Leibniz Institute of Virology (LIV)

Understanding RNA synthesis and dynamics in cells requires efficient labeling strategies that are not only compatible with cellular environments but can be performed in living cells. We developed a robust, bio‐orthogonal approach for live‐cell RNA labeling using TriPPPro (triphosphate prodrug) chemistry. This strategy enables the intracellular delivery of sterically demanding nucleoside triphosphates modified with inverse electron‐demand Diels–Alder (IEDDA)‐reactive groups, specifically trans‐cyclooctene (2TCOa) and bicyclo[6.1.0]nonyne (BCN). Once hydrolyzed inside cells, these TriPPPro‐modified uridines and cytidines are metabolically incorporated into nascent RNA by endogenous RNA polymerases. Subsequent IEDDA reaction with a dual‐fluorogenic tetrazine‐cyanine styryl dye conjugate allows wash‐free, high‐contrast imaging of RNA synthesis in cells. We demonstrate efficient RNA labeling, including nucleolar localization and specificity for newly transcribed RNA, validated by transcriptional inhibition and colocalization with ribosomal RNA. Comparative analyses confirm that TriPPPro delivery surpasses conventional transporter‐based systems in both labeling efficiency and cellular compatibility. This platform offers a modular, non‐genetic, and highly specific method for real‐time RNA imaging, with broad applicability for RNA biology and antiviral research.

Human adenoviruses (HAdVs) are widespread pathogens with the capacity to manipulate host cellular pathways, including critical tumor suppressor networks. During oncogenic cell transformation, the adenoviral E1B-55K protein serves as a multifunctional viral regulator that, inter alia, modulates both p53-dependent and -independent pathways - though this function has been disputed in the context of viral infection. Here, we elucidate the dual role of E1B-55K in disrupting host defenses, focusing on its impact on p53 signaling and interferon-stimulated genes (ISGs) during infection. Using RNA-seq and follow-up experimental validation in A549 (p53 wildtype) and H1299 (p53-null) cells infected with wildtype HAdV-C5 or an E1B-55K-deficient mutant, we show that E1B-55K suppresses p53-mediated transcriptional responses. Concurrently, E1B-55K modulates ISG expression in a context-dependent manner. Our results reveal that E1B-55K leverages cellular context to optimize viral replication by targeting a host tumor suppressor and indicate interference with innate immune pathways. Our study thereby uncovers a previously underappreciated aspect of E1B-55K function during infection, offering insights into its repressive activity and solidifying its role as a multifunctional viral oncoprotein with broader implications for the HAdV replication cycle.

In an extensive proteomics analysis, we found that HMGB1, an important cellular chromatin protein, was enriched in adenovirus replication compartments. In this study, we aimed to better understand the role of HMGB1 in the infection process of a human DNA virus, HAdV-C5. We tested different virus types, including some with specific gene deletions and mutations. Our results showed that during infection, HMGB1 levels decreased because the virus suppressed its production. Despite this, even at lower levels, HMGB1 still helped the virus replicate by interacting with key viral proteins and DNA at sites where the virus is actively replicating. Overall, our findings highlight how HMGB1 plays a crucial role in facilitating efficient virus replication, making it an important factor in the infection process.

Abstract Influenza seasonality is influenced by environmental and ecological conditions as well as evolutionary processes shaping the transmission potential of airborne Influenza A virus (IAV) particles and subsequent infectious disease. Considering fluctuating environmental conditions with absolute humidity as a key driver, we analyzed environmental effects on the infection dynamic in Germany during the influenza seasons between 2010 and 2018. Despite the fact that Germany is a comparably homogenous country with regard to socio-economical, and environmental properties, influenza seasons differed significantly between individual German counties and cities, with a clear gradient in incidence and intensity between the Southeast and Northwest. Thus, the transmission of IAV influenced by slight differences in the predominant weather conditions throughout Germany, emphasizing the importance of air temperature and absolute humidity for airborne flu virus transmission.

