Biology subjects

Herod, M. R.

Publications and source records attributed to Herod, M. R..

15 recordsLinked to original sources

bioRxiv2026

The proteasome and ERAD pathways can regulate hepatitis E virus pORF1 proteostasis and genome replication

Hepatitis E virus (HEV) is a major cause of acute viral hepatitis, with an estimated 20 million human infections annually. Its diverse host range presents a significant zoonotic threat, yet the molecular mechanisms underpinning HEV-host interactions remain poorly characterised. Here, we used transposon-based insertion screening of the HEV pORF1 polyprotein to identify a novel region within the viral PCP domain that permits HA-epitope insertion, enabling the first epitope tagging of this domain. Combined with V5 tagging of the hypervariable region (HVR), this approach enabled the generation of a replication-competent stable cell line expressing dual-tagged ORF1. Co-immunoprecipitation and TMT-based LC-MS proteomics revealed that pORF1 interacts with host proteins involved in endoplasmic reticulum-associated degradation (ERAD) and the 26S proteasome. Pharmacological inhibition demonstrated that both pathways are required for efficient HEV replication. Treatment with the FDA-approved proteasome inhibitor bortezomib altered pORF1 precursor abundance and led to loss of full-length pORF1 through enhanced proteolysis. Importantly, the kinetics of ORF1 degradation following proteasome inhibition correlated with immediate loss of HEV replication, demonstrating a functional link between pORF1 proteostasis and viral replication. Collectively, our findings identify the ERAD network as a previously unrecognised requirement for HEV replication and establish a role for the 26S proteasome in maintaining pORF1 proteostasis.

microbiology↗

bioRxiv2026

Enhanced inter-chain hydrogen bonding in the murine norovirus VP1 capsid leads to increased particle stability and delayed viral uncoating

Capsid stability is vital for virion survival as the capsid must withstand varying environmental challenges such as pH and temperature to allow the virus to reach a target cell. Noroviruses are non-enveloped, icosahedral, positive-sense RNA viruses of importance to human health globally, with no approved vaccine or antiviral available. Despite this, the molecular mechanisms behind norovirus capsid stability and capsid rearrangement prior to RNA translocation are understudied. Using murine norovirus as a model, we utilised thermal stress to create a thermally stable virus population. By introducing three identified substitutions in the major capsid protein VP1 from this virus population into an infectious clone, we were able to create a heat and pH stable virus that had delayed viral uncoating during the infectious lifecycle. Cryo-EM reconstructions of the triple substitution virus demonstrated that enhanced inter-chain hydrogen bonding was vital for increased capsid stability. Finally, mutagenesis to remove the enhanced inter-chain hydrogen bonding reverted capsid stability back to wild-type levels. This work contributes to fundamental calicivirus biology by demonstrating areas of importance in capsid stability down to amino acid resolution. Furthermore, this work could inform vaccine design for a thermostable norovirus vaccine in the future. Author SummaryNoroviruses cause gastroenteritis and globally contribute to the death of up to 250,000 people worldwide per annum and an estimated healthcare cost of $4 billion. Despite this, there is no vaccine or antiviral treatment available, thus more work needs to be conducted to understand the mechanisms that underpin the viral life cycle. The norovirus capsid is a meta-stable shell-like structure evolved to protect the viral RNA from the harsh external environment, until cellular triggers allow genome release to initiate infection of a host cell. However, the molecular interactions that are key for controlling this balance are relatively unstudied. In this report, we identify that hydrogen bonding at the capsid protomer-protomer interface are vital for maintaining this balance, with increased inter-molecular hydrogen bonding at three specific amino acids able to increase viral stability whilst still permitting infectious genome release. Furthermore, reducing inter-molecular hydrogen bonding at these crucial amino acids was able to reverse this mechanism. These results should inform future norovirus vaccine studies, where thermostable virus-like-particles are needed to overcome issues with cold-chain storage.

microbiology↗

bioRxiv2025

Interactions between the 2C protein of FMDV and components of the viral replication machinery are mediated by ER-derived membranes

