Biology subjects

Tuthill, T. J.

Publications and source records attributed to Tuthill, T. J..

11 recordsLinked to original sources

bioRxiv2025

The assembly of stress granule-like foci during foot-and-mouth disease virus infection is uncoupled from activation of cellular intrinsic antiviral signalling

Foot-and-mouth disease virus (FMDV) is highly contagious among cloven-hoofed animals and poses a major threat to the livestock industry worldwide. A fundamental gap in knowledge for high consequence viruses such as FMDV is understanding how the virus evolved to evade cellular antiviral responses. FMDV belongs to the Picornaviridae, a family of positive-sense single-stranded RNA viruses. The detection of viral double-stranded viral RNA intermediates during infection can trigger both the assembly of cytoplasmic stress granules (SGs) and the activation of the RIG-I-like receptors (RLR)-mediated innate immune response (IIR). FMDV has been proposed to antagonize these mechanisms, suggesting that both can limit viral replication. In this study, we investigate the dynamic and importance of SG assembly for IIR activation upon dsRNA stimulation or FMDV replication in porcine epithelial kidney cells. First, we show that the formation of SG following a challenge with poly(I:C), a viral dsRNA mimic, does not modulate the activation of IIR. Our data further reveal transient assembly of SG during FMDV infection followed by virus-induced cleavage of G3BP1, a core SG protein. While SG assembly does not impact viral replication or antiviral response activation, we demonstrate that preventing their disassembly negatively impacts FMDV replication. Overall, our data suggests that while SGs assembly is uncoupled from IIR activation, manipulating their disassembly limits replication and might serve as a potential therapeutic target to prevent FMDV infection. ImportanceBiomolecular condensates, including stress granules, are key regulatory compartments that control fundamental cellular processes. By condensing RNAs and proteins, these structures enable cells to rapidly adapt to stress, such as viral infection. Different biomolecular condensates with specific dynamics or compositions have been described during infection, and many viruses are known to disrupt or highjack their components. Moreover, the cell-autonomous innate immune response is proposed to be regulated by biomolecular condensates. However, the molecular mechanisms underpinning these functions remain unclear and controversial. Here we investigated the interplay between stress granules and the innate response upon the stimulation with a viral dsRNA mimic or foot-and-mouth disease virus infection. We demonstrate that infection triggers the formation of stress granules independently from activation of innate signalling pathways. We also show that stress granules persistence attenuates viral replication posing them as part of the cells response that viruses must overcome or subvert to replicate.

microbiology↗

bioRxiv2025

ITAF45 is a Pervasive Trans Acting Factor for Picornavirus Type II IRES Elements

Viruses have evolved elaborate mechanisms to hijack the host mRNA translation machinery to direct viral protein synthesis. Picornaviruses, whose RNA genomes lack a cap structure, inhibit cap-dependent mRNA translation, and utilize an internal ribosome entry site (IRES) in the RNA 5'-UTR to recruit the 40S ribosomal subunit. IRES activity is stimulated by a set of host proteins termed IRES trans-acting factors (ITAFs). The cellular protein ITAF45 (also known as PA2G4 and EBP1) was identified as an essential ITAF for foot-and-mouth disease virus (FMDV), with no apparent role in cell-free systems for the closely related viruses harboring similar IRES elements such as encephalomyocarditis virus (EMCV) and Theilers murine encephalomyelitis virus (TMEV). Here, we demonstrate that ITAF45 is a pervasive host factor within cells for picornaviruses containing a Type II IRES. CRISPR/Cas9 knockout of ITAF45 in several human cell lines conferred resistance to infection with FMDV, EMCV, TMEV, and equine rhinitis A virus (ERAV). We show that ITAF45 enhances initiation of translation on type II IRESs in cell line models. This is mediated by the C-terminal lysine-rich region of ITAF45 known to enable binding to viral RNA. These findings challenge previous reports of a unique role for ITAF45 in FMDV infection, positioning ITAF45 as a promising antiviral target for various animal viruses and emerging human cardioviruses.

