Biology subjects

Graham, S. C.

Publications and source records attributed to Graham, S. C..

At least 19 recordsLinked to original sources

bioRxiv2026

Herpes simplex virus pUL56 abolishes neuronal activity by removing voltage-gated ion channels from the plasma membrane

Herpes simplex virus 1 (HSV-1) infections of the central nervous system cause encephalitis and are associated with increased risk of neurodegeneration, yet the molecular consequences of lytic infection in human neurones remain incompletely defined. We map the transcriptomic, proteomic and surface-proteome changes induced by HSV-1 across the lytic infection cycle in human iPSC-derived cortical glutamatergic neurones. HSV-1 drives extensive plasma-membrane remodelling, including the removal of voltage-gated sodium, potassium and calcium channels, resulting in a profound loss of synchronous calcium signalling. We identify the viral ubiquitin-ligase adaptor pUL56 as the principal effector of this process: pUL56-dependent depletion of ion channels abolishes coordinated calcium signalling, whereas deletion or mutation of its E3-ligase binding motifs preserves synchrony. Furthermore, expression of pUL56 alone is sufficient to abolish neuronal electrical activity. These findings establish pUL56 as a potent viral suppressor of neuronal excitability and provide potential mechanistic links between HSV-1 infection and neurodegenerative pathology.

microbiology↗

bioRxiv2026

Human iPSC Models of Ganglioside Deficiency Reveal a Sialylated Lipid Requirement for Plasma-Membrane Organization and Neuronal Activity

Gangliosides are abundant neuronal glycosphingolipids, yet their roles in organizing the plasma membrane and supporting neuronal function remain poorly defined. Mutations in the biosynthetic enzymes ST3GAL5 or B4GALNT1 cause severe neurodevelopmental disorders, yet their cellular consequences are unclear. Using isogenic human iPSC-derived cortical neurons, we show that loss of these enzymes eliminates major neuronal gangliosides but produces strikingly divergent outcomes. ST3GAL5 deficiency reprograms the glycosphingolipid repertoire toward non-neuronal species and abolishes network-level electrical activity. In contrast, B4GALNT1-deficient neurons retain near-normal excitability, supported by accumulation of simple sialylated precursors (GM3/GD3). Quantitative proteomics reveals a profound loss of plasma membrane proteins, including ion channels and synaptic organizers, only in ST3GAL5-deficient neurons. These findings identify sialylated glycosphingolipids as essential scaffolds for plasma membrane organization and neuronal excitability, providing a mechanistic basis for the severe phenotype caused by loss of GM3 synthase in humans. TEASERHuman neuronal models reveal why loss of GM3 synthase causes severe neurodevelopmental disease

cell biology↗

bioRxiv2025

Optimisation of lytic herpes simplex virus infection in human induced pluripotent stem cell derived cortical neurones

Herpes simplex virus (HSV)-1 infection of cortical neurones is a leading cause of encephalitis. While we have substantial knowledge about the molecular virology of HSV-1 lytic infection in cells of the periphery, like keratinocytes or fibroblasts, we know much less about infection of human neurones owing to the challenges of working with neuronal cell-based models. Here we demonstrate the use of a human induced pluripotent stem cell (iPSC)-derived cortical neurone model (i3Neurones) for HSV-1 infection. i3Neurones are highly scalable and can be rapidly and efficiently differentiated into an isogenic population of cortical glutamatergic neurones. We show that i3Neurones support the full HSV-1 lytic replication cycle. We present an optimised protocol for the infection of i3Neurones with HSV-1 that allows their synchronous infection at near-100% efficiency, and optimised fixation methods that preserves organelle and neurite structure for immunocytochemistry analysis. Our study highlights i3Neurones as a robust, scalable platform for microscopy and biochemical studies of HSV-1 and other neurotropic pathogens. Data summaryThe authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

microbiology↗

bioRxiv2025

Alphaherpesvirus pUL21 homologues use non-canonical motifs to compete with cellular adaptors for protein phosphatase 1 binding

