Biology subjects

Pietschmann, T.

Publications and source records attributed to Pietschmann, T..

11 recordsLinked to original sources

bioRxiv2026

The actin motor protein MYO10 facilitates post-entry spread of respiratory syncytial virus

Human respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract infections in infants. However, host factors that influence disease severity remain incompletely defined. While clinical risk factors are known, identifying genetic susceptibility has been challenging. In this study, we combined human genetics with functional virology to identify host factors that modulate RSV infection and spread. Starting from a cohort of infants hospitalized with severe RSV disease, we prioritized rare coding variants present in homozygous form and predicted to cause strong functional impairment, and selected candidate genes for mechanistic follow-up. Functional interrogation of 23 candidates by CRISPR/Cas9 knockout screening in human lung epithelial cells identified unconventional myosin-X (MYO10), encoding the actin-based motor protein myosin-X, as a critical host factor for RSV. Genetic disruption or siRNA-mediated depletion of MYO10 significantly reduced RSV infectivity, with the strongest effects at post-entry stages of the viral life cycle. Loss of MYO10 impaired filopodia formation, cell migration, and wound healing, leading to altered cell-cell connectivity and restricted viral dissemination. MYO10 depletion reduced both short-range cell-to-cell transmission and longer-distance extracellular spread, resulting in fewer infected cells and diminished accumulation of progeny virus in culture supernatants. In contrast, RSV entry, early gene expression, and interferon responses were unaffected. Finally, a rare homozygous MYO10 motor-domain variant (rs7737765; H148Y), enriched in severe cases, also reduced RSV replication in cell culture--opposite to expectations for a risk allele--yet underscoring biological relevance and suggesting that MYO10 variation may influence disease in vivo through additional effects on epithelial function. ImportanceRespiratory syncytial virus (RSV) is a major cause of severe respiratory illness in infants, yet it remains unclear why some children develop more serious disease than others. In this study, we combined patient genetic data with laboratory experiments to identify host factors that influence how RSV spreads in the lung. We found that the human protein MYO10, which mediates the formation of small cell protrusions, plays a key role in enabling the virus to spread from cell to cell. When MYO10 was disrupted, viral spread was strongly reduced, even though early steps of infection were unaffected. Interestingly, a rare genetic variant in MYO10 found in patients also altered viral replication, highlighting potential clinical relevance. These findings provide new insight into how host cell architecture contributes to RSV infection and suggest that targeting host pathways involved in viral spread could complement existing antiviral strategies.

microbiology↗

bioRxiv2026

Small aberrant viral genomes induce the innate immune response to arenaviruses

Arenaviruses are a family of negative-sense RNA viruses mainly found in rodents in which they cause chronic asymptomatic infections. Some arenaviruses, however, can occasionally infect humans and cause pathogenic viral haemorrhagic fevers. These viral haemorrhagic fevers are associated with a massively dysregulated immune response characterized by excessive release of pro-inflammatory cytokines resulting in hyperinflammatory immunopathology. Here, we systematically characterize the immune response of human cells to a wide panel of mammalian arenaviruses and identify a clear distinction between New World viruses, which uniformly induce a robust interferon response, and Old World viruses, which do not induce any significant host response. This innate immune response is primarily driven by RIG-I-mediated RNA sensing and might be aided by differences in viral interferon antagonist expression patterns. Furthermore, we identified differences in quantity and type of non-standard viral genomes produced in unpassaged Old World and New World virus infections. Finally, we demonstrate that small non-standard viral RNA expression by New World viruses are key drivers of the innate immune response and might help explain how the host response can shape viral disease phenotypes and inform triage of patients.

