Biology subjects

Butcher, S. J.

Publications and source records attributed to Butcher, S. J..

11 recordsLinked to original sources

bioRxiv2026

Rapid plastid isolation reveals the chloroplast proteome and structures of the chlororibosome large subunit and RuBisCO in Marchantia polymorpha

Plastids house the biology of eukaryotic photosynthesis. While 1000s of plastid genomes have been sequenced, the availability of less than ten proteomes and only two species with full 70S plastid ribosomal structures limit our understanding of plant evolution. We optimized a protocol for the rapid isolation of Marchantia polymorpha plastids that provides a highly enriched and intact organelle fraction from gradient volumes as little as 2 mL. The approach was successfully applied to six other species. Focusing on M. polymorpha, we determined the proteome of the plastid fraction, identifying 1337 nuclear-encoded proteins with a high confidence, where 83% belong to orthologs shared with angiosperms. We further isolated protein complexes by RNA affinity purification using poly-lysine and provide the high-resolution structures of the 50S subunit of the chloroplast ribosome and RuBisCO using cryogenic EM and image reconstruction to 2.23 and 2.12 [A] resolution, respectively. For plastids, our data show that the genome reduction event experienced by the bryophyte common ancestor has had little impact on the organelles complexity and they underscore a high level of structural conservation of key components. Our data provide novel resources to explore the functional evolution of plastid proteomes and major macromolecular complexes of cyanobacterial origin.

plant biology↗

bioRxiv2026

The R2T(P) complex orchestrates the SHQ1 driven early steps of box H/ACA snoRNP maturation

The chaperones RuvBL1 and RuvBL2 are members of the AAA+ ATPase family and participate in diverse cellular processes, including DNA repair, transcriptional regulation, and assembly of macromolecular complexes such as snoRNPs. The biogenesis of box H/ACA snoRNPs additionally requires the assembly factor SHQ1. These protein-RNA complexes are essential for ribosome biogenesis and telomerase stability and are linked to diseases such as dyskeratosis congenita and cancer. Despite detailed knowledge of mature complexes, their assembly mechanisms and how they can be modulated remain unclear. We characterize a trimeric interaction between SHQ1 and RuvBL1:RuvBL2, providing insight into early maturation of the protein-only precursor of box H/ACA snoRNPs. SHQ1 binds the flexible domain II of RuvBL1:RuvBL2, corresponding to the dodecamerization interface, suggesting disruption of this interface and promotion of hexamer formation. We further purified a complex containing RuvBL1:RuvBL2, SHQ1, and DKC1, the catalytic component of H/ACA snoRNPs and a client of SHQ1. This demonstrates that SHQ1 and DKC1 can simultaneously associate with RuvBL1:RuvBL2, potentially facilitating DKC1 release and subsequent snoRNA binding. Additionally, we identified a direct interaction between SHQ1 and RPAP3, a co-chaperone of RuvBL1:RuvBL2 (within the R2TP). Hence, RPAP3 may be responsible for recruiting SHQ1:DKC1 to hexameric RuvBL1:RuvBL2 and/or assist in the AAA+ mediated dissociation of the dimer. Since SHQ1 shares a domain with PIH1D1, an integral member of the R2TP complex, our findings suggest that early H/ACA snoRNP maturation may involve the R2T instead of the previously proposed R2TP complex.

biophysics↗

bioRxiv2026

Structure determination and dual targeting of a plant TACO1 identifies its ancient role as an organelle translation regulator

Ribosome stalling caused by polyproline (PPs) motifs is common. Their translation is enhanced by accessory proteins such as YebC in bacteria, whose homolog, TRANSLATIONAL ACTIVATOR OF CYTOCHROME C OXIDASE 1 (TACO1), aids the translation of mitochondria-encoded proteins. The prevalence of PP motifs across plastid-encoded genes and their impact on the translation of photosynthesis-relevant proteins remains unexplored. Equally, a translation-enhancer of PP motifs equivalent to TACO1 for plastid ribosomes has not been reported. Here, we show that plastid genomes encode 24 proteins with a minimum of one PP motif on average, half of which are conserved in their cyanobacterial homologs, and that the vast majority of eukaryotes, including plants, encode a single TACO1 that we demonstrate to be dually targeted to mitochondria and plastids of Marchantia polymorpha. We resolved the MpTACO1 structure at 2.34 [A] by X-ray crystallography and the flexibility by small-angle X-ray scattering. Through modelling, we demonstrate that MpTACO1 can fit into the peptidyl transfer centre of plant chlororibosomes in a similar manner as human TACO1 in the mitoribosome. The identification and structure determination of the first plastid-targeted YebC/TACO1 allows us to sketch a unified model for the function and evolution of this ancient family of ribosomal accessory proteins, underscoring their indispensable role in the translation of bioenergetic membrane proteins reaching back almost 4 billion years. HighlightsO_LIDozens of GC-rich polyproline (PP) encoding regions are retained by AT-rich genomes C_LIO_LIPP motif conservation hints at regulatory mechanisms and required translation pauses C_LIO_LIChloroplast targeting of a (mitochondrial) translation enhancer of PP motifs C_LIO_LIMpTACO1 structure at 2.34 [A] resolution demonstrates its high level of conservation C_LI

plant biology↗

bioRxiv2024

The influence of the pre-membrane and envelope proteins on structure, pathogenicity and tropism of tick-borne encephalitis virus