Protein phosphatase 1 (PP1) is a key regulator of cellular phosphorylation and its activity is regulated via binding to cellular regulatory proteins via conserved short linear motifs (SLiMs). The herpes simplex virus (HSV)-1 protein pUL21 binds PP1 via the TROPPO motif, which lacks sequence similarity to canonical PP1-binding SLiMs. Here, we combine structure prediction, mutagenesis, and biophysical assays to elucidate the molecular basis of this interaction. AlphaFold2-Multimer structural models suggest that the TROPPO motifs of pUL21 and of pORF38, the varicella-zoster virus homologue of pUL21, bind the same hydrophobic groove on PP1 as RVxF and {phi}{phi}[xF] motifs, forming an extended {beta}-sheet that bridges PP1 and the N-terminal domain of pUL21 or pORF38. Site-directed mutagenesis of both pUL21 and PP1 confirms key predicted interactions. Competition fluorescence polarisation confirms that pUL21 competes directly with cellular PP1 RvXF motifs for PP1 binding, albeit with lower affinity. Substituting key residues of the pUL21 TROPPO motif to resemble a canonical RVxF sequence increases PP1 binding, suggesting that alphaherpesviruses have evolved suboptimal motifs to fine-tune phosphatase recruitment that may balance viral kinase and phosphatase activities during infection. Our findings reveal a novel mechanism of PP1 recruitment by viral proteins and suggest that many other PP1 regulators may possess non-canonical binding motifs and thus remain undiscovered.

biochemistry↗

bioRxiv2025

A new protein-dependent riboswitch activates ribosomal frameshifting

Programmed -1 ribosomal frameshifting (PRF) is a translational control mechanism used by RNA viruses to regulate the relative abundance of proteins encoded in different reading frames. Cardioviruses exhibit the highest known PRF efficiency, with ~85% of ribosomes shifting into the -1 frame. This unusual event requires an interaction between the viral 2A protein and a stimulatory element in the RNA genome, but the basis for protein-dependence is unclear. To address this, here we investigate structure and dynamics of the PRF signal in Theiler's murine encephalitis virus (TMEV). By combining X-ray crystallography, small angle X-ray scattering (SAXS) and single-molecule fluorescence resonance energy transfer (smFRET), we show that 2A binding switches the RNA from a stem-loop conformation into a pseudoknot, and we demonstrate that pseudoknot formation is essential for efficient PRF in vitro and in cells. Together, these findings illustrate how the cardiovirus PRF element behaves as a protein-dependent riboswitch, defining the molecular mechanism by which frameshifting is conditionally activated.

molecular biology↗

bioRxiv2025

Flexibility and modulation of translation initiation in enterovirus genomes

Enteroviruses comprise a large group of mammalian pathogens that often utilize two open reading frames (ORFs) to encode their proteins: the upstream protein (UP) and the main polyprotein. In some enteroviruses, in addition to the canonical upstream AUG (uAUG), there is another AUG that may represent an alternative upstream initiation site. An analysis of enterovirus sequences containing additional upstream AUGs identified several clusters, including strains of pathogenic Enterovirus alphacoxsackie and E. coxsackiepol. Using ribosome profiling on coxsackievirus CVA-13 (E. coxsackiepol), we demonstrate that both upstream AUG codons can be used for translation initiation in infected cells. Moreover, we confirm translation from both upstream AUGs using a reporter system. Mutating the additional upstream AUG in the context of CVA-13 did not result in phenotypic changes in immortalized cell lines. However, the wild-type virus outcompeted this mutant in human intestinal organoids and differentiated neuronal systems, representing an advantage in physiologically relevant infection sites. Mutation of the stop codon of the shorter upstream ORF led to dysregulated translation of the other ORFs in the reporter system, suggesting a potential role for the additional uORF in modulating the expression level of the other ORFs. These findings demonstrate the remarkable plasticity of enterovirus IRES-mediated initiation and the competitive advantage of double-upstream-AUG-containing viruses in terminally differentiated intestinal organoids and neuronal systems.

microbiology↗

bioRxiv2024

Whole Virion Proteomics of HCMV Reveals that gpUL141 is a Virion Component that Regulates Envelope Glycoprotein Composition to Protect Against Humoral Immunity

