Biology subjects

Fodor, E.

Publications and source records attributed to Fodor, E..

3 recordsLinked to original sources

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↗