Tick-borne encephalitis virus (TBEV) is a neurotropic flavivirus that causes thousands of human infections annually. Viral tropism in the brain is determined by the presence of necessary receptors, entry factors and the ability of the virus to overcome host defenses. The viral structural proteins, pre-membrane (prM) and envelope (E), play an important role in receptor binding, membrane fusion, particle maturation, and antibody neutralization. To understand how these proteins influence virus distribution and tropism in the brain, we generated a chimeric virus harboring the prM and ectodomain of E from TBEV in the background of the low pathogenic Langat virus (LGTV). We solved the atomic structures of both the chimeric virus and LGTV to compare them to the known TBEV structure. We show that this chimeric virus remains low-pathogenic, while being structurally and antigenically similar to TBEV. Using 3D optical whole brain imaging combined with immunohistochemistry, we found that both LGTV and the chimeric virus primarily infect cerebral cortex, with no significant differences in their localization or tropism. In contrast, TBEV shows high infection of the cerebellum and strong preference towards Purkinje cells, indicating that the non-structural proteins are important for determining TBEV tropism in the brain. Together, this provides new insights into the roles of the structural and non-structural proteins of tick-borne flaviviruses.

microbiology↗

bioRxiv2023

The α-dystroglycan N-terminus is a broad-spectrum antiviral agent against SARS-CoV-2 and enveloped viruses

The COVID-19 pandemic has shown the need to develop effective therapeutics in preparedness for further epidemics of virus infections that pose a significant threat to human health. As a natural compound antiviral candidate, we focused on -dystroglycan, a highly glycosylated basement membrane protein that links the extracellular matrix to the intracellular cytoskeleton. Here we show that the N-terminal fragment of -dystroglycan (-DGN), as produced in E. coli in the absence of post-translational modifications, blocks infection of SARS-CoV-2 in cell culture, human primary gut organoids and the lungs of transgenic mice expressing the human receptor angiotensin I-converting enzyme 2 (hACE2). Prophylactic and therapeutic administration of -DGN reduced SARS-CoV-2 lung titres and protected the mice from respiratory symptoms and death. Recombinant -DGN also blocked infection of a wide range of enveloped viruses including the four Dengue virus serotypes, influenza A virus, respiratory syncytial virus, tick-borne encephalitis virus, but not human adenovirus, a non-enveloped virus in vitro. This study establishes soluble recombinant -DGN as a broad-band, natural compound candidate therapeutic against enveloped viruses.

microbiology↗

bioRxiv2023

The structure of immature tick-borne encephalitis virus

Tick-borne encephalitis virus (TBEV) is a medically important flavivirus that poses a significant health threat in Europe and Asia. However, the structure of the immature form of TBEV remains unknown. Here, we employed state-of-the-art cryogenic electron microscopy (cryoEM) to determine the structure of the immature TBEV particle. The immature TBEV particle has a diameter of 56 nm and its surface glycoproteins are organised into spikes characteristic of immature flaviviruses. The cryoEM reconstructions of the whole virus and of the individual spike enabled us to build atomic models of the major viral components, the E and prM proteins. The insights obtained from our study provide a foundation for understanding the early stages of TBEV assembly and maturation. The pr domains of prM have a critical role in holding the heterohexameric prM3E3 spikes in metastable conformation. Destabilisation of the prM furin-sensitive loop at acidic pH facilitates its processing. The prM cleavage, the collapse of E protein ectodomains onto the virion surface concurrent with significant movement of the membrane domains of both E and M, and release of the pr fragment from the particle render the virus mature and infectious. This knowledge contributes to our understanding of the flavivirus life cycle.

microbiology↗

bioRxiv2022

Simultaneous Membrane and RNA Binding by Tick-Borne Encephalitis Virus Capsid Protein

