Biology subjects

Rosenthal, M.

Publications and source records attributed to Rosenthal, M..

12 recordsLinked to original sources

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

Quinolines interfere with heme-mediated activation of artemisinins

Artemisinin-based combination therapies (ACTs) remain the mainstay of treatment for Plasmodium falciparum malaria, despite reports of ACT treatment failure. ACTs consist of an artemisinin and a longer-lived partner drug, which is often a quinoline. Given that heme is central to the mechanism of action of artemisinins and some quinolines, we hypothesized that these antimalarials would exhibit strong drug-drug interactions. Previous studies using standard 48 h or 72 h assays identified additive to mildly antagonistic interactions between artemisinins and quinolines. Here, we sought to re-evaluate these interactions using a pulsing assay that better mimics the short in vivo half-life of artemisinins. We found that chloroquine (CQ), piperaquine (PPQ), and amodiaquine substantially antagonize dihydroartemisinin (DHA), the active metabolite of artemisinins. CQ-DHA antagonism was notably exacerbated in CQ-resistant parasites, resulting in a superantagonistic phenotype in isobolograms. Further, we found that CQ co-treatment conferred artemisinin resistance to Kelch 13 wild type parasites in the ring stage survival assay. Using a small molecule probe to measure chemically reactive heme in live parasites, we determined that quinolines block artemisinin activation by rendering cytosolic heme inert. Finally, we probed beyond traditional ACTs, evaluating interactions of the proposed triple ACT, DHA-PPQ-Mefloquine, as well as OZ439-quinoline combinations, which were all found to be antagonistic. Collectively, these in vitro data suggest that peroxide-quinolines may have liabilities as combination therapies.

pharmacology and toxicology↗

bioRxiv2023

Structural snapshots of phenuivirus cap-snatching and transcription

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a human pathogen that is now endemic to several East Asian countries. The viral large (L) protein catalyzes viral transcription by stealing host mRNA caps via a process known as cap-snatching. Here, we establish an in vitro cap-snatching assay and present three high-quality electron cryo-microscopy (cryo-EM) structures of the SFTSV L protein in biologically relevant, transcription-specific states. In a priming-state structure, we show capped RNA bound to the L protein cap-binding domain (CBD). The L protein conformation in this priming structure is significantly different from published replication-state structures, in particular the N- and C-terminal domains. The capped-RNA is positioned in a way that it can feed directly into the RNA-dependent RNA polymerase (RdRp) ready for elongation. We also captured the L protein in an early-elongation state following primer-incorporation demonstrating that this priming conformation is retained at least in the very early stages of primer extension. This structural data is complemented by in vitro biochemical and cell-based assays. Together, these insights further our mechanistic understanding of how SFTSV and other bunyaviruses incorporate stolen host mRNA fragments into their viral transcripts thereby allowing the virus to hijack host cell translation machinery.

biochemistry↗

bioRxiv2023

An RNA to rule them all: Critical steps in Lassa virus ribonucleoparticle assembly and recruitment

Lassa virus is a negative-strand RNA virus with only four structural proteins that causes periodic outbreaks in West Africa. The nucleoprotein (NP) encapsidates the viral genome, forming the ribonucleoprotein complexes (RNPs) together with the viral RNA and the L protein. RNPs have to be continuously restructured during viral genome replication and transcription. The Z protein is important for membrane recruitment of RNPs, viral particle assembly and budding, and has also been shown to interact with the L protein. However, the interaction of NP, viral RNA and Z is poorly understood. Here, we characterize the interactions between Lassa virus NP, Z and RNA using structural mass spectrometry. We identify the presence of RNA as the driver for disassembly of ring-like NP trimers, a storage form, into monomers to subsequently form higher order RNA-bound NP assemblies. We locate the interaction site of Z and NP and demonstrate that while NP binds Z independently of the presence of RNA, this interaction is pH-dependent. These data improve our understanding of RNP assembly, recruitment and release in Lassa virus.

biophysics↗

bioRxiv2022

Structural insights into viral genome replication by the Severe fever with thrombocytopenia syndrome virus L protein