microbiology↗

bioRxiv2025

Hantavirus stability and inactivation

Hantaviruses are zoonotic viruses that can cause highly pathogenic disease, including hantavirus cardiopulmonary syndrome (HCPS) and haemorrhagic fever with renal syndrome (HFRS), in humans with case-fatality rates of up to 50%. However, our understanding of the basic viral life cycle and the underlying causes of viral pathogenesis remains sparse, in large part due to a lack of molecular biology tools for hantaviruses and the need to work in high-containment laboratory facilities with these viruses. Here, we investigated the kinetics of infectious Tula virus (TULV) particle production in Vero E6 cells and subsequent stability in cell culture media. In addition, we evaluated the stability of infectious virus particles in response to different physical and environmental stresses, including heat, freezing, dehydration and UV exposure, answering key questions about the environmental transmission potential of hantaviruses. Interestingly, we observed a remarkable stability of TULV when stored at room temperature or colder, as well as after dehydration, which suggests that hantaviruses could remain infectious for a sustained period of time after being secreted by their host species. Subsequently, we determined the ability of commonly used virus inactivation methods, including RNA and protein extraction buffers, to inactivate TULV both in a cell-free and cell-associated context and found that TULV was efficiently inactivated by all these methods similar to other enveloped RNA viruses. Finally, we successfully validated the complete inactivation using these inactivation methods using the highly pathogenic HCPS-causing New World Andes virus (ANDV) and the HFRS-causing Old World Hantaan virus (HTNV). These results provide valuable information about safe and effective inactivation methods of viral samples and about the environmental risk potential of hantaviruses. Author summaryHantaviruses are ubiquitous rodent viruses and contain some of the most lethal known zoonotic viruses, including Andes virus (ANDV) and Hantaan virus (HTNV), with no FDA-or EMA-approved antiviral treatment or prevention options available. However, studying the molecular biology and pathogenesis of these viruses is significantly impeded by the need for biosafety level (BSL) 3 (or higher) containment facilities for most hantaviruses and a general lack of molecular biology tools. Our study provides a comprehensive analysis of the stability of infectious hantavirus particles in response to different physical and chemical stresses. We demonstrate that a diverse range of hantaviruses are effectively and quickly inactivated by commonly used generic viral inactivation methods. However, we also provide evidence for an inherent stability under environmental conditions that could enable prolonged transmission potential of hantaviruses, even after secretion from their host species. These data are essential information to design safe inactivation methods of infectious hantavirus material and offers insights into the environmental risks of hantavirus infections with implications for laboratory safety and public health measures.

microbiology↗

bioRxiv2025

Respiratory syncytial virus load-dependent ciliated cell dedifferentiation and downregulation of immune response genes

Respiratory syncytial virus (RSV) can cause severe lower respiratory tract infections in infants, older adults, and individuals with chronic or immunocompromising conditions. To understand how RSV affects airway cells, we infected primary human airway epithelial cultures with RSV and analyzed infected and bystander cells using time-resolved single-cell RNA sequencing and imaging. RSV mainly infected ciliated cells, triggering a virus load-dependent shutdown of genes involved in ciliogenesis, antigen presentation, and innate sensing, including key interferon (IFN) and pattern recognition pathways. Only a subset of infected cells produced type I and III IFNs, while bystander cells exhibited strong IFN-stimulated gene (ISG) signatures. Neither IFN treatment nor ISG induction eliminated infection, but IRF1, an antiviral transcription factor not suppressed by RSV, remained robustly expressed. Ectopic IRF1 expression in vitro reduced viral replication. These findings reveal how RSV evades antiviral defenses and highlight IRF1 as a potential target for therapeutic intervention.

microbiology↗

bioRxiv2024

Cystobactamid off-target profiling reveals favorable safety, superoxide reduction, and SCARB1 inhibition in eukaryotes

