Biology subjects

Fritzlar, S.

Publications and source records attributed to Fritzlar, S..

5 recordsLinked to original sources

bioRxiv2025

Customizable host and viral transcript enrichment using CRISPR-Cas9 long-read sequencing for isoform discovery and validation

Long-read RNA sequencing has been broadly utilized to examine the diversity of transcriptomes, understand differential expression and discover novel transcript isoforms. One of the major limitations of whole transcriptome sequencing is the difficulty in obtaining sufficient depth for low abundant transcripts. Methods which address this are either difficult to scale or customize: long- range PCR is customizable but difficult to scale beyond a few targets; probe hybridization panels are suited for scaling but require substantial investment to customize. In this study, we adopted RNA-guided CRISPR-Cas9 nuclease-based enrichment to target specific human and SARS-CoV-2 transcripts followed by long-read sequencing, utilizing minimal number of guide RNAs per target isoform. Our findings demonstrate that the CRISPR-Cas system is a highly effective method for customizable long-read sequencing of target transcripts while maintaining the accuracy of relative gene expression levels. The results highlight a valuable method for future research on transcript enrichment for isoform identification and low abundance transcript detection in infectious disease diagnosis.

genomics↗

bioRxiv2023

Influenza infected macrophages release viral ribonucleoproteins that shape the local host response

Airway epithelial cells and macrophages (M{Phi}) represent cellular targets of infection by influenza A virus (IAV). Epithelial cells support IAV infection and replication by enabling the generation and release of new viral particles (productive replication). In contrast, M{Phi} are susceptible to initial IAV infection but the release of infectious viral particles is inhibited through abortive replication. Despite the lack of infectious virions released from infected M{Phi}, we detected newly synthesised viral RNA and nucleoprotein (NP) in M{Phi} supernatants. We show that viral RNA is released from infected M{Phi} as viral ribonucleoprotein (vRNP) complexes which elicits potent inflammatory responses when exposed to uninfected cells. These vRNPs specifically induced IL-1{beta}, CXCL13, IL-32, CCL4 and CXCL10 in uninfected cells and were at least partially sensed through the RIG-I/MDA5 pathway. While M{Phi} represent a dead-end for IAV infection through abortive replication, the release of vRNPs shapes immune repsonses of uninfected cells in the local microenvironment.

microbiology↗

bioRxiv2023

Norovirus NS3 protein induces apoptosis through translation repression and dysregulation of BCL-2 pro-survival proteins

Norovirus infection is characterised by a rapid onset of disease and the development of debilitating symptoms including projectile vomiting and diffuse diarrhoea. Vaccines and antivirals are sorely lacking and developments in these areas are hampered by the lack of an adequate cell culture system to investigate human norovirus replication and pathogenesis. Herein, we describe how the model norovirus, Mouse norovirus (MNV), produces a viral protein, NS3, with the functional capacity to attenuate host protein translation which invokes the activation cell death via apoptosis. We show that this function of NS3 is conserved between human and mouse viruses and map the protein domain attributable to this function. Our study highlights a critical viral protein that mediates crucial activities during replication, potentially identifying NS3 as a worthy target for antiviral drug development.

microbiology↗

bioRxiv2022

ACE2 is necessary for SARS-CoV-2 infection and sensing by macrophages but not sufficient for productive viral replication

Macrophages are key cellular contributors to COVID-19 pathogenesis. Whether SARS-CoV-2 can enter macrophages, replicate and release new viral progeny remains controversial. Similarly, whether macrophages need to sense replicating virus to drive cytokine release is also unclear. Macrophages are heterogeneous cells poised to respond to their local microenvironment, and accordingly, the SARS-CoV-2 entry receptor ACE2 is only present on a subset of macrophages at sites of human infection. Here, we use in vitro approaches to investigate how SARS-CoV-2 interacts with ACE2-negative and ACE2-positive human macrophages and determine how these macrophage populations sense and respond to SARS-CoV-2. We show that SARS-CoV-2 does not replicate within ACE2-negative human macrophages and does not induce pro-inflammatory cytokine expression. By contrast, ACE2 expression in human macrophages permits SARS-CoV-2 entry, replication, and virion release. ACE2-expressing macrophages sense replicating virus to trigger pro-inflammatory and anti-viral programs that limit virus release. These combined findings resolve several controversies regarding macrophage-SARS-CoV-2 interactions and identify a signaling circuit by which macrophages sense SARS-CoV-2 cell entry and respond by restricting viral replication. One sentence summaryLack of macrophage ACE2 expression precludes SARS-CoV-2 entry and sensing, while ACE2-expressing macrophages sense intramacrophage SARS-CoV-2 replication to induce rapid anti-viral responses that limit new virion release.

immunology↗

bioRxiv2019

Mouse Norovirus infection arrests host cell translation uncoupled from the stress granule-PKR-eIF2α axis

The integrated stress response (ISR) is a cellular response system activated upon different types of stresses, including viral infection, to restore cellular homeostasis. However, many viruses manipulate this response for their own advantage. In this study we investigated the association between murine norovirus (MNV) infection and the ISR and demonstrate that MNV regulates the ISR by activating and recruiting key ISR host factors. We observed that during MNV infection, there is a progressive increase in phosphorylated eukaryotic initiation factor 2 alpha (p-eIF2) resulting in the suppression of host translation, yet MNV translation still progresses under these conditions. Interestingly, the shutoff of host translation also impacts the translation of key signalling cytokines such as IFN{beta}, IL-6 and TNF. Our subsequent analyses revealed that the phosphorylation of eIF2 was mediated via Protein kinase-R (PKR), but further investigation revealed that PKR activation, phosphorylation of eIF2 and translational arrest were uncoupled during infection. We further observed that stress granules (SGs) are not induced during MNV infection, and MNV has the capacity to restrict SG nucleation and formation. We observed that MNV recruited the key SG nucleating protein G3BP1 to its replication sites and intriguingly the silencing of G3BP1 negatively impacts MNV replication. Thus, it appears, MNV utilises G3BP1 to enhance replication, but equally to prevent SG formation, intimating an anti-MNV property of SGs. Overall, thus study highlights MNV manipulation of SGs, PKR and translational control to regulate cytokine translation and to promote viral replication. ImportanceViruses hijack host machinery and regulate cellular homeostasis to actively replicate their genome, propagate and cause disease. In retaliation, cells possess various defence mechanisms to detect, destroy and clear infecting viruses as well as signal to neighbouring cells to inform them of the imminent threat. In this study, we demonstrate that the murine norovirus (MNV) infection stalls host protein translation and the production of antiviral and pro-inflammatory cytokines. However, virus replication and protein translation still ensues. We show that MNV further prevents the formation of cytoplasmic RNA granules, called stress granules (SG), by recruiting the key host protein G3BP1 to the MNV replication complex; a recruitment that is crucial to establishing and maintaining virus replication. Thus MNV promotes immune evasion of the virus by altering protein translation. Together, this evasion strategy delays innate immune responses to MNV infection and accelerates disease onset.

microbiology↗