Foot-and-mouth disease virus (FMDV) remains an ever-present threat to the economic stability of the livestock industry and global trade. Despite this, questions remain regarding the fundamental biology underpinning the replication of this virus. Here, we examine components of the FMDV replication machinery (focussing on the viral 2C protein) and investigate the conditions under which they interact. Using a novel 2C antibody in co-immunoprecipitation experiments under different conditions followed by mass spectrometry, we identify membrane-associated proteins (such as the viral proteins 2B and precursors of 3A, which are poorly-characterised proteins involved in viral replication) along with ER-associated host proteins. In addition, our analysis shows that a number of nuclear factors interact with 2C in a membrane-independent manner, potentially being co-opted to support RNA replication of the virus. Furthermore, we demonstrate that the interaction of 2C with several viral proteins (including key members of the replication machinery and viral RNA) is maintained following ultracentrifugation, suggesting that these co-sediment as part of a complex. Our data suggest that the replication complex is ER-derived and highlight several new avenues of investigation for the disruption of the FMDV lifecycle.

microbiology↗

bioRxiv2025

Integrative Structure of Norovirus NS3 Suggests a Role in RNA Transport

Human noroviruses (HuNoVs) are the leading global cause of non-bacterial gastroenteritis, yet no vaccines or antiviral therapies are currently approved1. The viral non-structural protein NS3 is a membrane-bound AAA+ ATPase of superfamily 3 (SF3) with multiple proposed roles in the norovirus replication cycle. However, the structure of NS3, and the mechanisms by which it contributes to genome replication and membrane remodeling, have remained unknown. We engineered a soluble, hexameric, and catalytically active form of NS3 and determined its cryo-EM structure in the presence of a nucleotide analogue at 2.9 [A] resolution. The structure adopts a split lock-washer architecture characteristic of AAA+ motors that operate via a hand-over-hand translocation mechanism2. Modeling of the nucleotide-binding site reveals conserved features governing ATP binding and hydrolysis. Using integrative modeling with AlphaFold3, we generated a full-length, membrane-associated model of NS3, in which an N-terminal transmembrane domain, central helical bundle, and AAA+ motor form a continuous conduit. This model supports a role for NS3 as a candidate membrane-spanning RNA translocase that couples ATP hydrolysis to genome movement. This structural framework helps address long-standing gaps in our understanding of norovirus replication and establishes a basis for mechanistic studies and structure-guided antiviral design.

microbiology↗

bioRxiv2024

Select autosomal dominant DFNA11 deafness mutations activate Myo7A in epithelial cells

Myosin-7A (Myo7A) is a motor protein crucial for the organization and function of stereocilia, specialized actin-rich protrusions on the surface of inner ear hair cells that mediate hearing. Mutations in Myo7A cause several forms of genetic hearing loss, including autosomal dominant DFNA11 deafness. Despite its importance, the structural elements of Myo7A that control its motor activity within cells are not well understood. In this study, we used cultured kidney epithelial cells to screen for mutations that activate the motor-dependent targeting of Myo7A to the tips of apical microvilli on these cells. Our findings reveal that Myo7A is regulated by specific IQ motifs within its lever arm, and that this regulation can function at least partially independent of its tail sequence. Importantly, we demonstrate that many of the DFNA11 deafness mutations reported in patients activate Myo7A targeting, providing a potential explanation for the autosomal dominant genetics of this form of deafness.

cell biology↗

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↗

bioRxiv2023

Amino acid substitutions in norovirus VP1 dictate cell tropism via an attachment process dependent on membrane mobility.