Antimicrobial resistance (AMR) poses a fundamental global threat, necessitating new strategies for effective therapies. Cystobactamids (CYS), a class of antibacterial agents targeting bacterial gyrase and topoisomerase IV, represent a non-traditional chemical scaffold with broad-spectrum activity. For toxicological de-risking, we performed a comprehensive profiling on eukaryotic cells, focusing on cytotoxicity, genotoxicity, and mitochondrial toxicity, demonstrating cellular safety and superoxide scavenging properties. Studies in zebrafish embryos assessed developmental, cardiovascular, and hepatic toxicity, indicating a favorable in vivo safety profile. Metabolism studies revealed glucuronidation and amide bond hydrolysis as key pathways, whereby CYS metabolic stability substantially improved by cobicistat co-treatment. Affinity-based protein profiling identified the cholesterol- and HCV-receptor scavenger receptor class B member 1 (SCARB1) as a primary eukaryotic off-target protein, with cystobactamids shown to inhibit SCARB1s function, preventing hepatitis C virus pseudoparticle entry into cells. These findings suggest a high therapeutic potential for cystobactamids and highlight SCARB1 as a primary eukaryotic target.

pharmacology and toxicology↗

bioRxiv2023

Hepatitis C virus cell culture adaptive mutations enhance cell culture propagation by multiple mechanisms but boost antiviral responses in primary human hepatocytes

Hepatitis C virus (HCV) infection progresses to chronicity in the majority of infected individuals. Its high intra-host genetic variability enables HCV to evade the continuous selection pressure exerted by the host, contributing to persistent infection. Utilizing a cell culture adapted HCV population (p100pop) which exhibits increased replicative capacity in various liver cell lines, this study investigated virus and host determinants which underlie enhanced viral fitness. Characterization of a panel of molecular p100 clones revealed that cell culture adaptive mutations optimize a range of virus-host interactions, resulting in expanded cell tropism, altered dependence on the cellular co-factor micro-RNA 122 and increased rates of virus spread. On the host side, comparative transcriptional profiling of hepatoma cells infected either with p100pop or its progenitor virus revealed that enhanced replicative fitness correlated with activation of endoplasmic reticulum stress signaling and the unfolded protein response. In contrast, infection of primary human hepatocytes with p100pop led to a mild attenuation of virion production which correlated with a greater induction of cell-intrinsic antiviral defense responses. In summary, long-term passage experiments in cells where selective pressure from innate immunity is lacking improves multiple virus-host interactions, enhancing HCV replicative fitness. However, this study further indicates that HCV has evolved to replicate at low levels in primary human hepatocytes to minimize innate immune activation, highlighting that an optimal balance between replicative fitness and innate immune induction is key to establishing persistence. Author SummaryHCV infection remains a global health burden with 58 million people currently chronically infected. However, the detailed molecular mechanisms which underly persistence are incompletely defined. We utilized a long-term cell culture adapted HCV, exhibiting enhanced replicative fitness in different human liver cell lines, in order to identify molecular principles by which HCV optimizes its replication fitness. Our experimental data revealed that cell culture adaptive mutations confer changes in the host response and usage of various host factors. The latter allows functional flexibility at different stages of the viral replication cycle. However, increased replicative fitness resulted in an increased activation of the innate immune system, which likely poses boundary for functional variation in authentic hepatocytes, explaining the observed attenuation of the adapted virus population in primary hepatocytes.

molecular biology↗

bioRxiv2022

Hepatitis D virus infection of stem cell-derived hepatocytes triggers an IFN- and NFkB-based innate immune response unable to clear infection.

