Biology subjects

Groves, D.

Publications and source records attributed to Groves, D..

4 recordsLinked to original sources

bioRxiv2026

Molecular and Structural Characterisation of the MERS-Coronavirus Polymerase

MERS-CoV (Middle East respiratory syndrome coronavirus) belongs to the Betacoronavirus genus, which also includes SARS-CoV-2, the causative agent of the COVID-19 pandemic. MERS-CoV is the pathogen responsible for Middle East Respiratory Syndrome (MERS), a respiratory disease with a 36.5% case fatality rate. Given the capacity of coronaviruses for genetic recombination and mutation, MERS-CoV remains a pathogen of significant pandemic potential, with viral evolution potentially facilitating enhanced adaptation and transmission in humans. There are no approved treatments for MERS-CoV infection. The MERS-CoV replication-transcription complex (RTC), a multimeric protein complex key to genome amplification, has no cellular analogues and a highly conserved sequence, making it a useful target for antiviral compounds; however, structural and biochemical characterisation of the MERS-CoV RTC is limited. Here, we characterise the minimal MERS-CoV RTC using in vitro and single-molecule assays and show that it is susceptible to the RNA-dependent-RNA-polymerase inhibitor suramin. We present a structure of the minimal MERS-CoV RTC in complex with an RNA duplex, which shows strong structural similarity to the SARS-CoV-2 RTC. We use cross-subunit swapping experiments to show that the MERS-CoV NSP12 core can efficiently utilise SARS-CoV-2 cofactors NSP7 and NSP8, whereas SARS-CoV-2 NSP12 exhibits reduced in vitro activity with the MERS-CoV NSP8. This asymmetric cross-species compatibility highlights both the evolutionary conservation and virus-specific adaptation of coronavirus polymerase-cofactor interactions. Together, these findings provide a structural and mechanistic framework for understanding MERS-CoV RNA synthesis and establish its conserved RTC as a potential target for the development of broadly acting coronavirus antivirals.

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↗

bioRxiv2022

Microtubule severing enzymes oligomerization and allostery: a tale of two domains

Severing proteins are nanomachines from the AAA+ (ATPases associated with various cellular activities) superfamily whose function is to remodel the largest cellular filaments, microtubules. The standard AAA+ machines adopt hexameric ring structures for functional reasons, while being primarily monomeric in the absence of the nucleotide. Both major severing proteins, katanin and spastin, are believed to follow this trend. However, studies proposed that they populate lower-order oligomers in the presence of co-factors, which are functionally relevant. Our simulations show that the preferred oligomeric assembly is dependent on the binding partners, and on the type of severing protein. Essential dynamics analysis predicts that the stability of an oligomer is dependent on the strength of the interface between the helical bundle domain (HBD) of a monomer and the convex face of the nucleotide binding domain (NBD) of a neighboring monomer. Hot spots analysis found that the region consisting of the HBD tip and the C-terminal (CT) helix is the only common element between the allosteric networks responding to nucleotide, substrate, and inter-monomer binding. Clustering analysis indicates the existence of multiple pathways for the transition between the secondary structure of the HBD tip in monomers and the structure(s) it adopts in oligomers.

biophysics↗

bioRxiv2020

periscope: sub-genomic RNA identification in SARS-CoV-2 ARTIC Network Nanopore Sequencing Data

We have developed periscope, a tool for the detection and quantification of sub-genomic RNA (sgRNA) in SARS-CoV-2 genomic sequence data. The translation of the SARS-CoV-2 RNA genome for most open reading frames (ORFs) occurs via RNA intermediates termed "sub-genomic RNAs". sgRNAs are produced through discontinuous transcription which relies on homology between transcription regulatory sequences (TRS-B) upstream of the ORF start codons and that of the TRS-L which is located in the 5 UTR. TRS-L is immediately preceded by a leader sequence. This leader sequence is therefore found at the 5 end of all sgRNA. We applied periscope to 1,155 SARS-CoV-2 genomes from Sheffield, UK and validated our findings using orthogonal datasets and in vitro cell systems. Using a simple local alignment to detect reads which contain the leader sequence we were able to identify and quantify reads arising from canonical and non-canonical sgRNA. We were able to detect all canonical sgRNAs at expected abundances, with the exception of ORF10. A number of recurrent non-canonical sgRNAs are detected. We show that the results are reproducible using technical replicates and determine the optimum number of reads for sgRNA analysis. In VeroE6 ACE2+/- cell lines, periscope can detect the changes in the kinetics of sgRNA in orthogonal sequencing datasets. Finally, variants found in genomic RNA are transmitted to sgRNAs with high fidelity in most cases. This tool can be applied to all sequenced COVID-19 samples worldwide to provide comprehensive analysis of SARS-CoV-2 sgRNA.

bioinformatics↗