Biology subjects

Fodor, E.

Publications and source records attributed to Fodor, E..

11 recordsLinked to original sources

bioRxiv2026

Influenza A virus initiates genome selection through promoter-dependent nuclear export

Influenza A virus replicates its segmented RNA genome in the nucleus in the context of viral ribonucleoprotein (vRNP) complexes. Newly synthesised vRNPs are exported to the cytoplasm for virion assembly, whereas structurally similar complementary RNPs (cRNPs), which function as replication intermediates, are not incorporated into virions. The molecular basis for this selectivity remains unclear. Here, combining promoter mutagenesis, single-molecule fluorescence in situ hybridisation (smFISH), virus-like particle (VLP) assays and cryo-electron microscopy (cryo-EM), we find that cRNPs are retained in the nucleus and that export competence is encoded by promoter architecture, with the length of the single-stranded 3' promoter region as the principal contributing feature and promoter sequence and duplex architecture further modulating export efficiency. cRNPs carrying export-competent promoters are incorporated into virus-like particles, demonstrating a functional link between nuclear export and packaging competence. Cryo-EM analyses reveal that both 3' vRNA and cRNA promoters bind a common polymerase surface site but are associated with distinct polymerase conformational landscapes, with only the vRNA promoter being compatible with an encapsidase conformation of the polymerase. Together, our findings identify a promoter-dependent checkpoint that links RNA promoter architecture and polymerase conformation to RNP export and packaging. These results demonstrate that influenza virus initiates genome selection in the nucleus and reveal nuclear export as an early step in genome selection.

microbiology↗

bioRxiv2025

High-throughput Single-Virion DNA-PAINT Reveals Structural Diversity, Cooperativity and Flexibility during Selective Packaging in Influenza

Influenza A, a negative-sense RNA virus, has a genome that consists of eight single-stranded RNA segments. Influenza co-infections can result in re-assortant viruses that contain gene segments from multiple strains, causing pandemic outbreaks with severe consequences for human health. The outcome of re-assortment is likely influenced by a selective sequence-specific genome packaging mechanism. To uncover the contributions of individual segment pairings to selective packaging, we set out to statistically analyse packaging defects and inter-segment distances in individual A/Puerto Rico/8/34 (H1N1) (PR8) virus particles. To enable such analysis, we developed a multiplexed DNA-PAINT approach capable of assessing the segment stoichiometry of more than 10,000 individual virus particles in one experiment; our approach can also spatially resolve the individual segments inside complete virus particles with a localisation precision of ~10 nm. Our results show the influenza genome can be assembled through multiple pathways in a redundant and cooperative process guided by preferentially interacting segment pairs and aided by synergistic effects that enhance genome assembly, driving it to completion. Our structural evidence indicates that the interaction strength of segment pairs affects the spatial configuration of the gene segments, which appears to be preserved in mature virions. As our method quantified the interactions of whole influenza segments instead of identifying individual sequence-based interactions, our results can serve as a template to quantify the contributions of individual sequence motifs to selective packaging.

microbiology↗

bioRxiv2024

Mechanistic insights into the activity of SARS-CoV-2 RNA polymerase inhibitors using single-molecule FRET

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has resulted in significant global mortality and disruption. Despite extensive research, the precise molecular mechanisms underlying SARS-CoV-2 replication remain unclear. To address this, we developed a single-molecule Forster resonance energy transfer (smFRET) assay to directly visualize and analyse in vitro RNA synthesis by the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). We purified the minimal replication complex, comprising nsp12, nsp7, and nsp8, and combined it with fluorescently labelled RNA substrates, enabling real-time monitoring of RNA primer elongation at the single-molecule level. This platform allowed us to investigate the mechanisms of action of key inhibitors of SARS-CoV-2 replication. In particular, our data provides evidence for remdesivirs mechanism of action, which involves polymerase stalling and subsequent chain termination dependent on the concentration of competing nucleotide triphosphates. Our study demonstrates the power of smFRET to provide dynamic insights into SARS-CoV-2 replication, offering a valuable tool for antiviral screening and mechanistic studies of viral RdRp activity.

microbiology↗

bioRxiv2023

Structural and functional characterisation of the interaction between the influenza A virus RNA polymerase and the CTD of host RNA Polymerase II

