Biology subjects

Gerold, G.

Publications and source records attributed to Gerold, G..

9 recordsLinked to original sources

bioRxiv2026

Encephalitic alphavirus infection reduces neurite outgrowth and downregulates Rho GTPase pathways in human iPSC-derived neurospheres

Venezuelan equine encephalitis virus (VEEV) is a re-emerging neurotropic arbovirus, which affects the developing human brain, potentially leading to miscarriage, stillbirth, or long-term neurological sequelae. Currently, the mechanisms underlying these sequelae remain largely elusive. Using human induced pluripotent stem cell (iPSC)-derived neurospheres as a model for brain development, we show that both human neurons and glial cells are susceptible to VEEV infection. Microscopy-based quantification reveals that VEEV impairs neurite outgrowth in the human neurospheres. To mechanistically assess these observations, we performed multi-omics analyses of VEEV-infected neurospheres and demonstrate translational shutoff including downregulation of Rho GTPase pathways. The transcriptional and proteome profile is indicative of reduced actin cytoskeleton dynamics and consequently migratory defects of cells in the neurospheres. Shotgun secretomics moreover revealed downmodulation of neurite outgrowth associated proteins including midkine (MDK), amyloid-beta precursor protein (APP) and microtubule-associated protein 1B (MAP1B). Our findings reveal mechanistic insights into how VEEV infection impairs neuronal development and demonstrate that neurospheres are a powerful platform for assessing teratogenic effects of viruses on brain development.

neuroscience↗

bioRxiv2026

A pro-apoptotic selection strategy enables CRISPR screening in mosquitoes and identifies Lachesin as a chikungunya virus entry factor

Chikungunya virus (CHIKV) is a re-emerging mosquito-borne alphavirus that is transmitted primarily by Aedes aegypti and Aedes albopictus mosquitoes. CHIKV infection can result in debilitating arthritis-like symptoms in humans. While determinants of CHIKV host cell entry into mammalian cells are known, their equivalents in mosquito cells remain largely elusive. To identify CHIKV entry factors, we performed a membrane-focused genome-scale CRISPR loss-of-function screen in Aedes albopictus mosquito cells. To enable selection of refractory cells, we engineered CHIKV to express the pro-apoptotic Drosophila Reaper protein. CHIKV-Reaper induced robust cell death in mosquito cells with only modest viral fitness loss. Using this selection strategy together with an Aedes albopictus C6/36-based CRISPR screening platform, we identified glycosylphosphatidylinositol (GPI)-anchored cell surface proteins and multiple enzymes involved in the GPI-anchor biosynthesis pathway as proviral candidates. Ectopic expression of the GPI-anchored cell-adhesion protein Lachesin rendered refractory mammalian cells susceptible to CHIKV and the related arthritogenic alphaviruses Semliki Forest virus and Ross River virus. In contrast, Lachesin-expressing cells remained refractory to the encephalitic alphavirus Venezuelan equine encephalitis virus. We demonstrate that Lachesin is essential for CHIKV infection in both Aedes aegypti and Aedes albopictus cells, as confirmed by gene silencing. Here, we identify Lachesin as a critical candidate entry receptor for CHIKV and establish pro-apoptotic arboviruses as a powerful and versatile strategy for functional CRISPR screening in mosquito cells.

microbiology↗

bioRxiv2026

A novel dual-chimeric surrogate virus to select, test and characterize mutants of protease and glycoprotein inhibitors.

Antivirals are life-saving medications. However, pathogens develop resistances, mitigating their effect. Resistance studies (gain-of-function, GOF) are therefore paramount before and while antivirals are used clinically to characterize resistances and ideally study how to counteract them with other antivirals. The ability to switch from one antiviral, against which the virus is resistant, to another, depends on the availability of alternative drugs. Furthermore, targeting different parts of the virus synergistically with multiple drugs decreases the odds of simultaneous resistance. However, studying resistance, cross-resistance and remaining susceptibility requires working with the actual pathogen. Especially when introducing resistances into dangerous viruses, performing GOF studies would raise safety concerns and require firm biological containment. In this study we developed a novel dual-chimeric virus encoding both SARS-CoV-2 spike glycoprotein and main protease (Mpro) based on the model virus, vesicular stomatitis virus (VSV). We applied selection pressure with spike and Mpro inhibitors, generated mutants of these viruses and determined their resistance and cross-resistance profiles by performing dose-response experiments with live and pseudotyped viruses. Additionally, we conducted in silico calculations to determine the impact of spike substitutions to the observed resistance phenotype. With the new technology presented here, we demonstrate that it is possible to carry out GOF studies more safely and highlight their importance in the future challenges of virological research.

