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Biology subjects

Steiner, S.

Publications and source records attributed to Steiner, S..

11 recordsLinked to original sources

Human AUTS2 regulates neurodevelopmental pathways via dual DNA/RNA binding.

The AUTS2 gene is implicated in neurodevelopmental and psychiatric disorders, with patient mutations leading to intellectual disability, microcephaly, and autistic behavior. While AUTS2s chromatin-and RNA-related functions are recognized, its direct binding to RNA in human neural progenitors has not been previously demonstrated. Here, we used ChIP-seq and eCLIP-seq in human neural progenitor cells (NPCs) to map AUTS2s chromatin targets and, for the first time, its direct RNA interactome. AUTS2 knockdown in NPCs led to widespread gene expression changes and impaired cell proliferation, migration, and neurite outgrowth. Integrated analysis revealed downregulation of Wnt pathway genes, notably WNT7A, among targets directly bound by AUTS2 at both chromatin and RNA levels. Supplementation with WNT7A rescued cellular phenotypes in AUTS2-deficient NPCs, underscoring the significance of Wnt signaling. These findings highlight AUTS2s central role in human neurodevelopment and provide mechanistic insight into how its disruption may contribute to the pathology of neurodevelopmental disorders.

neuroscience↗

Stress granules formed during different RNA virus infections show remarkable plasticity and substantial virus-specific differences in their formation and composition

Eukaryotic cells evolved a cellular stress response to cope with extrinsic and intrinsic stress stimuli including virus infections. The major result of this response is the shutdown of bulk translation to prevent damage and allow the reprogramming of translation towards stress-resolving pathways. The resulting translationally stalled mRNA and associated proteins are accumulated in membrane-less cytosolic condensates called stress granules (SG). While the inhibitory effect of translation arrest on virus growth is well established, the role of SGs in the cellular defense against viruses is still unclear. The observation of specific interference with SG formation during various virus infections led to the hypothesis that SGs could serve as antiviral signaling platforms. In this study, we used mouse hepatitis virus (MHV) to characterize SGs formed during coronavirus infection. By applying APEX2-mediated proximity labelling in combination with quantitative proteomics, we dissected the proteome of MHV-induced granules and compared it to canonical SGs formed during oxidative stress. Our data revealed substantial differences in protein abundance and composition indicating stressor-specific SG characteristics. To assess if the observed differences are a general feature of virus-induced SGs or rather virus-specific, we extended our investigations to the Semliki Forest virus (SFV), a member of the alphavirus family known to induce SGs. An initial comparison of SG formation kinetics by live-cell imaging showed distinct time points of SG induction between both viruses. A comprehensive comparison of the SG protein compositions revealed profound differences in the SG proteome between SFV and MHV. A further subcellular localization of SG components by microscopy not only confirmed a reduced abundance of several translation initiation factors in MHV-induced granules, but surprisingly, revealed the presence of SFV RNA and the absence of MHV RNA in virus-induce SGs. The reduced connection to canonical SG themes observed for MHV-induced granules compared to SFV- and oxidative stress-induced ones indicates a different impact of these condensates on MHV replication and further raises the question whether they should be considered SGs. The surprising plasticity of SGs concerning induction kinetics, protein composition and abundance, and inclusion or exclusion of viral RNA provide a base for future investigations of the role(s) of SGs in the context of viral infection and how they may impact virus replication.

microbiology↗

Three-dimensional quantitative characterization of Coxiella burnetii infection using focused ion beam-scanning electron microscopy