Viruses interact with receptors on the cell surface to initiate and co-ordinate infection. The distribution of receptors on host cells can be a key determinant of viral tropism and host infection. Unravelling the complex nature of virus-receptor interactions is, therefore, of fundamental importance to understanding viral pathogenesis. Noroviruses are non-enveloped, icosahedral, positive-sense RNA viruses of global importance to human health, with no approved vaccine or antiviral agent available. Here we use murine norovirus as a model for the study of molecular mechanisms of virus-receptor interactions. We show that variation at a single amino acid residue in the major viral capsid protein had a key impact on the interaction between virus and receptor. This variation did not affect virion production or virus growth kinetics, but a specific amino acid was rapidly selected through evolution experiments, and significantly improved cellular attachment when infecting immune cells in suspension. However, reducing plasma membrane mobility counteracted this phenotype, providing insight into for the role of membrane fluidity and receptor recruitment in norovirus cellular attachment. When the infectivity of a panel of recombinant viruses with single amino acid variations was compared in vivo, there were significant differences in the distribution of viruses in a murine model, demonstrating a role in cellular tropism in vivo. Overall, these results highlight the importance of lipid rafts and virus-induced receptor recruitment in viral infection, as well as how capsid evolution can greatly influence cellular tropism, within-host spread and pathogenicity. ImportanceAll viruses initiate infection by utilising receptors to attach to target host cells. These virus-receptor interactions can therefore dictate viral replication and pathogenesis. Understanding the nature of virus-receptor interactions could also be important to developing novel therapies. Noroviruses are non-enveloped icosahedral viruses of medical importance. They are a common cause of acute gastroenteritis with no approved vaccine or therapy and are a tractable model for studying fundamental virus biology. In this study, we utilise the murine norovirus model system to show that variation in a single amino acid of the major capsid protein can alone can affect viral infectivity through improved attachment to suspension cells. Reducing plasma membrane mobility reduced infectivity, providing an insight into the importance of membrane mobility for receptor recruitment. Furthermore, variation at this site was able to change viral distribution in a murine model, illustrating how in-host capsid evolution can influence viral infectivity and immune evasion.

microbiology↗

bioRxiv2023

Insights into polyprotein processing and RNA-protein interactions in foot-and-mouth disease virus genome replication.

Foot-and-mouth disease affects cloven hoofed animals and is caused by foot-and-mouth disease virus (FMDV), a picornavirus with a positive-sense RNA genome. The FMDV genome contains a single open reading frame, which is translated to produce a polyprotein that is cleaved by viral proteases to produce the viral structural and non-structural proteins. Initial processing of the polyprotein occurs at three main junctions to generate four primary products; Lpro and the P1, P2 and P3 precursors (also termed 1ABCD, 2BC and 3AB1,2,3CD). The 2BC and 3AB1,2,3CD precursors undergo subsequent proteolysis to generate non-structural proteins that are required for viral replication, including the enzymes 2C, 3Cpro and 3Dpol. These precursors can be processed through both cis and trans (i.e., intra- and inter-molecular proteolysis) pathways, which are thought to be important for controlling virus replication. Our previous studies suggested that a single residue in the 3B3-3C junction had an important role in controlling 3AB1,2,3CD processing. Here, we use in vitro based assays to show that a single point mutation at the 3B3-3C boundary increases the rate of proteolysis to generate a novel 2C-containing precursor. Complementation assays showed that while this point mutation permitted production of some non-enzymatic non-structural proteins, those with enzymatic functions were inhibited. Interestingly, replication could only be supported by complementation with mutations in cis acting RNA elements, providing genetic evidence for a functional interaction between replication enzymes and RNA elements. ImportanceFoot-and-mouth disease virus (FMDV) is an economically important pathogen of animals that is responsible for foot-and-mouth disease (FMD). FMD is endemic in many parts of the world and can results in major economic losses. Replication of the virus is a highly coordinated event that occurs within membrane-associated compartments in infected cells and requires the viral non-structural proteins. These are all initially produced as a polyprotein that undergoes proteolysis likely through both cis and trans pathways (i.e., intra- and inter-molecular proteolysis). Alternative processing pathways can provide a mechanism to help coordinate viral replication by providing temporal control to protein production. Here, we analyse the consequences of mutations that change temporal control of FMDV polyprotein processing. Our data suggests that correct processing is required to produce key enzymes for replication in an environment in which they can interact with essential viral RNA elements. These data further the understanding of FMDV genome replication.

microbiology↗

bioRxiv2023

A highly discriminatory RNA strand-specific assay to facilitate analysis of the role of cis-acting elements in foot-and-mouth disease virus replication

