirtszelle nehmen könnte. Die Analyse mittels RNA-Sequenzierung ergab, dass bei Expression von AnkG im Vergleich zu einer Kontrolle, vermehrt anti-apoptotische Gene hochreguliert und pro- apoptotische herunterreguliert werden. Zudem zeigten die Daten der RNA-Sequenzierung, dass auch Gene differentiell reguliert werden, die in das Zytoskelett und das Trafficking involviert sind oder für Transkriptionsfaktoren kodieren sowie in das Signaling involviert sind. Mittels Analysen mit der AnkG Mutante AnkG1-28 R27S, die auch nicht mehr mit der 7SK RNA und LARP7 interagiert, konnte festgestellt werden, dass diese Interaktionen kritisch für die anti-apoptotische Aktivität von AnkG sind, da AnkG1-28 R27S nicht mehr anti-apoptotisch wirkt. Zudem konnte mit Hilfe von CDK9-Inhibitoren gezeigt werden, dass auch eine Depletion von CDK9 einen Verlust der anti-apoptotischen Wirkung von AnkG zur Folge hat. Insgesamt konnte also gezeigt werden, dass AnkG mit DDX21 und dem 7SK snRNP interagiert und dabei die Freisetzung des Transkriptionsfaktors P-TEFb beziehungsweise CDK9 fördert. Dieser Mechanismus ist kritisch für die anti-apoptotische Aktivität von AnkG, da eine Depletion von CDK9 zu einem Verlust der anti-apoptotischen Wirkung führt. Es wurde auch die Rolle von AnkG während der Infektion untersucht, um festzustellen, ob die anti-apoptotische Wirkung auch hierfür notwendig ist. Mit Hilfe einer AnkG-Deletionsmutante (∆ankG) konnte festgestellt werden, dass ∆ankG und der Wildtyp gleich gut infizieren. Allerdings liegt offenbar ein Defekt in der Etablierung beziehungsweise Reifung der Coxiella-enthaltenden Vakuole (CCV) vor. Die CCVs von ∆ankG sind kleiner und es liegen mehr Vakuolen pro Zelle vor. Dies zeigt, dass eine Deletion von AnkG einen Defekt bei den Fusionsprozessen zur Bildung der reifen CCV zur Folge hat. Dies hat einen Einfluss auf die intrazelluläre Replikation, die bei ∆ankG im Vergleich zum Wildtyp deutlich reduziert ist. Dieser Defekt tritt unabhängig von der anti-apoptotischen Aktivität auf, da auch bei einer Infektion von Zellen mit inhibiertem intrinsischen Apoptoseweg mit ∆ankG den wildtypischen Phänotyp nicht wiederherstellt. Insgesamt konnte hier gezeigt werden, dass AnkG neben der anti-apoptotischen Aktivität auch eine Rolle bei der intrazellulären Replikation beziehungsweise der Reifung der CCV spielt. Weiterhin konnte im Rahmen dieser Arbeit gezeigt werden, dass auch das Wirtszellprotein MTA2 mit dem anti-apoptotischen Bereich von AnkG interagiert. Von MTA2 ist bekannt, dass es verantwortlich für die Deacetylierung und damit Inaktivierung des Transkriptionsfaktors p53 ist. Gerade die Deacetylierung des Lysins 120 von p53 verhindert eine pro-apoptotische Aktivität dieses Proteins. Vorläufige Massenspektrometrie-Analysen konnten zeigen, dass unter dem Einfluss von AnkG die Acetylierung von p53 an Lysin 120 hemmt. Auch eine Interaktion mit weiteren RNAs, insbesondere nicht kodierenden RNAs, konnte nachgewiesen werden. Die Konsequenzen dieser Interaktion müssen allerdings noch näher analysiert werden. Insgesamt konnte gezeigt werden, dass AnkG über die Interaktion mit DDX21 und dem 7SK snRNP anti-apoptotisch wirkt. Zudem spielt AnkG auch eine Rolle bei der Reifung der CCV und damit bei der intrazellulären Replikation

A Genetically Encoded Dark-to-Bright Biosensor for Visualisation of Granzyme-Mediated Cytotoxicity