Coxiella burnetii is a highly virulent intracellular pathogen that causes acute and chronic Q fever in humans. The bacterium utilizes a Dot/Icm type IV secretion system (T4SS) to translocate over 100 effectors into host cells to facilitate biogenesis of Coxiella-containing vacuoles (CCVs), which are specialized lysosome-derived organelles that support bacterial replication. During replication in CCVs, C. burnetii undergoes a unique biphasic developmental cycle defined by two distinct cellular forms: the infectious and metabolically dormant small cell variant (SCV) that converts to a replicative large cell variant (LCV). This developmental cycle was believed to be intimately coupled with biogenesis of CCVs, but the underlying mechanisms remained unclear. Here, we combine advanced focused ion beam-scanning electron microscopy (FIB-SEM) with machine learning-based data analyses to visualize host cells infected by C. burnetii. We reveal significant pleiomorphism of both the bacteria and CCVs in three dimensions. We show that this technology can be leveraged to characterize CCV biogenesis defects displayed by C. burnetii mutants that are unable to generate the large CCV displayed by wild-type C. burnetii. Analysis of HeLa cells infected with a cig2::Tn mutant confirmed that this mutant creates vacuoles that have a defect in homotypic fusion but that the proportions of SCV to LCV in vacuoles in which this mutant resides are nearly identical to CCVs containing wild-type C. burnetii, which indicates the vacuole biogenesis defect displayed by the cig2::Tn mutant did not impact the developmental cycle intracellularly. Collectively, this study provides unprecedented three-dimensional images of the complex intracellular lifestyle of C. burnetii. This imaging technology also provides unique insights into the biphasic developmental cycle and will be a powerful approach to dissect CCV biogenesis defects displayed by mutant C. burnetii.

microbiology↗

Mpox virus spreads from cell-to-cell and leads to neuronal injury in human cerebral organoids

In 2022-23, the world experienced the largest recorded monkeypox virus (MPXV) outbreak outside of endemic regions. Remarkably, cases of neurological manifestations were reported, some of which fatal. MPXV DNA and MPXV-specific antibodies were detected in the cerebrospinal fluid of encephalitis-affected patients, suggesting neuroinvasive potential of MPXV. We explored the susceptibility of neural tissue to MPXV infection using human neural organoids (hNOs) exposed to a primary isolate belonging to clade IIb lineage. The virus efficiently replicates in hNOs as indicated by the exponential increase of infectious viral loads and the elevated frequency of MPXV-positive cells over time. Electron microscopy imaging revealed the presence of viral particles as well as perinuclear viral factories. We observed susceptibility of several cell types to the virus, including neural progenitor cells and neurons. Furthermore, we detected the presence of viral antigen in neurites and in foci of grouped cells distributed throughout the tissue. In line with this, we documented significantly more cell-associated than released infectious virus, suggesting viral spread by cell-to-cell contact. Using an mNeonGreen-expressing recombinant MPXV, we confirmed cell-associated virus transmission through live-cell imaging. While hNOs displayed no evident outer morphological changes upon infection, we detected the formation of beads in neurites, a phenomenon commonly associated with neurodegenerative disorders. Live-cell imaging further confirmed the recurrent formation of neuritic beads in neurons in the days following MPXV infection, with bead formation preceding neurite-initiated cell death. Notably, treatment of MPXV infected hNOs with the antiviral drug tecovirimat resulted in a significant reduction of infectious viral loads by several orders of magnitude. Taken together, our findings suggest viral manipulation of axonal transport driving neuronal degeneration and identify a mechanism potentially contributing to MPXV-mediated neuropathology that may have therapeutic implications.

microbiology↗

Developmental transitions coordinate assembly of the Coxiella burnetii Dot/Icm type IV secretion system

Coxiella burnetii is an obligate intracellular bacterial pathogen that has evolved a unique bi-phasic developmental cycle. The infectious form of C. burnetii is the dormant small cell variant (SCV) that transitions to a metabolically active large cell variant (LCV) that replicates inside the lysosome-derived host vacuole. A Dot/Icm type IV secretion system (T4SS), which can deliver over 100 effector proteins to host cells, is essential for the biogenesis of the vacuole and for intracellular replication. How the distinct C. burnetii life cycle impacts the assembly and function of the Dot/Icm T4SS has remained unknown. Here, we combine advanced cryo-focused ion beam (cryo-FIB) milling and cryo-electron tomography (cryo-ET) imaging to visualize all developmental transitions and assembly of the Dot/Icm T4SS. Importantly, assembled Dot/Icm T4SSs were not present in the infectious SCV. The appearance of an assembled Dot/Icm machine correlated with the transition of the SCV to the LCV intracellularly. Furthermore, temporal characterization of C. burnetii morphological changes revealed regions of the inner membrane that invaginated to form tightly packed stacks during the LCV to SCV transition at late stages of infection, which could enable the SCV to LCV transition that occurs upon infection of a new host cell. Overall, these data establish how C. burnetii developmental transitions control critical bacterial processes to promote intracellular replication and transmission.