The type I interferon (IFN) response is crucial for cells to restrict viral replication during infection. Many viruses, including human cytomegalovirus (HCMV), have evolved mechanisms to antagonise the type I IFN response. We have previously observed an increase in protein expression of certain interferon stimulated genes (ISG) when comparing the high passage HCMV strain AD169 compared to the low passage strain HCMV Merlin, suggesting that AD169 is defective in its ability to inhibit type I IFN function. To better understand HCMV interaction with the type I IFN response, we examined expression of cellular and viral proteins expressed in Merlin- and AD169- infected cells associated with IFN production and signalling. HCMV IFN antagonists expressed by both viruses had differences in amino acids throughout their protein sequences, although analysis using AlphaFold revealed no obvious differences in the total structure of these proteins. Analysis of quantitative mass spectrometry datasets showed modest differences in the expression of cellular IFN associated proteins between strains. Contrary to previously reported data, we found no obvious loss of IRF3 expression, though this may be due to experimental differences between studies. These data revealed that multiplicity of infection was an important factor in IRF3 expression. We found little or no difference in the production of IFN-β RNA between Merlin- and AD169- infected cells in RT-qPCR assays and little or no difference in replication of AD169 and Merlin in virus replication assays. Overall, these data suggest different strains of HCMV had different, albeit modest, abilities to influence the expression of type I IFN pathway proteins during infection, but this had no overall impact on the ability of different strains to produce a type I IFN or to replicate.

Atypical teratoid rhabdoid tumors (ATRTs) are aggressive central nervous system tumors mainly affecting young children. Extensive molecular characterization based on gene expression and DNA methylation patterns has solidly established three major ATRT subtypes (MYC, SHH and TYR), which show distinct clinical features, setting the basis for more effective, targeted treatment regimens. Transcriptional activity of transposable elements (TEs), like LINE1s and LTRs, is tightly linked with human cancers as a direct consequence of lifting epigenetic repression over TEs. The sole recurrent biallelic loss-of-function mutation in SMARCB1 in ATRTs, a core component of the SWI/SNF chromatin remodeling complex, raises the question of how TE transcription contributes to ATRT development. Here, we comprehensively investigate the transcriptional profiles of 1.9M LINE1 and LTR elements across ATRT subtypes in primary human samples. We find TE transcription profiles allow sample stratification into ATRT subtypes. The TE activity signature in the ATRT-MYC subtype is unique, setting these tumors apart from SHH and TYR ATRTs. More specifically, ATRT-MYC show broadly reduced transcript levels of LINE1 and ERVL-MaLR subfamilies. ATRT-MYC also displayed significantly less LTR and LINE1 loci with bidirectional promoter activity. Furthermore, we identify 849 differentially transcribed TEs in primary samples, which are predictive towards established ATRT-SHH and -MYC cell line models. In summary, including TE transcription profiles into the molecular characterization of ATRTs might reveal new tumor vulnerabilities leading to novel therapeutic interventions, such as immunotherapy.Supplementary InformationThe online version contains supplementary material available at 10.1186/s40478-025-02078-w.

IntroductionNecrotizing enterocolitis (NEC) is a leading cause of neonatal death and long-term morbidity, involving complex pathophysiology including prematurity, abnormal bacterial colonization, and ischemia-reperfusion injury, partially mediated by neutrophils. However, the limitations of current animal models hinder the development of targeted therapies for NEC. Thus, this study aimed to develop a human intestinal organoid model for NEC to investigate its pathophysiology, understand neutrophil involvement, and bridge animal and human research.MethodsOrganoid cultures were established from human neonatal intestinal samples with NEC (n=7) and without gut inflammation (controls, n=7), treated with lipopolysaccharides (LPS), and/or cocultured with neutrophils. Flow cytometry quantified neutrophil survival (propidium iodide/Annexin-V), activation (CD11b/CD66b), and TLR-4 expression, as well as organoid TLR-4 expression and apoptosis markers. NEC status and neutrophil recruitment were analyzed using immunofluorescence.ResultsAfter LPS administration, NEC organoids showed significantly increased TLR-4 expression, intestinal apoptosis markers, and NEC scores compared to controls, with more pronounced differences after neutrophil addition. Neutrophil activation markers were elevated when cocultured with both NEC and control organoids, but TLR-4 expression increased only with NEC organoids.DiscussionThe findings suggest that epithelial cells from NEC patients have a heightened innate TLR-4 expression upon LPS stimulation, potentially contributing to NEC development. LPS stimulation resulted in more pronounced NEC-like lesions in NEC organoids, which were exacerbated by neutrophils. This model demonstrates that neutrophils might contribute to NEC manifestation and maintenance and that NEC organoids can reflect disease aspects, potentially aiding in the development of targeted therapies.