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a phenuivirus that has rapidly become endemic in several East Asian countries. The large (L) protein of SFTSV, which includes the RNA-dependent RNA polymerase (RdRp), is responsible for catalysing viral genome replication and transcription. Here, we present 5 cryo-electron microscopy (cryo-EM) structures of the L protein in several states of the genome replication process, from pre-initiation to late-stage elongation, at a resolution of up to 2.6 [A]. We identify how the L protein binds the 5' viral RNA in a hook-like conformation and show how the distal 5' and 3' RNA ends form a duplex positioning the 3' RNA terminus in the RdRp active site ready for initiation. We also observe the L protein stalled in the early- and late-stages of elongation with the RdRp core accommodating a 9-bp product-template duplex. This duplex ultimately splits with the template binding to a designated 3' secondary binding site. The structural data and observations are complemented by in vitro biochemical and cell-based mini-replicon assays. Altogether, our data provide novel key insights into the mechanism of viral genome replication by the SFTSV L protein and will aid drug development against segmented negative-strand RNA viruses.

biochemistry↗

bioRxiv2022

AlphaPulldown - a Python package for protein-protein interaction screens using AlphaFold-Multimer

SummaryThe Artificial Intelligence-based structure prediction program AlphaFold-Multimer enabled structural modelling of protein complexes with unprecedented accuracy. Increasingly, AlphaFold-Multimer is also used to discover new protein-protein interactions. Here, we present AlphaPulldown, a Python package that streamlines protein-protein interaction screens and high-throughput modelling of higher-order oligomers using AlphaFold-Multimer. It provides a convenient command line interface, a variety of confidence scores, and a graphical analysis tool. Availability and implementationAlphaPulldown is freely available at https://www.embl-hamburg.de/AlphaPulldown.

bioinformatics↗

bioRxiv2021

Pancreatic cancer risk predicted from disease trajectories using deep learning

Pancreatic cancer is an aggressive disease that typically presents late with poor patient outcomes. There is a pronounced medical need for early detection of pancreatic cancer, which can be addressed by identifying high-risk populations. Here we apply artificial intelligence (AI) methods to a dataset of 6 million patient records with 24,000 pancreatic cancer cases in the Danish National Patient Registry (DNPR) and, for comparison, a dataset of three million records with 3,900 pancreatic cancer cases in the United States Department of Veterans Affairs (US-VA) healthcare system. In contrast to existing methods that do not use temporal information, we explicitly train machine learning models on the time sequence of diseases in patient clinical histories and test the ability to predict cancer occurrence in time intervals of 3 to 60 months after risk assessment. For cancer occurrence within 36 months, the performance of the best model (AUROC=0.88, DNPR), trained and tested on disease trajectories, exceeds that of a model without longitudinal information (AUROC=0.85, DNPR). Performance decreases when disease events within a 3 month window before cancer diagnosis are excluded from training (AUROC[3m]=0.83). Independent training and testing on the US-VA dataset reaches comparable performance (AUROC=0.78, AUROC[3m]=0.76). These results raise the state-of-the-art level of performance of cancer risk prediction on real-world data sets and provide support for the design of prediction-surveillance programs based on risk assessment in a large population followed by affordable surveillance of a relatively small number of patients at highest risk. Use of AI on real-world clinical records has the potential to shift focus from treatment of late-stage to early-stage cancer, benefiting patients by improving lifespan and quality of life.

bioinformatics↗

bioRxiv2021

Conformational changes in Lassa virus L protein associated with promoter binding and RNA synthesis activity

Lassa virus, which causes annual outbreaks in West Africa with increasing case numbers in recent years, is recognized by the WHO R&D blueprint as a significant threat for public health with high epidemic potential and no effective countermeasures. The viral large (L) protein, which contains the RNA-dependent RNA polymerase, is a key player for transcription of viral mRNA and genome replication. Here we present nine cryo-EM structures of Lassa virus L protein in the apo-, promoter-bound pre-initiation and active RNA synthesis states. We characterize distinct binding pockets for the conserved genomic 3 and 5 promoter RNAs and show how full-promoter binding induces a distinct pre-initiation conformation. In the apo- and elongation states, the endonuclease is inhibited by the binding of two distinct L protein peptides in the active site, respectively, whereas in the pre-initiation state, the endonuclease is uninhibited. In the stalled, early elongation state, a template-product duplex is bound in the active site cavity together with an incoming non-hydrolysable nucleotide. In this configuration, the full C-terminal region of the L protein, including the putative cap-binding domain, is highly ordered. The structural data are complemented by in vitro and cell-based studies testing a broad range of L protein mutants to probe functional relevance. These data advance our mechanistic understanding of how this flexible and multifunctional molecular machine is activated and will underpin antiviral drug development targeting the arenavirus L protein.

biochemistry↗

bioRxiv2020

An inability to maintain the ribonucleoprotein genomic structure is responsible for host detection of negative-sense RNA viruses

