Biology subjects

Urban, C.

Publications and source records attributed to Urban, C..

7 recordsLinked to original sources

bioRxiv2023

mRNA N1-2'O-methylation by CMTR1 affects NVL2 mRNA splicing

Cap0-mRNA is characterized by a 5-5triphosphate-linked N7-methylated guanosine(m7G). In higher eukaryotes, the methyltransferase CMTR1 additionally methylates the 2O-position of the penultimate mRNA nucleotide(N1) ribose (cap1-mRNA). While the m7G cap is essential for mRNA export and translation initiation by the eIF4F complex, the N1-2O-methylation prevents recognition of cap1-mRNA by the antiviral RNA receptors RIG-I and IFIT1, but a function beyond immunotolerance remained elusive. Here, we generated CMTR1-knockout(CMTR1-/-) cells and found that type-I-interferon(IFN-I) treatment resulted in IFIT1-mediated reduction of cell viability and broad mRNA translation. Consequently, stimulation of the antiviral receptor RIG-I in CMTR1-/- cells revealed an IFIT1 dependent dramatic reduction of IFN-I and chemokine protein induction, demonstrating the importance of N1-2O-methylation for antiviral responses. Additionally, IFN-I- and IFIT1-independent effects were observed: CMTR1-/- cells were smaller, divided slower, and exhibited a reduced transcription of mRNAs coding ribosomal proteins (RP), 5TOP-RNA and snoRNA host genes(SNHG). Additionally, proteome and transcriptome analysis revealed that expression of NVL2, an essential factor in ribosome biogenesis, is strongly suppressed by an alternative-splicing event of NVL2 mRNA in CMTR1-/- cells. This reduction could only be rescued by catalytically active CMTR1. Altogether, besides antiviral immunity N1-2O-methylation by CMTR1 has broad effects on cellular physiology and controls splicing of NVL2.

molecular biology↗

bioRxiv2023

hnRNPM and ELAVL1 control type I interferon induction by promoting IRF3 phosphorylation downstream of both cGAS and RIG-I

RIG-I and cGAS are crucial sensors of viral nucleic acids and induce type I IFNs via TBK1/IKK and IRF3. Here, we have identified hnRNPM as a novel positive regulator of IRF3 phosphorylation and type I IFN induction downstream of both cGAS and RIG-I. Combining interactome analysis and genome editing, we further identified ELAVL1 as an immune-relevant interactor of hnRNPM. Depletion of hnRNPM or ELAVL1 impaired type I IFN induction by HSV-1 and SeV. In addition, we found that hnRNPM and ELAVL1 interact with TBK1 and NF-kB p65. Confocal microscopy revealed cytosolic and perinuclear interactions between hnRNPM, ELAVL1, and TBK1. To our knowledge, hnRNPM and ELAVL1 represent the first non-redundant signaling components merging the cGAS-STING and RIG-I-MAVS pathways, thus representing a novel platform that fuels antiviral defense.

immunology↗

bioRxiv2023

RNase L activating 2'-5' oligoadenlyates bind ABCF1, -3 and Decr-1

A notable signaling mechanism employed by mammalian innate immune signaling pathways uses nucleotide based second messengers such as 2'-3'-cGAMP and 2'-5'-oligoadenylates (2'-5' OA), which bind and activate STING and RNase L, respectively. Interestingly, the involvement of nucleotide second messengers to activate antiviral responses is evolutionary conserved, evidenced by the identification of an antiviral cGAMP-dependent pathway in Drosophila. Using a mass spectrometry approach, we identified several members of the ABCF family in human, mouse, and Drosophila cell lysates as 2'-5' OA binding proteins, suggesting an evolutionary conserved function. Biochemical characterization of these interactions demonstrates high-affinity binding of 2'-5' OA to ABCF1, which depended on phosphorylated 2'-5' OA and an intact Walker A/B motif of the ABC cassette of ABCF1. As further support for species-specific interactions with 2'-5' OA, we additionally identified that the metabolism enzyme Decr1 from mouse, but not human or Drosophila cells forms a high-affinity complex with 2'-5' OA. A 1.4 [A] co-crystal structure of the mouse Decr1-2'-5' OA complex explains high-affinity recognition of 2'-5' OA and the mechanism of species-specificity. Despite clear evidence of physical interactions, we could not identify profound antiviral functions of ABCF1, ABCF3 or Decr1 or 2'-5' OA-dependent regulation of cellular translation rates as suggested by the engagement of ABCF proteins. Thus, although the biological consequences of the here identified interactions need to be identified, our data suggests that 2'-5' OA can serve as signaling hub to distribute a signal to different recipient proteins.

microbiology↗

bioRxiv2022

High Resolution Kinetic Characterization and Dynamic Mathematical Modeling of the RIG-I Signaling Pathway and the Antiviral Responses