microbiology↗

bioRxiv2025

Development of an ex vivo model to study the zoonotic transmission of noroviruses

Human noroviruses are the most common cause of viral gastroenteritis worldwide, causing sporadic cases and outbreaks. Genetically related animal noroviruses infect wild and domestic animals including pigs and dogs. To investigate the potential of noroviruses to jump the species barrier, we used porcine, canine, and avian precision-cut intestinal slices. We show that human norovirus can bind to pig and dog intestinal tissue and be taken up with a similar efficiency as the porcine and canine noroviruses, respectively. In contrast, no binding or internalization of human, porcine, or canine noroviruses was detected in chicken tissue. We further showed that human norovirus can replicate to some degree in pig intestinal tissue, infecting single cells. In contrast, while animal noroviruses attached to human intestinal cells, intracellular uptake was limited. This suggests that human-to-animal transmission is more likely than animal-to-human transmission and that viral uptake likely presents a species barrier.

microbiology↗

bioRxiv2025

NRF2 activators restrict coronaviruses by targeting a network involving ACE2, TMPRSS2, and XPO1

Nuclear factor erythroid 2-related factor 2 (NRF2) is a master regulator of anti-oxidative and detoxifying cell responses. In addition, it plays important roles in host cell defenses against pathogenic viruses, and small molecules that activate NRF2 signaling can exert potent antiviral effects. We recently found that the NRF2 activators 4-octyl itaconate (4OI), bardoxolone (BARD), and sulforaphane (SFN) interfere with influenza A virus replication by blocking the nuclear export factor exportin 1 (XPO1), which did not require NRF2 signaling. Here, we have assessed their potential to inhibit highly pathogenic (SARS-CoV-2) and seasonal (hCoV-229E) coronaviruses and begun to elucidate the involved mechanisms of action. Using human cell lines and iPSC-derived vascular endothelial cells, we find that NRF2 knock-out or knock-down enhances infection by both viruses, indicating that physiologic NRF2 signaling restricts human coronavirus infection. 4OI, BARD, SFN, as well as the XPO1 blocker Selinexor (SEL), greatly limit infection by both viruses, but in an NRF2-independent manner. Strikingly, the compounds (particularly 4OI) downregulate ACE2 and TMPRSS2 mRNA and protein in Calu3 cells, leading to a >10-fold reduction in viral cell entry by 4OI and SEL, as assessed using SARS-CoV-1 and -2 spike protein VSV pseudotypes. A cycloheximide chase experiment revealed that 4OI dramatically reduces ACE2 half-life, which requires the E3 ligases NEDD4L and MCM1, suggesting that 4OI targets ACE2 for destruction by the proteasome. Moreover, 4OI and SEL reduce XPO1 protein levels, and all compounds reduce XPO1 mRNA levels. Co-incubation experiments of 4OI and the transcription blocker actinomycin D in A549 cells suggest that 4OI acts primarily by interfering with transcription of the XPO1 gene. XPO1 knock-down markedly reduces 229E replication. All four compounds interfere with 229E infection, but do not alter expression of ANPEP, the cellular receptor for this virus. Their anti-229E efficacy depends on expression of XPO1 in host cells in the order of SEL (most dependent) >4OI >SFN >BARD (least dependent), suggesting that especially BARD interferes with 229E infectivity via yet another, unknown, target. Taken together, these results suggest that "NRF2 activators" act as potent antivirals against human coronaviruses by targeting diverse host factors which are critical for viral infectivity. Author summaryHost-directed antiviral compounds act by a variety of mechanisms. For instance, they stimulate cellular antiviral immune responses and target host cell factors which are required for the viral life cycle. Pharmacologic activation of the NRF2 signaling pathway is a particularly attractive antiviral strategy, as this pathway restricts replication of a variety of viruses and also protects cells from excessive inflammation and oxidative stress resulting from accumulation of reactive oxygen species. In our previous study of the NRF2 activators bardoxolone, sulforaphane, and 4-octyl itaconate as host-directed treatments for influenza A virus infection, we found that these compounds interfered with replication of the virus. Unexpectedly, this antiviral activity was completely independent of NRF2 signaling, but resulted from blocking the nuclear export factor XPO1. In the present study, we find that these compounds limit infection by SARS-CoV-2 and hCoV-229E and that, again, this antiviral effect is NRF-independent. Instead, it depends to a large extent on downregulating ACE2 and TMPRSS2 (the major host cell receptors for SARS-CoV-1 and 2) and blocking/downregulating XPO1. Our results underscore the potential of "NRF2 activators" as adjunct treatments for viral infections, as they protect the host by anti-oxidative, anti-inflammatory, and cytoprotective mechanisms and also interfere with diverse host factors required for the viral life cycle.