microbiology↗

The spike gene is a major determinant for the SARS-CoV-2 Omicron-BA.1 phenotype

Variant of concern (VOC) Omicron-BA1 has achieved global predominance in early 2022. Therefore, surveillance and comprehensive characterization of Omicron-BA.1 in advanced primary cell culture systems and multiple animal models is urgently needed. Here, we characterized Omicron-BA.1 and recombinant Omicron-BA.1 spike gene mutants in comparison with VOC Delta in well-differentiated primary human nasal and bronchial epithelial cells in vitro, followed by in vivo fitness characterization in naive hamsters, ferrets and hACE2-expressing mice, and in immunized hACE2-mice. We demonstrate a spike-mediated enhancement of early replication of Omicron-BA.1 in nasal epithelial cultures, but limited replication in bronchial epithelial cultures. In Syrian hamsters, Delta showed dominance over Omicron-BA.1 and in ferrets, Omicron-BA.1 infection was abortive. In mice expressing the authentic hACE2-receptor, Delta and a Delta spike clone also showed dominance over Omicron-BA.1 and an Omicron-BA.1 spike clone, respectively. Interestingly, in naive K18-hACE2 mice, we observed Delta spike-mediated increased replication and pathogenicity and Omicron-BA.1 spike-mediated reduced replication and pathogenicity, suggesting that the spike gene is a major determinant of both Delta and Omicron-BA.1 replication and pathogenicity. Finally, the Omicron-BA.1 spike clone was less well controlled by mRNA-vaccination in K18-hACE2-mice and became more competitive compared to the progenitor and Delta spike clones, suggesting that spike gene-mediated immune evasion is another important factor that led to Omicron-BA.1 dominance.

microbiology↗

Susceptibility of well-differentiated airway epithelial cell cultures from domestic and wildlife animals to SARS-CoV-2

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has spread globally, and the number of cases continues to rise all over the world. Besides humans, the zoonotic origin, as well as intermediate and potential spillback host reservoirs of SARS-CoV-2 are unknown. To circumvent ethical and experimental constraints, and more importantly, to reduce and refine animal experimentation, we employed our airway epithelial cell (AEC) culture repository composed of various domesticated and wildlife animal species to assess their susceptibility to SARS-CoV-2. In this study, we inoculated well-differentiated animal AEC cultures of monkey, cat, ferret, dog, rabbit, pig, cattle, goat, llama, camel, and two neotropical bat species with SARS-CoV-2. We observed that SARS-CoV-2 only replicated efficiently in monkey and cat AEC culture models. Whole-genome sequencing of progeny virus revealed no obvious signs of nucleotide transitions required for SARS-CoV-2 to productively infect monkey and cat epithelial airway cells. Our findings, together with the previously reported human-to-animal spillover events warrants close surveillance to understand the potential role of cats, monkeys, and closely related species as spillback reservoirs for SARS-CoV-2.

microbiology↗

SARS-CoV-2 spike D614G variant confers enhanced replication and transmissibility

During the evolution of SARS-CoV-2 in humans a D614G substitution in the spike (S) protein emerged and became the predominant circulating variant (S-614G) of the COVID-19 pandemic1. However, whether the increasing prevalence of the S-614G variant represents a fitness advantage that improves replication and/or transmission in humans or is merely due to founder effects remains elusive. Here, we generated isogenic SARS-CoV-2 variants and demonstrate that the S-614G variant has (i) enhanced binding to human ACE2, (ii) increased replication in primary human bronchial and nasal airway epithelial cultures as well as in a novel human ACE2 knock-in mouse model, and (iii) markedly increased replication and transmissibility in hamster and ferret models of SARS-CoV-2 infection. Collectively, our data show that while the S-614G substitution results in subtle increases in binding and replication in vitro, it provides a real competitive advantage in vivo, particularly during the transmission bottle neck, providing an explanation for the global predominance of S-614G variant among the SARS-CoV-2 viruses currently circulating.