Granzyme B (GZMB) is a key enzyme released by cytotoxic T lymphocytes (CTL) and natural killer (NK) cells to induce apoptosis in target cells. We designed a novel fluorogenic biosensor which is able to assess GZMB activity in a specific and sensitive manner. This cleavage-responsive sensor for T cell activity level (CRSTAL) is based on a fluorescent protein that is only activated upon cleavage by GZMB or caspase-8. CRSTAL was tested in stable cell lines and demonstrated a strong and long-lasting fluorescence signal upon induction with GZMB. It can detect GZMB activity not only by overexpression of GZMB in target cells but also following transfer of GZMB and perforin from effector cells during cytotoxicity. This feature has significant implications for cancer immunotherapy, particularly in monitoring the efficacy of chimeric antigen receptor (CAR)-T cells. CAR-T cells are a promising therapy option for various cancer types, but monitoring their activity in vivo is challenging. The development of biosensors like CRSTAL provides a valuable tool for monitoring of CAR-T cell activity. In summary, CRSTAL is a highly sensitive biosensor that can detect GZMB activity in target cells, providing a means for evaluating the cytotoxic activity of immune cells and monitoring T cell activity in real time.Deutsche Forschungsgemeinschaf

Cloning of a Passage-Free SARS-CoV-2 Genome and Mutagenesis Using Red Recombination

The ongoing pandemic coronavirus (CoV) disease 2019 (COVID-19) by severe acute respiratory syndrome CoV-2 (SARS-CoV-2) has already caused substantial morbidity, mortality, and economic devastation. Reverse genetic approaches to generate recombinant viruses are a powerful tool to characterize and understand newly emerging viruses. To contribute to the global efforts for countermeasures to control the spread of SARS-CoV-2, we developed a passage-free SARS-CoV-2 clone based on a bacterial artificial chromosome (BAC). Moreover, using a Lambda-based Red recombination, we successfully generated different reporter and marker viruses, which replicated similar to a clinical isolate in a cell culture. Moreover, we designed a full-length reporter virus encoding an additional artificial open reading frame with wild-type-like replication features. The virus-encoded reporters were successfully applied to ease antiviral testing in cell culture models. Furthermore, we designed a new marker virus encoding 3xFLAG-tagged nucleocapsid that allows the detection of incoming viral particles and, in combination with bio-orthogonal labeling for the visualization of viral RNA synthesis via click chemistry, the spatiotemporal tracking of viral replication on the single-cell level. In summary, by applying BAC-based Red recombination, we developed a powerful, reliable, and convenient platform that will facilitate studies answering numerous questions concerning the biology of SARS-CoV-2

Identification of Rosellinia species as producers of cyclodepsipeptide PF1022 A and resurrection of the genus Dematophora as inferred from polythetic taxonomy

Rosellinia (Xylariaceae) is a large, cosmopolitan genus comprising over 130 species that have been defined based mainly on the morphology of their sexual morphs. The genus comprises both lignicolous and saprotrophic species that are frequently isolated as endophytes from healthy host plants, and important plant pathogens. In order to evaluate the utility of molecular phylogeny and secondary metabolite profiling to achieve a better basis for their classification, a set of strains was selected for a multi-locus phylogeny inferred from a combination of the sequences of the internal transcribed spacer region (ITS), the large subunit (LSU) of the nuclear rDNA, beta-tubulin (TUB2) and the second largest subunit of the RNA polymerase II (RPB2). Concurrently, various strains were surveyed for production of secondary metabolites. Metabolite profiling relied on methods with high performance liquid chromatography with diode array and mass spectrometric detection (HPLC-DAD/MS) as well as preparative isolation of the major components after re-fermentation followed by structure elucidation using nuclear magnetic resonance (NMR) spectroscopy and high resolution mass spectrometry (HR-MS). Two new and nine known isopimarane diterpenoids were identified during our mycochemical studies of two selected Dematophora strains and the metabolites were tested for biological activity. In addition, the nematicidal cyclodepsipeptide PF1022 A was purified and identified from a culture of Rosellinia corticium, which is the first time that this endophyte-derived drug precursor has been identified unambiguously from an ascospore-derived isolate of a Rosellinia species. While the results of this first HPLC profiling were largely inconclusive regarding the utility of secondary metabolites as genus-specific chemotaxonomic markers, the phylogeny clearly showed that species featuring a dematophora-like asexual morph were included in a well-defined clade, for which the genus Dematophora is resurrected. Dematophora now comprises all previously known important plant pathogens in the genus such as D. arcuata, D. bunodes, D. necatrix and D. pepo, while Rosellinia s. str. comprises those species that are known to have a geniculosporium-like or nodulisporium-like asexual morph, or where the asexual morph remains unknown. The extensive morphological studies of L.E. Petrini served as a basis to transfer several further species from Rosellinia to Dematophora, based on the morphology of their asexual morphs. However, most species of Rosellinia and allies still need to be recollected in fresh state, cultured, and studied for their morphology and their phylogenetic affinities before the infrageneric relationships can be clarified.Fil: Wittstein, Kathrin. Helmholtz-Zentrum für Infektionsforschung GmbH; Alemania. German Centre for Infection Research; AlemaniaFil: Cordsmeier, Arne. Helmholtz-Zentrum für Infektionsforschung GmbH; Alemania. Universitat Erlangen Nuremberg; AlemaniaFil: Lambert, Christopher. Helmholtz-Zentrum für Infektionsforschung GmbH; Alemania. German Centre for Infection Research; AlemaniaFil: Wendt, L.. Helmholtz-Zentrum für Infektionsforschung GmbH; Alemania. German Centre for Infection Research; AlemaniaFil: Sir, Esteban Benjamin. Universidad Nacional de Tucumán. Instituto de Bioprospección y Fisiología Vegetal. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet Noa Sur. Instituto de Bioprospección y Fisiología Vegetal; ArgentinaFil: Weber, J.. Helmholtz-Zentrum für Infektionsforschung GmbH; Alemania. German Centre for Infection Research; AlemaniaFil: Wurzler, N.. Helmholtz-Zentrum für Infektionsforschung GmbH; Alemania. German Centre for Infection Research; AlemaniaFil: Petrini, Liliane E.. Via al Perato 15c; SuizaFil: Stadler, Marc. Helmholtz-Zentrum für Infektionsforschung GmbH; Alemania. German Centre for Infection Research; Alemani