Tick-borne encephalitis virus is an enveloped, pathogenic, RNA virus in the family Flaviviridae, genus Flavivirus. Viral particles are formed when the nucleocapsid, consisting of an RNA genome and multiple copies of the capsid protein, buds through the endoplasmic reticulum membrane and acquires the viral envelope and the associated proteins. The coordination of the nucleocapsid components to the sites of assembly and budding are poorly understood. Here, we investigate nucleocapsid assembly by characterizing the interactions of the wild-type and truncated capsid proteins with membranes by using biophysical methods and model membrane systems. We show that capsid protein initially binds membranes via electrostatic interactions with negatively-charged lipids which is followed by membrane insertion. Additionally, we show that membrane-bound capsid protein can recruit viral genomic RNA. We confirm the biological relevance of the biophysical findings by using mass spectrometry to show that purified virions contain negatively-charged lipids. Our results suggest that nucleocapsid assembly is coordinated by negatively-charged membrane patches on the endoplasmic reticulum and that the capsid protein mediates direct contacts between the nucleocapsid and the membrane.

biochemistry↗

bioRxiv2022

Structural studies reveal that endosomal cations promote formation of infectious CVA9 A particles, facilitating RNA and VP4 release.

Coxsackievirus A9, an enterovirus, is a common cause of paediatric aseptic meningitis and neonatal sepsis. During cell entry, enterovirus capsids undergo conformational changes leading to expansion, formation of large pores, externalization of VP1 N-termini and loss of the lipid factor from VP1. Factors such as receptor binding, heat, and acidic pH can trigger capsid expansion in some enteroviruses. Here we show that fatty-acid free bovine serum albumin or neutral endosomal ionic conditions can independently prime CVA9 for expansion and genome release. Our results show that CVA9 treatment with albumin or endosomal ions generates a heterogeneous population of virions, which could be physically separated by asymmetric flow field flow fractionation and computationally by cryo-EM and image processing. We report cryo-EM structures of CVA9 A-particles obtained by albumin or endosomal ion treatment and a control non-expanded virion to 3.5, 3.3 and 2.9 [A] resolutions, respectively. Where albumin promotes stabile expanded virions, the endosomal ionic concentrations induce unstable CVA9 virions which easily disintegrate losing their genome. Loss of most of the VP4 molecules and exposure of negatively-charged amino acid residues in the capsids interior after expansion, create a repulsive viral RNA-capsid interface, aiding genome release. ImportanceCoxsackievirus A9 (CVA9) is a common cause of meningitis and neonatal sepsis. The triggers and mode of action of RNA release into the cell unusually do not require receptor interaction. Rather, a slow process in the endosome, independent of low pH is required. Here, we show by biophysical separation, cryogenic electron microscopy and image reconstruction that albumin and buffers mimicking the endosomal ion composition can separately and together expand and prime CVA9 for uncoating. Furthermore, we show in these expanded particles that VP4 is present at only ~10% of the occupancy found in the virion, VP1 is externalised and the genome is repelled by the negatively-charged, repulsive inner surface of the capsid that occurs due to the expansion. Thus, we can now link observations from cell biology of infection with the physical processes that occur in the capsid to promote genome uncoating.

microbiology↗

bioRxiv2021

Proteome-wide cross-linking mass spectrometry to identify specific virus capsid-host interactions between tick-borne encephalitis virus and neuroblastoma cells

Virus-host protein-protein interactions are central to viral infection, but are challenging to identify and characterise, especially in complex systems involving intact viruses and cells. In this work, we demonstrate a proteome-wide approach to identify virus-host interactions using chemical cross-linking coupled with mass spectrometry. We adsorbed tick-borne encephalitis virus onto metabolically-stalled neuroblastoma cells, covalently cross-linked interacting virus-host proteins, and performed limited proteolysis to release primarily the surface-exposed proteins for identification by mass spectrometry. Using the intraviral protein cross-links as an internal control to assess cross-link confidence levels, we identified 22 high confidence unique intraviral cross-links and 59 high confidence unique virus-host protein-protein interactions. The identified host proteins were shown to interact with eight distinct sites on the outer surface of the virus. Notably, we identified an interaction between the substrate-binding domain of heat shock protein family A member 5, an entry receptor for four related flaviviruses, and the hinge region of the viral envelope protein. We also identified host proteins involved in endocytosis, cytoskeletal rearrangement, or located in the cytoskeleton, suggesting that entry mechanisms for tick-borne encephalitis virus could include both clathrin-mediated endocytosis and macropinocytosis. Additionally, cross-linking of the viral proteins showed that the capsid protein forms dimers within tick-borne encephalitis virus, as previously observed with purified C proteins for other flaviviruses. This method enables the identification and mapping of transient virus-host interactions, under near-physiological conditions, without the need for genetic manipulation. Author summaryTick-borne encephalitis virus is an important human pathogen that can cause severe infection often resulting in life-long neurological complications or even death. As with other viruses, it fully relies on the host cells, and any successful infection starts with interactions between the viral structural proteins and cellular surface proteins. Mapping these interactions is essential both for the fundamental understanding of viral entry mechanisms, and for guiding the design of new antiviral drugs and vaccines. Here, we stabilise the interactions between tick-borne encephalitis virus and human proteins by chemical cross-linking. We then detect the interactions using mass spectrometry and analyse the data to identify protein-protein complexes. We demonstrate that we can visualise the protein interaction interfaces by mapping the cross-linked sites onto the host and viral protein structures. We reveal that there are eight distinct sites on the outer surface of the viral envelope protein that interact with host. Using this approach, we mapped interactions between the tick-borne encephalitis virus envelope protein, and 59 host proteins, identifying a possible new virus receptor. These results highlight the potential of chemical cross-linking coupled with mass spectrometry to identify and map interactions between viral and host proteins.