Influenza A viruses (IAV), causing seasonal epidemics and occasional pandemics, rely on interactions with host proteins for their RNA genome transcription and replication. The viral RNA polymerase utilizes host RNA polymerase II (Pol II) and interacts with the serine 5 phosphorylated (pS5) C-terminal domain (CTD) of Pol II to initiate transcription. Our study, using single-particle electron cryomicroscopy (cryo-EM), reveals the structure of the 1918 pandemic IAV polymerase bound to a synthetic pS5 CTD heptad repeat peptide. The structure shows that the CTD peptide binds at the C-terminal domain of the PA viral polymerase subunit (PA-C) and reveals a previously unobserved position of the 627 domain of the PB2 subunit near the CTD. We identify crucial residues of the CTD peptide mediating interactions with positively charged cavities on PA-C, explaining the preference of the viral polymerase for pS5 CTD. Functional analysis of mutants targeting the CTD-binding site within PA-C reveals reduced transcriptional function with normal replication, while other mutants display defects in both transcription and replication, highlighting the multifunctional role of PA-C in viral RNA synthesis. Our study provides insights into the structural and functional aspects of the influenza virus polymerase-host Pol II interaction and identifies a target for antiviral development.

microbiology↗

bioRxiv2023

The reference genome of the paradise fish (Macropodus opercularis)

Over the decades, a small number of model species, each representative of a larger taxa, have dominated the field of biological research. Amongst fishes, zebrafish (Danio rerio) has gained popularity over most other species and while their value as a model is well documented, their usefulness is limited in certain fields of research such as behavior. By embracing other, less conventional experimental organisms, opportunities arise to gain broader insights into evolution and development, as well as studying behavioral aspects not available in current popular model systems. The anabantoid paradise fish (Macropodus opercularis), an "air-breather" species from Southeast Asia, has a highly complex behavioral repertoire and has been the subject of many ethological investigations, but lacks genomic resources. Here we report the reference genome assembly of Macropodus opercularis using long-read sequences at 150-fold coverage. The final assembly consisted of {approx}483 Mb on 152 contigs. Within the assembled genome we identified and annotated 20,157 protein coding genes and assigned {approx}90% of them to orthogroups. Completeness analysis showed that 98.5% of the Actinopterygii core gene set (ODB10) was present as a complete ortholog in our reference genome with a further 1.2 % being present in a fragmented form. Additionally, we cloned multiple genes important during early development and using newly developed in situ hybridization protocols, we showed that they have conserved expression patterns.

genomics↗

bioRxiv2023

Structural Characterization of the Full-length Hantaan Virus Polymerase

Hantaviridae are a family of segmented negative-sense RNA viruses that contains important human and animal pathogens including Andes orthohantavirus and Hantaan orthohantavirus. Hantaviridae contain a viral RNA-dependent RNA polymerase that replicates and transcribes the viral genome in the cytoplasm of infected cells. Here we establish the expression and purification of the polymerase from the Old World Hantaan orthohantavirus and characterise the structure using single particle cryo electron microscopy. We determine a series of structures at resolutions between 2.7 and 3.3 [A] of RNA free polymerase comprising the core, core and endonuclease, and a full-length polymerase. The full-length polymerase structure depicts the location of the cap binding and C-terminal domains which are arranged in a conformation that is incompatible with transcription and in a novel conformation not observed in previous conformations of cap-snatching viral polymerase. We further describe structures with 5' vRNA promoter in the presence and absence of a nucleotide triphosphate. The nucleotide bound structure mimics a replication pre-initiation complex and the nucleotide stabilises the motif E in a conformation distinct from those previously observed. We observe motif E in four distinct conformations including {beta}-sheet, two helical arrangements, and an ordered loop. The insights gained here form a starting point for future detailed mechanistic studies of both the transcription and replication activities of the hantavirus polymerase and for the development of therapeutic targets.