Foot-and-mouth-disease virus (FMDV), the etiological agent responsible for foot-and-mouth disease (FMD), is a member of the genus Aphthovirus within the Picornavirus family. In common with all picornaviruses, replication of the single-stranded positive-sense RNA genome involves synthesis of a negative-sense complementary strand that serves as a template for the synthesis of multiple positive-sense progeny strands. We have previously employed FMDV replicons to examine viral RNA and protein elements essential to replication, however, the factors affecting differential strand production remain unknown. Replicon-based systems require transfection of high levels of RNA, which can overload sensitive techniques such as qPCR preventing discrimination of specific strands. Here, we describe a method in which replicating RNA is labelled in vivo with 5-ethynyl uridine. The modified base is then linked to a biotin tag using click chemistry, facilitating purification of newly synthesised viral genomes or anti-genomes from input RNA. This selected RNA can then be amplified by strand-specific qPCR, thus enabling investigation of the consequences of defined mutations on the relative synthesis of negative-sense intermediate and positive-strand progeny RNAs. We apply this new approach to investigate the consequence of mutation of viral cis-acting replication elements and provide direct evidence for their roles in negative-strand synthesis.

molecular biology↗

bioRxiv2022

Processing of the hepatitis E virus polyprotein can be mediated by a cellular protease

The genomes of positive-sense RNA viruses encode polyproteins that are essential for controlling viral replication. These viral polyproteins must undergo proteolysis (also termed polyprotein processing) to generate functional protein units. This proteolysis can be performed by virally-encoded proteases as well as host cellular proteases, and is generally believed to be a key step in regulating viral replication. Hepatitis E virus (HEV), a leading cause of acute viral hepatitis, translates its positive-sense RNA genome to generate a polyprotein, termed pORF1, which is necessary and sufficient for viral genome replication. However, the mechanism of polyprotein processing in HEV remains to be determined. In this study, we aimed to understand processing of this polyprotein and its role in viral replication using a combination of in vitro translation experiments and HEV sub-genomic replicons. Our data suggest no evidence for a virally-encoded protease or auto-proteolytic activity as in vitro translation predominantly generates unprocessed viral polyprotein precursors. However, seven cleavage sites within the polyprotein (suggested by bioinformatic analysis) are susceptible to the host cellular protease, thrombin. Using a sub-genomic replicon system, we demonstrate that mutagenesis of these sites prevents replication, as does pharmacological inhibition of serine proteases. Overall, our data supports a model where HEV uses host proteases to support its replication and could have uniquely evolved not to rely on a virally-encoded protease for replication. Author summaryPositive-strand RNA viruses produce polyproteins that are cleaved by proteases that control viral replication. The polyproteins of all well studied positive-strand viruses undergo proteolysis in a highly controlled manner to generate functional proteins and regulate the transition from translation to RNA replication. Proteolysis of viral polyproteins is generally performed by virally-encoded proteases, although host cell proteases are used by some viruses. In this report, we provide evidence that suggests that hepatitis E virus, a medically important human pathogen, does not encode a protease and unlike other viral polyproteins cannot undergo auto-catalytic processing. Instead, we provide evidence that the polyprotein is susceptible to proteolysis by host cell proteases and that this is essential for viral replication. Our data contradict the previous dogma of positive-sense viral replication and suggests a model where this virus has evolved to use a host protease to control viral replication and tropism.

microbiology↗

bioRxiv2022

Hepatitis E virus genome replication is independent of cyclophilins A and B

Hepatitis E virus (HEV) is an emerging pathogen responsible for more than 20 million cases of acute hepatitis globally per annum. Healthy individuals typically have a self-limiting infection, however, mortality rates in some populations such as pregnant women can reach 30%. A detailed understanding of the virus lifecycle is lacking, mainly due to limitations in experimental systems. In this regard, the cyclophilins are an important family of proteins that have peptidyl-prolyl isomerase activity and play roles in the replication of a number of positive-sense RNA viruses, including hepatotropic viruses such as hepatitis C virus (HCV). Cyclophilin A (CypA) and cyclophilin B (CypB) are the two most abundant human cyclophilins in hepatocytes and are therefore potential targets for pan-viral therapeutics. Here, we investigated the importance of CypA and CypB for HEV genome replication using a sub-genomic replicon system. This system removes the requirements for viral entry and packaging and therefore allows for the sensitive measurement of viral genome replication in isolation. Using pharmacological inhibition by cyclosporine A (CsA), known to suppress HCV replication, and silencing by shRNA we find that CypA and CypB are not essential for replication of genotype 1 or 3 HEV replication. However, we find that silencing of CypB reduces replication of genotype 1 HEV in some cells, but not genotype 3. These data suggests HEV is atypical in its requirements for cyclophilin for viral genome replication and that this phenomenon could be genotype specific.

