Biology subjects

Grimes, J.

Publications and source records attributed to Grimes, J..

6 recordsLinked to original sources

bioRxiv2026

Coronavirus spherules are supported by a novel triple-layer protein lattice

Coronaviruses (CoVs) cause significant disease in humans and animals, with a track-record of cross-species transmission. Despite their global significance, available control strategies are limited, especially for emerging CoVs. A deeper fundamental understanding of the virus life cycle is essential to underpin development of control measures. A critical step of the CoV life cycle is the rearrangement of host cell membranes into replication organelles (ROs). This is conserved across all CoVs and ROs comprise double membrane vesicles (DMVs), the site of viral RNA synthesis, and double membrane spherules. CoV spherules are complex structures requiring both positive and negative curvature of paired membranes so are predicted to have a high energy cost to maintain. To date they have an entirely unknown function. Interestingly, although always present, abundance of spherules in CoV infected cells varies with Betacoronavirus Middle East Respiratory Syndrome coronavirus, Gammacoronavirus infectious bronchitis virus (IBV) and porcine deltacoronavirus in particular producing a considerable number. Here, using cryo-bioimaging we identified for the first time that CoV spherules are embedded with a complex and organised triple-layered protein lattice and that spherules always contain a cargo that appears arranged as a shell inside the vesicle. In addition, we confirmed the presence of a double membrane spanning molecular pore in Gammacoronavirus DMVs and further identified a population of potential DMV formation intermediates. Together, this work provides a foundation for understanding a fundamental yet unresolved aspect of coronavirus replication.

microbiology↗

bioRxiv2025

The PP1 phosphatase complex coordinates pre-mRNA 3end processing and termination of RNA polymerase II transcription

Phosphorylation plays a central role in coordinating transcription with pre-mRNA maturation, yet the mechanistic understanding of how transcription is regulated by phosphorylation remains limited. The PP1 phosphatase removes CDK9-dependent phosphorylation of Pol II and Spt5, triggering transcription termination. Here, we demonstrate that PNUTS enhances PP1 activity, whereas WDR82 mediates an interaction between PP1 and the pre-mRNA 3' end processing machinery subunit Symplekin. We present the first structure of a PNUTS-WDR82-Symplekin-Ssu72 complex, revealing that PNUTS and Symplekin bind WDR82 through distinct interfaces using conserved short linear motifs. Mutations that inactivate PP1s catalytic activity or disrupt its interaction with either PNUTS or the 3' end processing machinery led to impaired transcription elongation, inefficient cleavage of nascent transcripts, altered poly(A) site selection, and widespread transcription termination defects. Our findings demonstrate that the PP1 complex is essential for coupling transcription to pre-mRNA 3' end maturation, ensuring production of functional mRNA.

molecular biology↗

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↗

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↗

bioRxiv2022

Single-molecule analysis of receptor-beta-arrestin interactions in living cells

{beta}-arrestin plays a key role in G protein-coupled receptor (GPCR) signaling and desensitization. Despite recent structural advances, the mechanisms that govern receptor-{beta}-arrestin interactions at the plasma membrane of living cells remain elusive. Here, we combine single-molecule microscopy with molecular dynamics simulations to dissect the complex sequence of events involved in {beta}-arrestin interactions with both receptors and the lipid bilayer. In contrast to the currently widely accepted model, we show that {beta}-arrestin spontaneously inserts into the lipid bilayer and transiently interacts with receptors via lateral diffusion on the plasma membrane. Moreover, we show that following receptor interaction, the plasma membrane stabilizes {beta}-arrestin in a membrane-bound, active-like conformation, allowing it to diffuse to clathrin coated pits separately from the activating receptor. These results challenge our current understanding of {beta}-arrestin function at the plasma membrane, revealing a new critical role for {beta}-arrestin pre-association with the lipid bilayer in facilitating its interactions with receptors and subsequent activation.

cell biology↗

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↗