biochemistry↗

bioRxiv2021

Identification of guanylyltransferase activity in the SARS-CoV-2 RNA polymerase

SARS-CoV-2 is a positive-sense RNA virus that is responsible for the ongoing Coronavirus Disease 2019 (COVID-19) pandemic, which continues to cause significant morbidity, mortality and economic strain. SARS-CoV-2 can cause severe respiratory disease and death in humans, highlighting the need for effective antiviral therapies. The RNA synthesis machinery of SARS-CoV-2 is an ideal drug target and consists of non-structural protein 12 (nsp12), which is directly responsible for RNA synthesis, and numerous co-factors that are involved in RNA proofreading and 5 capping of viral mRNAs. The formation of the 5 cap-1 structure is known to require a guanylyltransferase (GTase) as well as 5 triphosphatase and methyltransferase activities. However, the mechanism of SARS-CoV-2 mRNA capping remains poorly understood. Here we show that the SARS-CoV-2 RNA polymerase nsp12 functions as a GTase. We characterise this GTase activity and find that the nsp12 NiRAN (nidovirus RdRP-associated nucleotidyltransferase) domain is responsible for carrying out the addition of a GTP nucleotide to the 5 end of viral RNA via a 5 to 5 triphosphate linkage. We also show that remdesivir triphosphate, the active form of the antiviral drug remdesivir, inhibits the SARS-CoV-2 GTase reaction as efficiently as RNA polymerase activity. These data improve understanding of coronavirus mRNA cap synthesis and highlight a new target for novel or repurposed antiviral drugs against SARS-CoV-2. ImportanceSARS-CoV-2 is a respiratory RNA virus responsible for the Coronavirus Disease 2019 (COVID-19) pandemic. Coronaviruses encode an RNA polymerase which, in combination with other viral proteins, is responsible for synthesising capped viral mRNA. mRNA cap synthesis requires a guanylyltransferase enzyme; here we show that the SARS-CoV-2 guanylyltransferase is located in the viral RNA polymerase, and we identify the protein domain responsible for guanylyltransferase activity. Furthermore we demonstrate that remdesivir triphosphate, the active metabolite of remdesivir, inhibits both the guanylyltransferase and RNA polymerase functions of the SARS-CoV-2 RNA polymerase. These findings improve understanding of the coronavirus mRNA cap synthesis mechanism, in addition to highlighting a new target for the development of therapeutics to treat SARS-CoV-2 infection.

microbiology↗

bioRxiv2020

Enisamium is a small molecule inhibitor of the influenza A virus and SARS-CoV-2 RNA polymerases

Influenza A virus and coronavirus strains cause a mild to severe respiratory disease that can result in death. Although vaccines exist against circulating influenza A viruses, such vaccines are ineffective against emerging pandemic influenza A viruses. Currently, no vaccine exists against coronavirus infections, including pandemic SARS-CoV-2, the causative agent of the Coronavirus Disease 2019 (COVID-19). To combat these RNA virus infections, alternative antiviral strategies are needed. A key drug target is the viral RNA polymerase, which is responsible for viral RNA synthesis. In January 2020, the World Health Organisation identified enisamium as a candidate therapeutic against SARS-CoV-2. Enisamium is an isonicotinic acid derivative that is an inhibitor of multiple influenza B and A virus strains in cell culture and clinically approved in 11 countries. Here we show using in vitro assays that enisamium and its putative metabolite, VR17-04, inhibit the activity of the influenza virus and the SARS-CoV-2 RNA polymerase. VR17-04 displays similar efficacy against the SARS-CoV-2 RNA polymerase as the nucleotide analogue remdesivir triphosphate. These results suggest that enisamium is a broad-spectrum small molecule inhibitor of RNA virus RNA synthesis, and implicate it as a possible therapeutic option for treating SARS-CoV-2 infection. Unlike remdesivir, enisamium does not require intravenous administration which may be advantageous for the development of COVID-19 treatments outside a hospital setting. ImportanceInfluenza A virus and SARS-CoV-2 are respiratory viruses capable of causing pandemics, and the latter is responsible for the Coronavirus Disease 2019 (COVID-19) pandemic. Both viruses encode RNA polymerases which transcribe their RNA genomes and are important targets for antiviral drugs including remdesivir. Here, we show that the antiviral drug enisamium inhibits the RNA polymerases of both influenza A virus and SARS-CoV-2. Furthermore, we show that a putative metabolite of enisamium is a more potent inhibitor, inhibiting the SARS-CoV-2 RNA polymerase with similar efficiency to remdesivir. Our data offer insight into the mechanism of action for enisamium, and implicate it as a broad-spectrum antiviral which could be used in the treatment of SARS-CoV-2 infection.