Human pluripotent stem cell-derived hepatocyte-like cells (HLCs) are a valuable model to investigate host-pathogen interactions of hepatitis viruses in a mature and authentic environment. Here, we investigated the susceptibility of HLCs to the Hepatitis D Virus (HDV), a virus that in co-infection with HBV is responsible for the most severe form of viral hepatitis. Cells undergoing hepatic differentiation became susceptible to HDV infection after acquiring expression of the Na+-taurocholate cotransporting polypeptide (NTCP), the receptor mediating HBV and HDV entry. Inoculation of mature HLCs with HDV lead to increasing amounts of intracellular HDV RNA and accumulation of the HDV antigen in the cells. The infection was abrogated when using known entry inhibitors targeting NTCP or by disrupting genome replication using the nucleoside analogue Ribavirin. Upon infection, the HLCs mounted an innate immune response based on induction of the interferons IFNB and L, but not IFNA, and were associated with an upregulation of interferon-stimulated genes. The intensity of this immune response positively correlated with the level of viral replication and was dependant on both the JAK/STAT and NF{kappa}B pathway activation. Importantly, neither this innate immune response nor an exogenous treatment of IFN2b inhibited HDV replication. However, pre-treatment of the HLCs with IFN2b reduced viral infection, suggesting that ISGs may limit early stages of infection. This novel HDV in vitro infection model represents a valuable tool for studying HDV replication and investigating candidate antiviral drugs in cells displaying mature hepatic functions. Lay summaryHDV can infect stem cell-derived hepatocytes through an NTCP-mediated entry process. Infection triggers an IFN and NF{kappa}B dependent innate immune response. However, viral replication seems unaffected by this innate response or by exogenous IFN treatment. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/502443v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@44ca7forg.highwire.dtl.DTLVardef@4a7be3org.highwire.dtl.DTLVardef@b9c3corg.highwire.dtl.DTLVardef@3e66c3_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2021

Repurposing screen highlights broad-spectrum coronavirus antivirals and their host targets

Libraries composed of licensed drugs represent a vast repertoire of molecules modulating physiologic processes in humans, thus providing unique opportunities for discovery of host targeting antivirals. We interrogated the ReFRAME repurposing library with 12,993 molecules for broad-spectrum coronavirus antivirals and discovered 134 compounds inhibiting an alphacoronavirus, mapping to 59 molecular target categories. Dominant targets included the 5-hydroxytryptamine receptor and dopamine receptor and cyclin-dependent kinase inhibitors. Counter-screening with SARS-CoV-2 and validation in primary cells identified Phortress, an aryl hydrocarbon receptor (AHR) ligand, Bardoxolone and Omaveloxolone, two nuclear factor, erythroid 2 like 2 (NFE2L2) activators as inhibitors of both alpha- and betacoronaviruses. The landscape of coronavirus targeting molecules provides important information for the development of broad-spectrum antivirals reinforcing pandemic preparedness.

microbiology↗

bioRxiv2021

Differential interferon-α subtype immune signatures suppress SARS-CoV-2 infection

Type I interferons (IFN-I) exert pleiotropic biological effects during viral infections, balancing virus control versus immune-mediated pathologies and have been successfully employed for the treatment of viral diseases. Humans express twelve IFN-alpha () subtypes, which activate downstream signalling cascades and result in distinct patterns of immune responses and differential antiviral responses. Inborn errors in type I IFN immunity and the presence of anti-IFN autoantibodies account for very severe courses of COVID-19, therefore, early administration of type I IFNs may be protective against life-threatening disease. Here we comprehensively analysed the antiviral activity of all IFN subtypes against SARS-CoV-2 to identify the underlying immune signatures and explore their therapeutic potential. Prophylaxis of primary human airway epithelial cells (hAEC) with different IFN subtypes during SARS-CoV-2 infection uncovered distinct functional classes with high, intermediate and low antiviral IFNs. In particular IFN5 showed superior antiviral activity against SARS-CoV-2 infection. Dose-dependency studies further displayed additive effects upon co-administered with the broad antiviral drug remdesivir in cell culture. Transcriptomics of IFN-treated hAEC revealed different transcriptional signatures, uncovering distinct, intersecting and prototypical genes of individual IFN subtypes. Global proteomic analyses systematically assessed the abundance of specific antiviral key effector molecules which are involved in type I IFN signalling pathways, negative regulation of viral processes and immune effector processes for the potent antiviral IFN5. Taken together, our data provide a systemic, multi-modular definition of antiviral host responses mediated by defined type I IFNs. This knowledge shall support the development of novel therapeutic approaches against SARS-CoV-2.

cell biology↗

bioRxiv2021

Initial HCV infection of adult hepatocytes triggers a temporally structured transcriptional program containing diverse pro- and anti-viral elements