microbiology↗

bioRxiv2024

Induction of neutralising antibodies against conserved rhinovirus capsid protein VP4 depends on presenting VP4 in a virus-like conformation

Infection with rhinovirus (RV) is associated with significant morbidity and hospitalisation in people with chronic lung disease. At present there is no approved RV vaccine or antiviral. There are approximately 180 RV serotypes, classified into 3 species (RVA, RVB, RVC). There is no cross-protection between serotypes because of high diversity in immunodominant antigenic sites. This makes it impractical to create a broadly protective vaccine using traditional methods, involving whole capsids. A more feasible strategy is to direct the immune response towards conserved but less dominant epitopes. The capsid protein VP4 is highly conserved within each RV genotype and some antibodies that target VP4 are neutralising. This makes VP4 vulnerable to antibodies and a promising vaccine target. Here we investigate the ability of RV VP4 N-terminal peptides presented on different display systems to initiate a neutralising immune response in mice. We compared three different-sized VP4 peptides (spanning residues, 1-15, 1-30 and 1-45) displayed on two different display systems-SpyCatcher Virus-like particles (VLPs) or Keyhole Limpet Hemocyanin (KLH). Overlapping regions of VP4 were antigenically different when presented on different platforms and in peptides with different length. The conformation of the 1-15 region of VP4 was critical for inducing antibodies that were both neutralising and able to bind VP4 in the context of the virus particle. Therefore, correctly displayed VP4 peptides can recapitulate virus-like antigenicity. These findings improve our understanding of VP4 antigenicity and will inform the design of future RV vaccines. This work could also impact the design of other peptide vaccines, since a variable antigenic conformation is a common characteristic of pathogen-derived peptide targets. Impact statementRhinovirus (RV) is a highly prevalent respiratory virus that is a frequent cause of common cold symptoms. For people with chronic lung diseases such as asthma, infection can cause significant worsening of disease, leading to increased suffering and hospitalisation. There are currently no vaccines or anti-viral drugs that target RV infections. Development of a RV vaccine would be hugely beneficial to suffers of chronic lung diseases. Attempts to develop a broadly protective RV vaccine have been hindered by the presence of over 180 serotypes which have little or no natural cross-reactivity. Previous studies have identified the highly conserved VP4 epitope as a potential target for broadly protective vaccines. However, there has been little progress in evaluating its effectiveness as a vaccine candidate. In this study we assessed the ability of different RV VP4 immunogens to induce neutralising antibodies in mice. We demonstrated that antigenic conformation of VP4 varies greatly between immunogens and presentation of the first 15 N-terminal residues in a virus-like conformation is important for generating neutralising antibodies. This knowledge could help to maximise the production of neutralising VP4-specific antibodies in future studies which could ultimately lead to a universal RV vaccine.

microbiology↗

bioRxiv2024

The pseudoknot region and poly-(C) tract comprise an essential RNA packaging signal for assembly of foot-and-mouth disease virus