microbiology↗

bioRxiv2018

Mini viral RNAs act as innate immune agonists during influenza virus infection

Influenza A virus infection usually causes a mild to moderately severe respiratory disease in humans. However, infection with the 1918 H1N1 pandemic or highly pathogenic avian influenza viruses (HPAIV) of the H5N1 subtype, can lead to viral pneumonia, systemic disease and death. The molecular processes that determine the outcome of influenza virus infection are multifactorial and involve a complex interplay between host, viral, and bacterial factors1. However, it is generally accepted that a strong innate immune dysregulation known as cytokine storm contributes to the pathology of pandemic and avian influenza virus infections2-4. The RNA sensor Retinoic acid-inducible gene I (RIG-I) plays an important role in sensing viral infection and initiating a signalling cascade that leads to interferon (IFN) expression5. Here we show that short aberrant RNAs (mini viral RNAs; mvRNAs), produced by the viral RNA polymerase during the replication of the viral RNA genome, bind and activate the intracellular pathogen sensor RIG-I, and lead to the expression of interferon-{beta}. We find that erroneous polymerase activity, dysregulation of viral RNA replication, or the presence of avian-specific amino acids underlie mvRNA generation and cytokine expression in mammalian cells and propose an intramolecular copy-choice mechanism for mvRNA generation. By deep-sequencing RNA samples from lungs of ferrets infected with influenza viruses we show that mvRNAs are generated during infection of animal models. We propose that mvRNAs act as main agonists of RIG-I during influenza virus infection and the ability of influenza virus strains to generate mvRNAs should be considered when assessing their virulence potential.

microbiology↗

bioRxiv2017

The structure of the influenza A virus genome

Influenza A viruses (IAVs) are segmented single-stranded negative sense RNA viruses that constitute a major threat to human health. The IAV genome consists of eight RNA segments contained in separate viral ribonucleoprotein complexes (vRNPs) that are packaged together into a single virus particle1,2. While IAVs are generally considered to have an unstructured single-stranded genome, it has also been suggested that secondary RNA structures are required for selective packaging of the eight vRNPs into each virus particle3,4. Here, we employ high-throughput sequencing approaches to map both the intra and intersegment RNA interactions inside influenza virions. Our data demonstrate that a redundant network of RNA-RNA interactions is required for vRNP packaging and virus growth. Furthermore, the data demonstrate that IAVs have a much more structured genome than previously thought and the redundancy of RNA interactions between the different vRNPs explains how IAVs maintain the potential for reassortment between different strains, while also retaining packaging selectivity. Our study establishes a framework towards further work into IAV RNA structure and vRNP packaging, which will lead to better models for predicting the emergence of new pandemic influenza strains and will facilitate the development of antivirals specifically targeting genome assembly.

microbiology↗

bioRxiv2017

Crossing enhanced and high fidelity SpCas9 nucleases to optimize specificity and cleavage

BackgroundThe propensity for off-target activity of Streptococcus pyogenes Cas9 (SpCas9) has been considerably decreased by rationally engineered variants with increased fidelity (eSpCas9; SpCas9-HF1). However, a subset of targets still generate considerable off-target effects. To deal specifically with these targets, we generated new \"Highly enhanced Fidelity\" nuclease variants (HeFSpCas9s) containing mutations from both eSpCas9 and SpCas9-HF1 and examined these improved nuclease variants side-by-side, to decipher the factors that affect their specificities and to determine the optimal nuclease for applications sensitive to off-target effects.\n\nResultsThese three increased-fidelity nucleases can routinely be used only with perfectly matching 20 nucleotide-long spacers; a matching 5' G extension being more detrimental to their activities than a mismatching one. HeFSpCas9s exhibit substantially improved specificity specifically for those targets for which eSpCas9 and SpCas9-HF1 have higher off-target propensity. There is also a ranking among the targets by their cleavability and off-target effects manifested by the increased fidelity nucleases. Furthermore, we show that the mutations in these variants may diminish the cleavage, but not the DNA-binding, of SpCas9s.\n\nConclusionsNo single nuclease variant shows generally superior fidelity; instead, for highest specificity cleavage, each target needs to be matched with an appropriate high fidelity nuclease. We provide here a framework for generating new nuclease variants for targets that currently have no matching optimal nuclease, and offer a simple mean for identifying the optimal nuclease for targets in the absence of accurate target-ranking prediction tools.

bioengineering↗