Human cytomegalovirus (HCMV) is a clinically important herpesvirus that has co-evolved for millions of years with its human host, and establishes lifelong persistent infection. A substantial proportion of its 235kb genome is dedicated to manipulating host immunity through targeting antiviral host proteins for degradation or relocalisation. Quantitative proteomics of the infected cell has extensively characterised these processes, but the cell-free virion has been less well studied. We therefore carried out proteomic analysis of a clinical HCMV strain (Merlin) virion. This revealed 18 novel components, including the viral protein gpUL141, which is recognised as an NK immune-evasin that targets several host proteins (CD155, CD112, and TRAILR) when expressed within the cell. Co-Immunoprecipitation of gpUL141 from virions identified interactions with viral entry glycoproteins from the trimer (gH/gL/gO), pentamer (gH/gL/UL128/UL130/UL131A), and gH/gpUL116 complexes, as well as gB. Only interactions with gH/gB occurred in the absence of other viral proteins. Analysis supported a model in which gpUL141 homodimers independently interacted with separate gB/gH-containing complexes. gpUL141 encodes an ER retention domain that restricts trafficking through the ER/golgi, and limited the transport of glycoprotein complexes bound by gpUL141. As a result, gpUL141 reduced levels of multiple glycoprotein complexes on the infected cell surface as well as in the virion. This reduced syncytium formation, inhibited antibody-dependent cellular cytotoxicity (ADCC), and reduced susceptibility to neutralising antibodies. Thus, gpUL141 represents an immune-evasin that not only targets host proteins to limit NK-cell attack, but also alters the trafficking of multiple viral glycoprotein complexes in order to evade humoral immunity. SignificanceThe large number of immune-modulators encoded by HCMV has led to it becoming a paradigm for pathogen-mediated immune-modulation. These mechanisms have informed on virus pathogenesis, the evolution and function of host defences, and identified therapeutic targets. Previously discovered immune-evasins functioned by modifying host proteins. gpUL141 represents a novel strategy in which multiple immunological functions are targeted through manipulating eight different viral entry glycoproteins, via interactions with two proteins common to multiple complexes. Entry glycoproteins define virus tropism, mechanisms of infection and spread, and susceptibility to neutralising and non-neutralising antibody activities, yet UL141 is frequently mutated in passaged viruses. This demonstrates the need to work with virus strains encoding the complete repertoire of viral accessory proteins during preclinical therapeutic development.

microbiology↗

bioRxiv2024

Applying 3D correlative structured illumination microscopy and X-ray tomography to characterise herpes simplex virus-1 morphogenesis

Numerous viral genes are involved in assembly of herpes simplex virus-1 (HSV-1), but their relative importance and function remain poorly characterised. Transmission electron microscopy has been used to study viral protein function in cells infected with HSV-1 mutants; however, these studies were usually conducted without correlative light microscopy to identify specific viral components. In this study, fluorescent capsid (eYFP-VP26) and envelope (gM-mCherry) proteins were imaged by structured illumination microscopy under cryogenic conditions (cryoSIM) and cellular ultrastructure was captured from the same infected cells using cryo-soft-X-ray tomography (cryoSXT). Nine fluorescent HSV-1 mutants, each lacking a different viral protein, were compared to assess the importance of viral proteins in different stages of HSV-1 morphogenesis. The relative importance of five viral proteins to nuclear egress were ranked (pUL34 > pUL21 > VP16 > pUL16 > pUS3) according to the levels of attenuation observed for each virus. Correlative imaging also revealed the roles of five viral proteins in cytoplasmic envelopment. VP16 was found to be important in capsid delivery to envelopment compartments, while cytoplasmic clusters of virus particles plus features of stalled envelopment not previously described were observed in the absence of pUL11, pUL51, gK, and gE. Finally, this 3D imaging approach was used to capture different assembly stages during cytoplasmic envelopment and to determine that envelopment occurs by particle budding rather than wrapping. The findings demonstrate that tomographic 3D correlative imaging is an emerging technology that sheds new light on viral protein functions and virion morphogenesis. ImportanceTo date, the characterisation of HSV-1 mutants in the study of virus assembly has been limited to transmission electron microscopy (TEM) without the addition of correlative light microscopy to identify fluorescently labelled viral proteins. In addition, only a small number of mutants are typically used in each study. Herein, a comparative analysis of nine HSV-1 mutants lacking specific structural proteins was performed using correlative fluorescence microscopy and X-ray tomography for the first time, revealing the relative roles of each viral protein in virus assembly. pUL11, pUL51, gK, and gE were shown to have important roles in cytoplasmic envelopment, with the loss of their functions leading to various stalled cytoplasmic envelopment phenotypes involving polarised arrays of capsids at one side of cytoplasmic vesicles that to our knowledge have never been seen with TEM. This correlative imaging approach enabled the study of cytoplasmic envelopment in 3D, revealing an envelopment mechanism driven by capsid budding rather than membrane wrapping. Further cryoSIM experiments revealed that pUL16 and pUL21 are important in nuclear egress of HSV-1 and that VP16 promotes nuclear egress and delivery of capsids to cytoplasmic envelopment compartments. By providing novel and comparative insights into the roles of different viral proteins in various stages of HSV-1 assembly, these findings highlight the utility of correlative cryo-fluorescence plus cryo-soft-X-ray tomography for probing trajectories of intracellular pathogen assembly.

microbiology↗

bioRxiv2024

Human cytomegalovirus deploys molecular mimicry to recruit VPS4A to sites of virus assembly