microbiology↗

bioRxiv2020

Identification of a conserved virion-stabilizing network inside the interprotomer pocket of enteroviruses

Major efforts have been underway to develop broad-spectrum high potency capsid binders that inhibit the life cycle of enteroviruses, a large group (family Picornaviridae) whose members include poliovirus, coxsackieviruses, echoviruses, numbered enteroviruses, and rhinoviruses. These diverse viruses cause a wide variety of illnesses, ranging from the mild common cold to hand-foot-and-mouth disease, myocarditis, pancreatitis, aseptic meningitis, and encephalitis. So-called classical capsid binders target a surface exposed hydrophobic pocket in one of the viral coat proteins (VP1) to prevent the genome uncoating process. However, efficacy, toxicity, emergence of drug-resistant viruses, and existence of certain enteroviral species that lack the VP1 pocket limit their clinical benefit. Recently, we identified a new druggable site at a conserved interface formed by multiple capsid proteins, the VP1-VP3 interprotomer pocket. To further study the properties that confer druggability at this site, we have determined high-resolution cryo-electron microscopy structures of two enteroviruses, coxsackieviruses B3 and B4, complexed with interprotomer-targeting compounds, CP17 and CP48 respectively. Until now, there has been no structure available for Coxsackievirus B4 despite the fact that the virus has long been implicated in the development of insulin-dependent diabetes mellitus. At better than 3 Å resolution, we could identify the detailed interactions that facilitate ligand binding. Both compounds target the same three conserved residues, each from a different polypeptide chain, to form a virion-stabilizing network inside the pocket. We measured the in silico binding energy for both inhibitors when anchored to the network and found a global stabilizing effect on the order of thousands of kcal/mol under saturating conditions (60 total sites per virion). Intriguingly, a recent X-ray structure has revealed that glutathione targets the same network within the interprotomer site of bovine enterovirus F3, where it is thought to facilitate virus assembly. In summary, our findings provide the structural basis for how a newly designed class of capsid binders target and stabilize enteroviruses. Future efforts to chemically optimize drugs for enhanced targeting to the interprotomer pocket is a promising endeavor in the fight against enteroviruses, especially given the possibility of synergistic effects when used in combination with classical VP1 binders like pleconaril.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biophysics↗

bioRxiv2018

2.8 A resolution cryo-EM structure of human parechovirus 3 in complex with Fab from a neutralizing antibody

Human parechovirus 3 (HPeV3) infection is associated with sepsis in neonates characterized by significant immune activation and subsequent tissue damage. Strategies to limit infection have been unsuccessful due to inadequate molecular diagnostic tools for early detection and lack of a vaccine or specific antiviral therapy. Towards the latter, we present a 2.8 [A]-resolution structure of HPeV3 in complex with fragments from a neutralizing human monoclonal antibody AT12-015 using cryo-EM and image reconstruction. Modeling revealed that the epitope extends across neighboring asymmetric units with contributions from capsid proteins VP0, VP1, and VP3. Antibody decoration was found to block binding of HPeV3 to cultured cells. Additionally at high-resolution, it was possible to model a stretch of RNA inside the virion and from this identify the key features that drive and stabilize protein-RNA association during assembly.\n\nImportance\n\nHPeV3 is receiving increasing attention as a prevalent cause of sepsis-like symptoms in neonates, which despite the severity of disease, there are no effective treatments available. Structural and molecular insights into virus neutralization are urgently needed, especially as clinical cases are on the rise. Towards this goal, we present the first structure of HPeV3 in complex with fragments from a neutralizing monoclonal antibody. At high-resolution it was possible to precisely define the epitope that when targeted, prevents virions from binding to cells. Such an atomic-level description is useful for understanding host-pathogen interaction, viral pathogenesis mechanisms, and for finding potential cures for infection and disease.

microbiology↗