The pattern recognition receptor RIG-I is essential for the recognition of viral dsRNA and the activation of a cell-autonomous antiviral response. Upon stimulation, RIG-I triggers a signaling cascade leading to the expression of cytokines, most prominently type I and III interferons (IFNs). IFNs are secreted and signal in an auto- and paracrine manner to trigger the expression of a large variety of IFN-stimulated genes, which in concert establish an antiviral state of the cell. While the topology of this pathway has been studied quite intensively, the dynamics, particularly of the RIG-I-mediated IFN induction, is much less understood. Here, we employed electroporation-based transfection to synchronously activate the RIG-I signaling pathway, enabling us to characterize the kinetics and dynamics of cell-intrinsic innate immune signaling to virus infections. By employing an A549 IFNAR1/IFNLR deficient cell line, we could analyze the difference between the primary RIG-I signaling phase and the secondary signaling phase downstream of the IFN receptors. We further used our quantitative data to set up and calibrate a comprehensive dynamic mathematical model of the RIG-I and IFN signaling pathways. This model accurately predicts the kinetics of signaling events downstream of dsRNA recognition by RIG-I as well as the feedback and signal amplification by secreted IFN and JAK/STAT signaling. We have furthermore investigated the impact of various viral immune antagonists on the signaling dynamics experimentally, and we utilized the here described modelling approach to simulate and in silico study these critical virus-host interactions. Our work provides a comprehensive insight into the signaling events occurring early upon virus infection and opens up new avenues to study and disentangle the complexity of the host-virus interface.

immunology↗

bioRxiv2021

Archival influenza virus genomes from Europe reveal genomic and phenotypic variability during the 1918 pandemic

The 1918 influenza pandemic was the deadliest respiratory pandemic of the 20th century and determined the genomic make-up of subsequent human influenza A viruses (IAV). Here, we analyze the first 1918 IAV genomes from Europe and from the first, milder wave of the pandemic. 1918 IAV genomic diversity is consistent with local transmission and frequent long-distance dispersal events and in vitro polymerase characterization suggests potential phenotypic variability. Comparison of first and second wave genomes shows variation at two sites in the nucleoprotein gene associated with resistance to host antiviral response, pointing at a possible adaptation of 1918 IAV to humans. Finally, phylogenetic estimates based on extended molecular clock modelling suggests a pure pandemic descent of seasonal H1N1 IAV as an alternative to the hypothesis of an intrasubtype reassortment origin. One Sentence SummaryMuch can be learned about past pandemics by uncovering their footprints in medical archives, which we here demonstrate for the 1918 flu pandemic.

microbiology↗

bioRxiv2020

The zinc finger antiviral protein ZAP destabilises viral transcripts and restricts cytomegalovirus

Interferon-stimulated gene products (ISGs) play a crucial role in early infection control. The ISG zinc finger CCCH-type antiviral protein 1 (ZAP/ZC3HAV1) antagonises several RNA viruses by binding to CG-rich RNA sequences, whereas its effect on DNA viruses is largely unknown. Here, we decipher the role of ZAP in the context of human cytomegalovirus (HCMV) infection, a {beta}-herpesvirus that is associated with high morbidity in immunosuppressed individuals and newborns. We show that expression of the two major isoforms of ZAP, the long (ZAP-L) and short (ZAP-S), is induced during HCMV infection and that both negatively affect HCMV replication. Transcriptome and proteome analyses demonstrated that the expression of ZAP decelerates the progression of HCMV infection. SLAM-sequencing revealed that ZAP restricts HCMV at early stages of infection by destabilising a distinct subset of viral transcripts with low CG content. In summary, this report provides evidence of an important antiviral role for ZAP in host defense against HCMV infection and highlights its differentiated function during DNA virus infection.

molecular biology↗

bioRxiv2020

Multi-level proteomics reveals host-perturbation strategies of SARS-CoV-2 and SARS-CoV

The global emergence of SARS-CoV-2 urgently requires an in-depth understanding of molecular functions of viral proteins and their interactions with the host proteome. Several individual omics studies have extended our knowledge of COVID-19 pathophysiology1-10. Integration of such datasets to obtain a holistic view of virus-host interactions and to define the pathogenic properties of SARS-CoV-2 is limited by the heterogeneity of the experimental systems. We therefore conducted a concurrent multi-omics study of SARS-CoV-2 and SARS-CoV. Using state-of-the-art proteomics, we profiled the interactome of both viruses, as well as their influence on transcriptome, proteome, ubiquitinome and phosphoproteome in a lung-derived human cell line. Projecting these data onto the global network of cellular interactions revealed crosstalk between the perturbations taking place upon SARS-CoV-2 and SARS-CoV infections at different layers and identified unique and common molecular mechanisms of these closely related coronaviruses. The TGF-{beta} pathway, known for its involvement in tissue fibrosis, was specifically dysregulated by SARS-CoV-2 ORF8 and autophagy by SARS-CoV-2 ORF3. The extensive dataset (available at https://covinet.innatelab.org) highlights many hotspots that can be targeted by existing drugs and it can guide rational design of virus- and host-directed therapies, which we exemplify by identifying kinase and MMPs inhibitors with potent antiviral effects against SARS-CoV-2.

systems biology↗