Virus assembly is a crucial step for the completion of the viral replication cycle. In addition to ensuring efficient incorporation of viral genomes into nascent virions, high specificity is required to prevent incorporation of host nucleic acids. For picornaviruses, including FMDV, the mechanisms required to fulfil these requirements are not well understood. However, recent evidence has suggested that specific RNA sequences dispersed throughout picornavirus genomes are involved in packaging. Here, we have shown that such sequences are essential for FMDV RNA packaging and have demonstrated roles for both the pseudoknot (PK) region and the poly-(C) tract in this process, where the length of the poly-(C) tract was found to influence the efficiency of RNA encapsidation. Sub-genomic replicons containing longer poly-(C) tracts were packaged with greater efficiency in trans, and viruses recovered from transcripts containing short poly-(C) tracts were found to have greatly extended poly-(C) tracts after only a single passage in cells, suggesting that maintaining a long poly-(C) tract provides a selective advantage. We also characterised a critical packaging signal (PS) located in the pseudoknot (PK) region, adjacent to the poly-(C) tract, as well as several other non-essential but beneficial PSs elsewhere in the genome. Collectively, these PSs greatly enhanced encapsidation efficiency, with the poly-(C) tract possibly facilitating nearby PSs to adopt the correct conformation. Using these data, we have proposed a model where interactions with capsid precursors control a transition between two RNA conformations, directing the fate of nascent genomes to either be packaged or alternatively to act as templates for replication and/or for protein translation. Author summaryGenome packaging, whereby viral RNA is incorporated into protective protein capsids to produce more virus particles, is a crucial step in RNA virus life cycles. It is a stringent process as only viral RNA is encapsidated, while cellular RNA is excluded. This study reveals the essential role of packaging signals in FMDV RNA packaging, specifically those in the pseudoknot region and in a region that can contain >100 cytosines, termed the poly-(C) tract. We demonstrate that the length of the poly-(C) tract significantly affects packaging efficiency; genomes containing longer poly-(C) tracts are favoured. This is the first role that has been identified for the poly-(C) tract in FMDV. We have also found an essential packaging signal in the pseudoknot region, which is assisted by other packaging signals located throughout the genome, that together facilitate encapsidation of FMDV RNA. These results provide compelling evidence for the involvement of RNA packaging signals in FMDV assembly. Based on this, we propose a simple model for FMDV RNA packaging, which involves a transition from genome replication to genome packaging and is controlled by packaging signals. This knowledge could pave the way for future research and development of novel antiviral strategies targeting FMDV and other picornaviruses.

microbiology↗

bioRxiv2023

Genetic evidence of a functional linkage between the RNA-dependent RNA polymerase and the highly structured S fragment located at the 5' end of the genome of foot-and-mouth disease virus.

Secondary and tertiary RNA structures play key roles in genome replication of single stranded positive sense RNA viruses. Complex, functional structures are particularly abundant in the untranslated regions of picornaviruses, where they are involved in initiation of translation, priming of new strand synthesis and genome circularisation. The 5' UTR of foot-and-mouth disease virus (FMDV) is predicted to include a c. 360 nucleotide-long stem-loop, termed the short (S) fragment. This structure is highly conserved and essential for viral replication, but the precise function(s) are unclear. Here, we used selective 2' hydroxyl acetylation analysed by primer extension (SHAPE) to experimentally-determine aspects of the structure, alongside comparative genomic analyses to confirm structure conservation from a wide range of field isolates. To examine its role in virus replication, we introduced a series of deletions to the distal and proximal regions of the stem loop. These truncations affected genome replication in a size-dependent and, in some cases, host cell-dependent manner. Furthermore, during passage of viruses incorporating the largest tolerated deletion from the proximal region of the S fragment stem loop, an additional mutation was selected in the viral RNA-dependent RNA polymerase, 3Dpol.These data suggest that the S fragment and 3Dpol interact in the formation of the FMDV replication complex.

molecular biology↗

bioRxiv2022

Membrane interactions and uncoating of Aichi virus, a picornavirus that lacks a VP4

Kobuviruses are an unusual and poorly characterised genus within the picornavirus family, and can cause gastrointestinal enteric disease in humans, livestock and pets. The human Kobuvirus, Aichi virus (AiV) can cause severe gastroenteritis and deaths in children below the age of five years, however this is a very rare occurrence. During the assembly of most picornaviruses (e.g. poliovirus, rhinovirus and foot-and-mouth disease virus), the capsid precursor protein VP0 is cleaved into VP4 and VP2. However, Kobuviruses retain an uncleaved VP0. From studies with other picornaviruses, it is known that VP4 performs the essential function of pore formation in membranes, which facilitates transfer of the viral genome across the endosomal membrane and into the cytoplasm for replication. Here, we employ genome exposure and membrane interaction assays to demonstrate that pH plays a critical role in AiV uncoating and membrane interactions. We demonstrate that incubation at low pH alters the exposure of hydrophobic residues within the capsid, enhances genome exposure and enhances permeabilisation of model membranes. Furthermore, using peptides we demonstrate that the N-terminus of VP0 mediates membrane pore formation in model membranes, indicating that this plays an analogous function to VP4. ImportanceTo initiate infection, viruses must enter a host cell and deliver their genome into the appropriate location. The picornavirus family of small non-enveloped RNA viruses includes significant human and animal pathogens and are also models to understand the process of cell entry. Most picornavirus capsids contain the internal protein VP4, generated from cleavage of a VP0 precursor. During entry, VP4 is released from the capsid. In enteroviruses this forms a membrane pore, which facilitates genome release into the cytoplasm. Due to high levels of sequence similarity, it is expected to play the same role for other picornaviruses. Some picornaviruses, such as Aichi virus, retain an intact VP0, and it is unknown how these viruses re-arrange their capsids and induce membrane permeability in the absence of VP4. Here we have used Aichi virus as a model VP0 virus to test for conservation of function between VP0 and VP4. This could enhance understanding of pore function and lead to development of novel therapeutic agents that block entry.