Abstract Epithelial surfaces are the initial site of Herpes Simplex Virus 1 (HSV-1) infection. After latency establishment in the peripheral nervous system, reactivation can lead to skin pathologies. To reflect the complexity of skin we used complex human hair bearing skin organoids as a new model system for HSV-1 infection. We analyzed spread of the infection through the three-dimensional tissue and the cell type specific host response in a spatial temporal manner. Single-cell-resolution spatial transcriptomics and imaging shows a restricted viral infection in keratinocytes of the epidermis and hair follicles. The host factor IER3 is upregulated in papillary fibroblasts of the dermis as the infection progresses through the tissue. In keratinocytes of the stratum basale, we find IRF3 upregulated in infected but also virus negative cells, suggesting a signaling mechanism to initiate antiviral responses. Furthermore, we find a cell type specific inflammatory response with an upregulation of TNFSF9 in dermal fibroblasts and TNF in keratinocytes. TNFα target genes are induced in neighboring uninfected cells suggesting the induction of a localized inflammatory reaction to suppress viral infection. In summary, our findings position human hair-bearing skin organoids as a highly physiological model system for studying HSV-1 pathogenesis and reveal spatially organized host responses.

Up-regulation of Centrosomal Protein 55 (CEP55) in cancer cells increases malignancy, and the protein can be transferred via exosomes. However, the mechanism of how CEP55 is delivered to exosomes is unknown. In this study, we addressed this issue and analysed trafficking of EGFP-CEP55 from early to late endosomes by using high-resolution microscopy. Our data show that endogenous as well as EGFP-CEP55 appeared as dot-like structures in cancer cells. However, we did not find an internalization of CEP55 into early Rab5- and late Rab7-positive endosomes but only into secretory late CD63-positive endosomes. In addition, an association of the CEP55 dots with the endoplasmic reticulum and with ALG-2-interacting protein X (Alix) dots was detected. Moreover, mutation of the CEP55-Alix interaction site strongly reduced the formation of CEP55 dots as well as CEP55 localization in extracellular vesicles. In summary, our data indicate that delivery of CEP55 into exosomes does not occur by the canonical early-to-late endosome pathway but by Alix-mediated recruitment to secretory late secretory CD63 endosomes.

Upon infection, human papillomavirus (HPV) manipulates host cell gene expression to create an environment that is supportive of a productive and persistent infection. The virus-induced changes to the host cell's transcriptome are thought to contribute to carcinogenesis. Here, we show by RNA-sequencing that oncogenic HPV18 episome replication in primary human foreskin keratinocytes (HFKs) drives host transcriptional changes that are consistent between multiple HFK donors. We have previously shown that HPV18 recruits the host protein CTCF to viral episomes to control the differentiation-dependent viral transcriptional programme. Since CTCF is an important regulator of host cell transcription via coordination of epigenetic boundaries and long-range chromosomal interactions, we hypothesised that HPV18 may also manipulate CTCF to contribute to host transcription reprogramming. Analysis of CTCF binding in the host cell genome by ChIP-Seq revealed that while the total number of CTCF binding sites is not altered by the virus, there are a sub-set of CTCF binding sites that are either enriched or depleted of CTCF. Many of these altered sites are clustered within regulatory elements of differentially expressed genes, including the tumour suppressor gene cell adhesion molecule 1 (CADM1), which supresses epithelial cell growth and invasion. We show that HPV18 establishment results in reduced CTCF binding at the CADM1 promoter and upstream enhancer. Loss of CTCF binding is coincident with epigenetic repression of CADM1, in the absence of CpG hypermethylation, while adjacent genes including the transcriptional regulator ZBTB16 are activated. These data indicate that the CADM1 locus is subject to topological rearrangement following HPV18 establishment. We tested this hypothesis using 4C-Seq (circular chromosome confirmation capture-sequencing) and show that HPV18 establishment causes a loss of long-range chromosomal interactions between the CADM1 transcriptional start site and the upstream transcriptional enhancer. These data show that HPV18 manipulates host cell promoter-enhancer interactions to drive transcriptional reprogramming that may contribute to HPV-induced disease progression.