microbiology↗

Disparate temperature-dependent virus - host dynamics for SARS-CoV-2 and SARS-CoV in the human respiratory epithelium

Since its emergence in December 2019, SARS-CoV-2 has spread globally and become a major public health burden. Despite its close phylogenetic relationship to SARS-CoV, SARS-CoV-2 exhibits increased human-to-human transmission dynamics, likely due to efficient early replication in the upper respiratory epithelium of infected individuals. Since different temperatures encountered in the human respiratory tract have been shown to affect the replication kinetics of several viruses, as well as host immune response dynamics, we investigated the impact of temperatures during SARS-CoV-2 and SARS-CoV infection in the human airway epithelial cell culture model. SARS-CoV-2, in contrast to SARS-CoV, replicated more efficiently at temperatures encountered in the upper respiratory tract, and displayed higher sensitivity to type I and type III IFNs. Time-resolved transcriptome analysis highlighted a temperature-dependent and virus-specific induction of the IFN-mediated antiviral response. These data reflect clinical features of SARS-CoV-2 and SARS-CoV, as well as their associated transmission efficiencies, and provide crucial insight on pivotal virus - host interaction dynamics.

microbiology↗

Efficient inactivation of SARS-CoV-2 by WHO-recommended hand rub formulations and alcohols

The recent emergence of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing COVID-19 is a major burden for health care systems worldwide. It is important to address if the current infection control instructions based on active ingredients are sufficient. We therefore determined the virucidal activity of two alcohol-based hand rub solutions for hand disinfection recommended by the World Health Organization (WHO), as well as commercially available alcohols. Efficient SARS-CoV-2 inactivation was demonstrated for all tested alcohol-based disinfectants. These findings show the successful inactivation of SARS-CoV-2 for the first time and provide confidence in its use for the control of COVID-19. ImportanceThe current COVID-19 outbreak puts a huge burden on the worlds health care systems. Without effective therapeutics or vaccines being available, effective hygiene measure are of utmost importance to prevent viral spreading. It is therefore crucial to evaluate current infection control strategies against SARS-CoV-2. We show the inactivation of the novel coronavirus for the first time and endorse the importance of disinfectant-based hand hygiene to reduce SARS-CoV-2 transmission.

microbiology↗

Rapid reconstruction of SARS-CoV-2 using a synthetic genomics platform

Reverse genetics has been an indispensable tool revolutionising our insights into viral pathogenesis and vaccine development. Large RNA virus genomes, such as from Coronaviruses, are cumbersome to clone and to manipulate in E. coli hosts due to size and occasional instability1-3. Therefore, an alternative rapid and robust reverse genetics platform for RNA viruses would benefit the research community. Here we show the full functionality of a yeast-based synthetic genomics platform for the genetic reconstruction of diverse RNA viruses, including members of the Coronaviridae, Flaviviridae and Paramyxoviridae families. Viral subgenomic fragments were generated using viral isolates, cloned viral DNA, clinical samples, or synthetic DNA, and reassembled in one step in Saccharomyces cerevisiae using transformation associated recombination (TAR) cloning to maintain the genome as a yeast artificial chromosome (YAC). T7-RNA polymerase has been used to generate infectious RNA, which was then used to rescue viable virus. Based on this platform we have been able to engineer and resurrect chemically-synthetized clones of the recent epidemic SARS-CoV-24 in only a week after receipt of the synthetic DNA fragments. The technical advance we describe here allows to rapidly responding to emerging viruses as it enables the generation and functional characterization of evolving RNA virus variants - in real-time - during an outbreak.

microbiology↗