microbiology↗

bioRxiv2022

Positive strand RNA viruses differ in the constraints they place on the folding of their negative strand

Genome replication of positive strand RNA viruses requires the production of a complementary negative strand RNA that serves as a template for synthesis of more positive strand progeny. Structural RNA elements are important for genome replication, but while they are readily observed in the positive strand, evidence of their existence in the negative strand is more limited. We hypothesised that this was due to viruses differing in their capacity to allow this latter RNA to adopt structural folds. To investigate this, ribozymes were introduced into the negative strand of different viral constructs; the expectation being that if RNA folding occurred, negative strand cleavage and suppression of replication would be seen. Indeed this was what happened with hepatitis C virus (HCV) and feline calicivirus (FCV) constructs. However, little or no impact was observed for chikungunya virus (CHIKV), human rhinovirus (HRV), hepatitis E virus (HEV) and yellow fever virus (YFV) constructs. Reduced cleavage in the negative strand proved to be due to duplex formation with the positive strand. Interestingly, ribozyme-containing RNAs also remained intact when produced in vitro by the HCV polymerase, again due to duplex formation. Overall, our results show that there are important differences in the conformational constraints imposed on the folding of the negative strand between different positive strand RNA viruses.

microbiology↗

bioRxiv2020

Higher-order structures of the foot-and-mouth disease virus RNA-dependent RNA polymerase required for dynamic inter-molecular interactions involved in viral genome replication

Replication of many positive-sense RNA viruses occurs within intracellular membrane-associated compartments. These are believed to provide a favourable environment for replication to occur, concentrating essential viral structural and non-structural components, as well as protecting these components from host-cell pathogen recognition and innate immune responses. However, the details of the molecular interactions and dynamics within these structures is very limited. One of the key components of the replication machinery is the RNA-dependent RNA polymerase, RdRp. This enzyme has been shown to form higher-order fibrils in vitro. Here, using the RdRp from foot-and-mouth disease virus (termed 3Dpol), we report fibril structures, solved at ~7-9 [A] resolution by cryo-EM, revealing multiple conformations of a flexible assembly. Fitting high-resolution coordinates led to the definition of potential intermolecular interactions. We employed mutagenesis using a sub-genomic replicon system to probe the importance of these interactions for replication. We use these data to propose models for the role of higher order 3Dpol complexes as a dynamic scaffold within which RNA replication can occur.

microbiology↗

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

High-Resolution Cryo-EM Reveals Dynamics in the Murine Norovirus Capsid

Rather than acting as rigid symmetrical shells to protect and transmit their genomes, the capsids of non-enveloped, icosahedral viruses co-ordinate multiple, essential processes during the viral life-cycle, and undergo extensive conformational rearrangements to deliver these functions. Capturing conformational flexibility has been challenging, yet could be key in understanding and combating infections that viruses cause. Noroviruses are non-enveloped, icosahedral viruses of global importance to human health. They are a common cause of acute non-bacterial gastroenteritis, yet no vaccines or antiviral agents specific to norovirus are available. Here, we use cryo-electron microscopy to study the high-resolution solution structures of infectious, inactivated and mutant virions of murine norovirus (MNV) as a model for human noroviruses. Together with genetic studies, we show that the viral capsid is highly dynamic. While there is little change to the shell domain of the capsid, the protruding domains that radiate from this are flexible and adopt distinct states both independently and synchronously. In doing so the viral capsid is able to sample a defined range of conformational space, with implications for the maintenance of virion stability and infectivity. These data will aid in developing the first generation of effective control measures against this virus.

microbiology↗