Cellular biology has a uniformity not shared amongst viruses. This is perhaps best exemplified by negative-sense RNA viruses that encode their genetic material as a ribonucleoprotein complex composed of genome, RNA-dependent RNA polymerase, and the nucleoprotein. Here we demonstrate that limiting nucleoprotein availability not only universally culminates in a replicative catastrophe for negative-sense RNA viruses, but it results in the production of aberrant genomic material and induction of the interferon-based host defenses. This dynamic illustrates the tremendous stress imposed on negative-sense RNA viruses during replication as genomic products accumulate in an environment that requires an increasing demand on nucleoprotein availability. We show that limiting NP by RNA interference or drug targeting blocks replication and primes neighboring cells through the production of interferon. Together, these results demonstrate that the nucleoprotein represents the Achilles heel of the entire phylum of negative-sense RNA viruses. Here we establish this principle for a diverse collection of human pathogens and propose that the nucleoprotein should be a primary target for the development of future antiviral drugs. HIGHLIGHTSO_LILimited levels of NP result in production of defective viral genomes C_LIO_LIDefective viral genomes and viral antagonists are key determinants of the host antiviral response C_LIO_LIThe host response and defective viral genome generation further exasperate NP availability C_LIO_LINP is an optimal drug target for the whole phylum of negative-sense RNA viruses C_LI

microbiology↗

bioRxiv2020

Structural and functional characterization of the Severe fever with thrombocytopenia syndrome virus L protein

The Bunyavirales order contains several emerging viruses with high epidemic potential, including Severe fever with thrombocytopenia syndrome virus (SFTSV). The lack of medical countermeasures, such as vaccines and antivirals, is a limiting factor for the containment of any virus outbreak. To develop such antivirals a profound understanding of the viral replication process is essential. The L protein of bunyaviruses is a multi-functional and multi-domain protein performing both virus transcription and genome replication and, therefore, would be an ideal drug target. We established expression and purification procedures for the full-length L protein of SFTSV. By combining single-particle electron-cryo microscopy and X-ray crystallography, we obtained 3D models covering [~]70% of the SFTSV L protein in the apo-conformation including the polymerase core region, the endonuclease and the cap-binding domain. We compared this first L structure of the Phenuiviridae family to the structures of La Crosse peribunyavirus L protein and influenza orthomyxovirus polymerase. Together with a comprehensive biochemical characterization of the distinct functions of SFTSV L protein, this work provides a solid framework for future structural and functional studies of L protein-RNA interactions and the development of antiviral strategies against this group of emerging human pathogens.

biochemistry↗

bioRxiv2019

Rift Valley fever virus minigenome system for investigating the role of L protein residues in viral transcription and replication

Replicon systems are important molecular tools for investigating the function of virus proteins and regulatory elements involved in viral RNA synthesis. We developed an ambisense minigenome system for Rift Valley fever virus (RVFV) to dissect the effects of L gene mutations on viral transcription versus replication. The S RNA segment with regulatory elements for ambisense gene expression served as backbone for the minigenome. Expression of the luciferase reporter gene allowed the overall activity of the RVFV replication complex to be assessed, while northern blot analysis enabled differentiation between synthesis of viral mRNA and replication intermediates. The functionality of the system was demonstrated by probing residues predictably involved in the active site of the cap-snatching endonuclease in the N-terminus of the L protein. Corresponding mutations led to a selective defect in the viral mRNA synthesis as described for other viruses of the Bunyavirales order. The analysis of further L gene mutants revealed an essential and specific role of a C-terminal region in the RVFV L protein in viral transcription. In summary, the established minigenome system is suitable for functional testing of the relevance of residues for viral transcription and replication. It may be used to validate hypotheses arising from structural or biochemical investigations of the RVFV replication complex.

molecular biology↗

bioRxiv2018

Bayesian Estimation of 3D Chromosomal Structure from Single Cell Hi-C Data

The problem of 3D chromosome structure inference from Hi-C datasets is important and challenging. While bulk Hi-C datasets contain contact information derived from millions of cells, and can capture major structural features shared by the majority of cells in the sample, they do not provide information about local variability between cells. Single cell Hi-C can overcome this problem, but contact matrices are generally very sparse, making structural inference more problematic. We have developed a Bayesian multiscale approach, named SIMBA3D, to infer 3D structures of chromosomes from single cell Hi-C while including the bulk Hi-C data and some regularization terms as a prior. We study the landscape of solutions for each single-cell Hi-C dataset as a function of prior strength and demonstrate clustering of solutions using data from the same cell.

bioinformatics↗