The Interactive Complex between Cytomegalovirus Kinase vCDK/pUL97 and Host Factors CDK7–Cyclin H Determines Individual Patterns of Transcription in Infected Cells

The infection of human cytomegalovirus (HCMV) is strongly determined by the host–cell interaction in a way that the efficiency of HCMV lytic replication is dependent on the regulatory interplay between viral and cellular proteins. In particular, the activities of protein kinases, such as cyclin-dependent kinases (CDKs) and the viral CDK ortholog (vCDK/pUL97), play an important role in both viral reproduction and virus–host interaction. Very recently, we reported on the complexes formed between vCDK/pUL97, human cyclin H, and CDK7. Major hallmarks of this interplay are the interaction between cyclin H and vCDK/pUL97, which is consistently detectable across various conditions and host cell types of infection, the decrease or increase in pUL97 kinase activity resulting from cyclin H knock-down or elevated levels, respectively, and significant trans-stimulation of human CDK7 activity by pUL97 in vitro. Due to the fact that even a ternary complex of vCDK/pUL97–cyclin H–CDK7 can be detected by coimmunoprecipitation and visualized by bioinformatic structural modeling, we postulated a putative impact of the respective kinase activities on the patterns of transcription in HCMV-infected cells. Here, we undertook a first vCDK/pUL97-specific transcriptomic analysis, which combined conditions of fully lytic HCMV replication with those under specific vCDK/pUL97 or CDK7 drug-mediated inhibition or transient cyclin H knockout. The novel results were further strengthened using bioinformatic modeling of the involved multi-protein complexes. Our data underline the importance of these kinase activities for the C-terminal domain (CTD) phosphorylation-driven activation of host RNA polymerase in HCMV-infected cells. The impact of the individual experimental conditions on differentially expressed gene profiles is described in detail and discussed.</jats:p

Characterization of SARS-CoV-2 Escape Mutants to a Pair of Neutralizing Antibodies Targeting the RBD and the NTD

Mutations in the spike protein of SARS-CoV-2 can lead to evasion from neutralizing antibodies and affect the efficacy of passive and active immunization strategies. Immunization of mice harboring an entire set of human immunoglobulin variable region gene segments allowed to identify nine neutralizing monoclonal antibodies, which either belong to a cluster of clonally related RBD or NTD binding antibodies. To better understand the genetic barrier to emergence of SARS-CoV-2 variants resistant to these antibodies, escape mutants were selected in cell culture to one antibody from each cluster and a combination of the two antibodies. Three independently derived escape mutants to the RBD antibody harbored mutations in the RBD at the position T478 or S477. These mutations impaired the binding of the RBD antibodies to the spike protein and conferred resistance in a pseudotype neutralization assay. Although the binding of the NTD cluster antibodies were not affected by the RBD mutations, the RBD mutations also reduced the neutralization efficacy of the NTD cluster antibodies. The mutations found in the escape variants to the NTD antibody conferred resistance to the NTD, but not to the RBD cluster antibodies. A variant resistant to both antibodies was more difficult to select and only emerged after longer passages and higher inoculation volumes. VOC carrying the same mutations as the ones identified in the escape variants were also resistant to neutralization. This study further underlines the rapid emergence of escape mutants to neutralizing monoclonal antibodies in cell culture and indicates the need for thorough investigation of escape mutations to select the most potent combination of monoclonal antibodies for clinical use