microbiology↗

bioRxiv2021

Mutagenesis Mapping of RNA Structures within the Foot-and-Mouth Disease Virus Genome Reveals Functional Elements Localised in the Polymerase (3Dpol) Encoding Region.

AbstractRNA structure plays a crucial role in the replication of positive sense RNA viruses and can form functional elements within the untranslated regions (UTRs) and the protein coding sequences (or open reading frames (ORFs)). While RNA structures in the UTRs of several picornaviruses have been functionally characterised, the roles of putative RNA structures predicted for the ORF remain largely undefined. Here we have undertaken a bioinformatic analysis of the foot-and-mouth disease virus (FMDV) genome and predicted the existence of 53 evolutionarily conserved RNA structures within the ORF. Forty-five (45) of these structures were located in the regions encoding the non-structural proteins (nsps). To investigate if the structures in the regions encoding the nsps are required for FMDV replication we used a mutagenesis method, CDLR mapping, where sequential coding segments were shuffled to minimise RNA secondary structures while preserving protein coding, native dinucleotide frequencies and codon usage. To examine the impact of these changes on replicative fitness, mutated sequences were inserted into an FMDV sub-genomic replicon. We found that three of the RNA structures, all at the 3 termini of the FMDV ORF, were critical for replicon replication. Contrastingly, disruption of the other 42 conserved RNA structures that lie within the regions encoding the nsps had no effect on replicon replication, suggesting that these structures are not required for initiating translation or replication of viral RNA. Conserved RNA structures that are not essential for virus replication could provide ideal targets for the rational attenuation of a wide range of FMDV strains. ImportanceSome RNA structures formed by the genomes of RNA viruses are critical for viral replication. Our study shows that of 45 conserved RNA structures located within the regions of the foot-and-mouth disease virus (FMDV) genome that encode the non-structural proteins, only three are essential for replication of an FMDV sub-genomic replicon. Replicons replication is dependent on RNA translation and synthesis; thus, our results suggest that the three RNA structures are critical for either initiation of viral RNA translation and/or viral RNA synthesis. Although further studies are required to identify if the remaining 42 RNA structures have other roles in virus replication, they may provide targets for the rational large-scale attenuation of a wide range of FMDV strains. FMDV causes a highly contagious disease posing a constant threat to global livestock industries. Such weakened FMDV strains could be investigated as live-attenuated vaccines or could enhance biosecurity of conventional inactivated vaccine production.

evolutionary biology↗

bioRxiv2020

A COVID-19 vaccine candidate using SpyCatcher multimerization of the SARS-CoV-2 spike protein receptor-binding domain induces potent neutralising antibody responses

There is dire need for an effective and affordable vaccine against SARS-CoV-2 to tackle the ongoing pandemic. In this study, we describe a modular virus-like particle vaccine candidate displaying the SARS-CoV-2 spike glycoprotein receptor-binding domain (RBD) using SpyTag/SpyCatcher technology (RBD-SpyVLP). Low doses of RBD-SpyVLP in a prime-boost regimen induced a strong neutralising antibody response in mice and pigs that was superior to convalescent human sera. We evaluated antibody quality using ACE2 blocking and neutralisation of cell infection by pseudovirus or wild-type SARS-CoV-2. Using competition assays with a monoclonal antibody panel, we showed that RBD-SpyVLP induced a polyclonal antibody response that recognised all key epitopes on the RBD, reducing the likelihood of selecting neutralisation-escape mutants. The induction of potent and polyclonal antibody responses by RBD-SpyVLP provides strong potential to address clinical and logistic challenges of the COVID-19 pandemic. Moreover, RBD-SpyVLP is highly resilient, thermostable and can be lyophilised without losing immunogenicity, to facilitate global distribution and reduce cold-chain dependence.

