Biology subjects

Robb, N.

Publications and source records attributed to Robb, N..

3 recordsLinked to original sources

bioRxiv2025

Structural and functional characterisation of the Crimean-Congo Haemorrhagic Fever Virus RNA Dependent RNA Polymerase

Crimean-Congo Haemorrhagic Fever Virus (CCHFV) is found across Africa, Asia, and the Middle East where it can cause Haemorrhagic outbreaks with high case fatality rates. Central to the viral life cycle is the viral L-protein, a crucial and multifunctional protein which both transcribes and replicates the viral genome. Here, we present the cryoEM structures of an RNA free and a 5 promoter bound complex, describing the core catalytic RNA-dependent RNA polymerase (RdRp). We observe an RdRp that is substantially larger than related L-proteins and contains domain insertions unique to the nairovirus family. The 5 RNA promoter is found in a tight RNA hairpin stabilised by a single base pair, with 5 binding triggering the closure of protein over the RNA. Functional analysis of the endonuclease and RdRp activities reveals an enzyme which is capable of both activities and demonstrate RdRp inhibition by known antiviral nucleosides. These data advance our understanding of the molecular mechanisms behind genome replication and transcription, that will help inform future antiviral development.

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↗

bioRxiv2022

Reliability and accuracy of single-molecule FRET studies for characterization of structural dynamics and distances in proteins

Single-molecule FRET (smFRET) has become an established tool to study biomolecular structure and dynamics in vitro and in live cells. We performed a worldwide blind study involving 19 labs to assess the uncertainty of FRET experiments for proteins with respect to the measured FRET efficiency histograms, determination of distances, and the detection and quantification of structural dynamics. Using two protein systems that undergo distinct conformational changes, we obtained an uncertainty of the FRET efficiency of less than {+/-} 0.06, corresponding to an interdye distance precision of [≤] 0.2 nm and accuracy of [≤] 0.5 nm. We further discuss the limits for detecting distance fluctuations with sensitivity down to [lsim] 10% of the Forster distance and provide guidelines on how to detect potential dye perturbations. The ability of smFRET experiments to simultaneously measure distances and avoid averaging of conformational dynamics slower than the fluorescence lifetime is unique for dynamic structural biology.

biophysics↗