pharmacology and toxicology↗

bioRxiv2023

Efficient clathrin-mediated entry of enteric adenoviruses in human duodenal cells

Enteric adenovirus types F40 and 41 (EAdVs) are a leading cause of diarrhea and diarrhea-associated death in young children and have recently been proposed to cause acute hepatitis in children. Unlike other adenoviruses, EAdVs exhibit hitherto a strict tropism for gastrointestinal tissues with, to date, unknown infection mechanism and target cells. In this study, we turn to potentially limiting host factors by comparison of EAdV entry in cell lines with respiratory and intestinal origin by cellular perturbation, virus particle tracking and transmission electron microscopy. Our analyses highlight kinetic advantages in duodenal HuTu80 cell infection and reveal a larger fraction of mobile particles, faster virus uptake and infectious particle entry in intestinal cells. Moreover, EAdVs display a dependence on clathrin- and dynamin-dependent pathways in intestinal cells. Detailed knowledge of virus entry routes and host factor requirements is essential to understand pathogenesis and develop new countermeasures. Hence, this study provides novel insights into the entry mechanisms of a medically important virus with emerging tropism in a physiologically relevant cell line. Author SummaryEnteric adenoviruses have historically been difficult to grow in cell culture, which resulted in lack of knowledge of host factors and pathways required for infection of these medically relevant viruses. Previous studies in non-intestinal cell lines showed slow infection kinetics and generated comparatively low virus yields compared to other adenovirus types. We suggest duodenum derived HuTu80 cells as a superior cell line for studies to complement efforts using complex intestinal tissue models. We show that viral host cell factors required for virus entry differ between cell lines from distinct origins and demonstrate the importance of clathrin-mediated endocytosis.

microbiology↗

bioRxiv2022

Innate immune response to SARS-1 CoV-2 infection contributes to neuronal damage in human iPSC-derived peripheral neurons

Severe acute respiratory coronavirus 2 (SARS-CoV-2) infection causes neurological disease in some patients suggesting that infection can affect both the peripheral and central nervous system (PNS and CNS, respectively). It is not clear whether the outcome of SARS-CoV-2 infection of PNS and CNS neurons is similar, and which are the key factors that cause neurological disease: SARS-CoV-2 infection or the subsequent immune response. Here, we addressed these questions by infecting human induced-pluripotent stem cell-derived CNS and PNS neurons with the {beta} strain of SARS-CoV-2. Our results show that SARS-CoV-2 infects PNS neurons more efficiently than CNS neurons, despite lower expression levels of angiotensin converting enzyme 2. Infected PNS neurons produced interferon {lambda}1, several interferon stimulated genes and proinflammatory cytokines. They also displayed neurodegenerative-like alterations, as indicated by increased levels of sterile alpha and Toll/interleukin receptor motif-containing protein 1, amyloid precursor protein and -synuclein and lower levels of nicotinamide mononucleotide adenylyltransferase 2 and {beta}-III-tubulin. Interestingly, blockade of the Janus kinase and signal transducer and activator of transcription pathway by Ruxolitinib did not increase SARS-CoV-2 infection, but reduced neurodegeneration, suggesting that an exacerbated neuronal innate immune response contributes to pathogenesis in the PNS.

microbiology↗

bioRxiv2022

Pharmacological inhibition of bromodomain and extra-terminal proteins induces NRF-2-mediated inhibition of SARS-CoV-2 replication and is subject to viral antagonism

Inhibitors of bromodomain and extra-terminal proteins (iBETs), including JQ-1, have been suggested as potential therapeutics against SARS-CoV-2 infection. However, molecular mechanisms underlying JQ-1-induced antiviral activity and its susceptibility to viral antagonism remain incompletely understood. iBET treatment transiently inhibited infection by SARS-CoV-2 variants and SARS-CoV, but not MERS-CoV. Our functional assays confirmed JQ-1-mediated downregulation of ACE2 expression and multi-omics analysis uncovered induction of an antiviral NRF-2-mediated cytoprotective response as an additional antiviral component of JQ-1 treatment. Serial passaging of SARS-CoV-2 in the presence of JQ-1 resulted in predominance of ORF6-deficient variants. JQ-1 antiviral activity was transient in human bronchial airway epithelial cells (hBAECs) treated prior to infection and absent when administered therapeutically. We propose that JQ-1 exerts pleiotropic effects that collectively induce a transient antiviral state that is ultimately nullified by an established SARS-CoV-2 infection, raising questions on their clinical suitability in the context of COVID-19.

microbiology↗

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↗