immunology↗

bioRxiv2020

Evaluation of the immunogenicity of prime-boost vaccination with the replication-deficient viral vectored COVID-19 vaccine candidate ChAdOx1 nCoV-19

Clinical development of the COVID-19 vaccine candidate ChAdOx1 nCoV-19, a replication-deficient simian adenoviral vector expressing the full-length SARS-CoV-2 spike (S) protein was initiated in April 2020 following non-human primate studies using a single immunisation. Here, we compared the immunogenicity of one or two doses of ChAdOx1 nCoV-19 in both mice and pigs. Whilst a single dose induced antigen-specific antibody and T cells responses, a booster immunisation enhanced antibody responses, particularly in pigs, with a significant increase in SARS-CoV-2 neutralising titres.

immunology↗

bioRxiv2020

The RNA pseudoknots in foot-and-mouth disease virus are dispensable for genome replication but essential for the production of infectious virus.

The positive stranded RNA genomes of picornaviruses comprise a single large open reading frame flanked by 5' and 3' untranslated regions (UTRs). Foot-and-mouth disease virus (FMDV) has an unusually large 5' UTR (1.3 kb) containing five structural domains. These include the internal ribosome entry site (IRES), which facilitates initiation of translation, and the cis-acting replication element (cre). Less well characterised structures are a 5' terminal 360 nucleotide stem-loop, a variable length poly-C-tract of approximately 100-200 nucleotides and a series of two to four tandemly repeated pseudoknots (PKs). We investigated the structures of the PKs by selective 2' hydroxyl acetylation analysed by primer extension (SHAPE) analysis and determined their contribution to genome replication by mutation and deletion experiments. SHAPE and mutation experiments confirmed the importance of the previously predicted PK structures for their function. Deletion experiments showed that although PKs are not essential for replication, they provide genomes with a competitive advantage. However, although replicons and full-length genomes lacking all PKs were replication competent, no infectious virus was rescued from genomes containing less than one PK copy. This is consistent with our earlier report describing the presence of putative packaging signals in the PK region.

molecular biology↗

bioRxiv2019

Detection of antibodies against a conserved capsid epitope as the basis of a novel universal serological test for foot-and-mouth disease

Diagnostic tests for foot-and-mouth disease (FMD) include the detection of antibodies against either the viral non-structural proteins or the capsid. The detection of antibodies against the structural proteins (SP) of the capsid can be used to monitor seroconversion in both infected and vaccinated animals. However, SP tests need to be tailored to the individual FMD virus serotype and their sensitivity performances may be affected by antigenic variability within each serotype and mismatching between tests reagents. As a consequence, FMD Reference Laboratories need to maintain contingency to employ multiple type-specific assays for large-scale serological surveillance and post-vaccination monitoring in the event of FMD outbreaks. In this study, a highly conserved region in the N terminus of FMDV capsid protein VP2 (VP2N) was characterised using a panel of intertypic-reactive monoclonal antibodies. This revealed a universal epitope in VP2N which could be used as a peptide antigen to detect FMDV-specific antibodies against all types of the virus. A VP2-peptide ELISA (VP2-ELISA) was optimised using experimental and reference antisera from immunized, convalescent and negative animals (n=172). The VP2-ELISA is universal, simple and provided sensitive (98.6 %) and specific (93%) detection of antibodies to all FMDV strains used in this study. We anticipate that this SP test could have utility for sero-surveillance during virus incursions in FMD-free countries and as an additional screening tool to assess FMD virus circulation in endemic countries.

immunology↗