Spike residue 403 affects binding of coronavirus spikes to human ACE2

The bat sarbecovirus RaTG13 is a close relative of SARS-CoV-2, the cause of the COVID-19 pandemic. However, this bat virus was most likely unable to directly infect humans since its Spike (S) protein does not interact efficiently with the human ACE2 receptor. Here, we show that a single T403R mutation increases binding of RaTG13 S to human ACE2 and allows VSV pseudoparticle infection of human lung cells and intestinal organoids. Conversely, mutation of R403T in the SARS-CoV-2 S reduces pseudoparticle infection and viral replication. The T403R RaTG13 S is neutralized by sera from individuals vaccinated against COVID-19 indicating that vaccination might protect against future zoonoses. Our data suggest that a positively charged amino acid at position 403 in the S protein is critical for efficient utilization of human ACE2 by S proteins of bat coronaviruses. This finding could help to better predict the zoonotic potential of animal coronaviruses

Re-Analysis of the Widely Used Recombinant Murine Cytomegalovirus MCMV-m157luc Derived from the Bacmid pSM3fr Confirms Its Hybrid Nature

Murine cytomegalovirus (MCMV), and, in particular, recombinant virus derived from MCMV-bacmid pSM3fr, is widely used as the small animal infection model for human cytomegalovirus (HCMV). We sequenced the complete genomes of MCMV strains and recombinants for quality control. However, we noticed deviances from the deposited reference sequences of MCMV-bacmid pSM3fr. This prompted us to re-analyze pSM3fr and reannotate the reference sequence, as well as that for the commonly used MCMV-m157luc reporter virus. A correct reference sequence for this frequently used pSM3fr, containing a repaired version of m129 (MCK-2) and the luciferase gene instead of ORF m157, was constructed. The new reference also contains the original bacmid sequence, and it has a hybrid origin from MCMV strains Smith and K181

Analysis of SARS-CoV-2 Spike Protein Variants with Recombinant Reporter Viruses Created from a Bacmid System

SARS-CoV-2, the causative agent of COVID-19, has spread around the world with more than 700 million cases and 6.8 million deaths. Various variants of concern (VoC) have emerged due to mutations and recombination and concurrent selection for increased viral fitness and immune evasion. The viral protein that primarily determines the pathogenicity, infectivity, and transmissibility is the Spike protein. To analyze the specific impact of variant Spike proteins on infection dynamics, we constructed SARS-CoV-2 with a uniform B.1 backbone but with alternative Spike proteins. In addition, ORF6 was replaced by EYFP as a biological safety measure, and for use of this well-established reporter. We show that namely the delta variant Spike proteins cause a distinct phenotype from the wild type (B.1, D614G) and other variants of concern. Furthermore, we demonstrate that the omicron BA.1 Spike results in lower viral loads and a less efficient spread in vitro. Finally, we utilized viruses with the two different reporters EYFP and mCherry to establish a competitive growth assay, demonstrating that most but not all Spike variant viruses were able to outcompete wild type SARS-CoV-2 B.1

Evaluation of Bispecific T-Cell Engagers Targeting Murine Cytomegalovirus

Human cytomegalovirus is a ubiquitous herpesvirus that, while latent in most individuals, poses a great risk to immunocompromised patients. In contrast to directly acting traditional antiviral drugs, such as ganciclovir, we aim to emulate a physiological infection control using T cells. For this, we constructed several bispecific T-cell engager (BiTE) constructs targeting different viral glycoproteins of the murine cytomegalovirus and evaluated them in vitro for their efficacy. To isolate the target specific effect without viral immune evasion, we established stable reporter cell lines expressing the viral target glycoprotein B, and the glycoprotein complexes gN-gM and gH-gL, as well as nano-luciferase (nLuc). First, we evaluated binding capacities using flow cytometry and established killing assays, measuring nLuc-release upon cell lysis. All BiTE constructs proved to be functional mediators for T-cell recruitment and will allow a proof of concept for this treatment option. This might pave the way for strikingly safer immunosuppression in vulnerable patient groups