Transcriptional profiling provides global snapshots of virus-mediated cellular reprogramming, which can simultaneously encompass pro- and antiviral components. To determine early transcriptional signatures associated with HCV infection of authentic target cells, we performed ex vivo infections of adult primary human hepatocytes (PHHs) from seven donors. Longitudinal sampling identified minimal gene dysregulation at six hours post infection (hpi). In contrast, at 72 hpi, massive increases in the breadth and magnitude of HCV-induced gene dysregulation were apparent, affecting gene classes associated with diverse biological processes. Comparison with HCV-induced transcriptional dysregulation in Huh-7.5 cells identified limited overlap between the two systems. Of note, in PHHs, HCV infection initiated broad upregulation of canonical interferon (IFN)-mediated defense programs, limiting viral RNA replication and abrogating virion release. We further find that constitutive expression of IRF1 in PHHs maintains a steady-state antiviral program in the absence of infection, which can additionally reduce HCV RNA translation and replication. We also detected infection-induced downregulation of [~]90 genes encoding components of the EIF2 translation initiation complex and ribosomal subunits in PHHs, consistent with a signature of translational shutoff. As HCV polyprotein translation occurs independently of the EIF2 complex, this process is likely pro-viral: only translation initiation of host transcripts is arrested. The combination of antiviral intrinsic and inducible immunity, balanced against pro-viral programs, including translational arrest, maintains HCV replication at a low-level in PHHs. This may ultimately keep HCV under the radar of extra-hepatocyte immune surveillance while initial infection is established, promoting tolerance, preventing clearance and facilitating progression to chronicity. IMPORTANCEAcute HCV infections are often asymptomatic and therefore frequently undiagnosed. We endeavored to recreate this understudied phase of HCV infection using explanted PHHs and monitored host responses to initial infection. We detected temporally distinct virus-induced perturbations in the transcriptional landscape, which were initially narrow but massively amplified in breadth and magnitude over time. At 72 hpi, we detected dysregulation of diverse gene programs, concurrently promoting both virus clearance and virus persistence. On the one hand, baseline expression of IRF1 combined with infection-induced upregulation of IFN-mediated effector genes suppresses virus propagation. On the other, we detect transcriptional signatures of host translational inhibition, which likely reduces processing of IFN-regulated gene transcripts and facilitates virus survival. Together, our data provide important insights into constitutive and virus-induced transcriptional programs in PHHs, and identifies simultaneous antagonistic dysregulation of pro-and anti-viral programs which may facilitate host tolerance and promote viral persistence.

immunology↗

bioRxiv2020

Cell-intrinsic Innate Immune Responses against Chikungunya Virus in a Human Ex Vivo Synovial Fibroblast Model

In recent years, (re-)emerging arboviruses including Chikungunya virus (CHIKV) and Mayaro virus (MAYV) have caused growing concern due to expansion of insect vector ranges. No protective vaccine or specific antiviral strategies are currently available. Long-term morbidity after CHIKV infection includes debilitating chronic joint pain, which has associated health and economic impact. Here, we analyzed the early cell-intrinsic response to CHIKV and MAYV infection in primary human synovial fibroblasts. This interferon-competent cell type represents a potential source of polyarthralgia induced by CHIKV infection. Synovial fibroblasts from healthy and osteoarthritic donors were similarly permissive to CHIKV and MAYV infection ex vivo. Using RNA-seq, we defined a CHIKV infection-induced transcriptional profile with several hundred interferon-stimulated and arthralgia-mediating genes upregulated. Type I interferon was both secreted by infected fibroblasts and protective when administered exogenously. IL-6 secretion, which mediates chronic synovitis, however, was not boosted by infection. Single-cell RNA-seq and flow cytometric analyses uncovered an inverse correlation of activation of innate immunity and productive infection at the level of individual cells. In summary, primary human synovial fibroblasts serve as bona-fide ex vivo primary cell model of CHIKV infection and provide a valuable platform for studies of joint tissue-associated aspects of CHIKV immunopathogenesis.

microbiology↗