The AAA-type ATPase VPS4 is recruited by proteins of the endosomal sorting complex required for transport III (ESCRT-III) to catalyse membrane constriction and membrane fission. VPS4A accumulates at the cytoplasmic viral assembly complex (cVAC) of cells infected with human cytomegalovirus (HCMV), the site where nascent virus particles obtain their membrane envelope. Here we show that VPS4A is recruited to the cVAC via interaction with pUL71. Sequence analysis, deep-learning structure prediction, molecular dynamics and mutagenic analysis identify a short peptide motif in the C-terminal region of pUL71 that is necessary and sufficient for the interaction with VPS4A. This motif is predicted to bind the same groove of the N-terminal VPS4A Microtubule-Interacting and Trafficking (MIT) domain as the Type 2 MIT-Interacting Motif (MIM2) of cellular ESCRT-III components, and this viral MIM2-like motif (vMIM2) is conserved across {beta}-herpesvirus pUL71 homologues. However, recruitment of VPS4A by pUL71 is dispensable for HCMV morphogenesis or replication and the function of the conserved vMIM2 during infection remains enigmatic. VPS4-recruitment via a vMIM2 represents a previously unknown mechanism of molecular mimicry in viruses, extending previous observations that herpesviruses encode proteins with structural and functional homology to cellular ESCRT-III components.

cell biology↗

bioRxiv2022

Altered plasma membrane abundance of the sulfatide-binding protein NF155 links glycosphingolipid imbalances to demyelination

Myelin is a multi-layered membrane that tightly wraps neuronal axons enabling efficient, high-speed signal propagation. The axon and myelin sheath form tight contacts, mediated by specific plasma membrane proteins and lipids, and disruption of these contacts causes devastating demyelinating diseases. Using two cell-based models of demyelinating sphingolipidoses, we demonstrate that altered lipid metabolism changes the abundance of specific plasma membrane proteins. These altered membrane proteins have known roles in cell adhesion and signalling, with several implicated in neurological diseases. The cell surface abundance of the adhesion molecule Neurofascin, a protein critical for the maintenance of myelin-axon contacts, changes following disruption to sphingolipid metabolism. This provides a direct molecular link between altered lipid abundance and myelin stability. We show that the Neurofascin isoform NF155, but not NF186, interacts directly and specifically with the sphingolipid sulfatide via multiple binding sites and that this interaction requires the full-length extracellular domain of NF155. We demonstrate that NF155 adopts an S-shaped conformation and preferrentially binds sulfatide-containing membranes in cis, with important implications for protein arrangement in the tight axon-myelin space. Our work links glycosphingolipid imbalances to disturbance of membrane protein abundance and demonstrates how this may be driven by direct protein-lipid interactions, providing a mechanistic framework to understand the pathogenesis of galactosphingolipidoses.

neuroscience↗

bioRxiv2022

Attenuation hotspots in neurotropic human astroviruses

During the last decade, the detection of neurotropic astroviruses has increased dramatically. The MLB genogroup of astroviruses represents a genetically distinct group of zoonotic astroviruses associated with gastroenteritis and severe neurological complications in young children, the immunocompromised and the elderly. Using different virus evolution approaches, we identified dispensable regions in the 3' end of the capsid-coding region responsible for attenuation of MLB astroviruses in susceptible cell lines. To create recombinant viruses with identified deletions, MLB reverse genetics and replicon systems were developed. Recombinant truncated MLB viruses had wild type-like or enhanced growth and replication properties in permissive cells but were strongly attenuated in iPSC-derived neuronal cultures confirming the location of neurotropism determinants. This approach can be used for the development of vaccine candidates using attenuated astroviruses that infect humans, livestock animals and poultry.

microbiology↗

bioRxiv2022

Structural determinants of homophilic adhesion by receptor tyrosine phosphatases

The type IIB receptor protein tyrosine phosphatases (R2B RPTPs) are cell surface transmembrane proteins that engage in cell adhesion via their extracellular domains (ECDs) and cell signaling via their cytoplasmic phosphatase domains. The ECDs of R2B RPTPs form stable, homophilic, trans interactions between adjacent cell membranes. Previous work has demonstrated how one family member, PTPRM, forms homodimers; however, the determinants of homophilic specificity remain unknown. We have solved the X-ray crystal structure of the membrane-distal, N-terminal domains of PTPRK that form a head-to-tail dimer consistent with intermembrane adhesion. Comparison with the PTPRM structure demonstrates inter-domain conformational differences that may define homophilic specificity. Using small-angle X-ray scattering we determined the solution structures of the full-length ECDs of PTPRM and PTPRK, identifying that both are rigid, extended molecules that differ in their overall long-range conformation. Furthermore, we identify one residue, W351, within the interaction interface that differs between PTPRM and PTPRK and show that mutation to glycine, the equivalent residue in PTPRM, abolishes PTPRK dimer formation in vitro. This comparison of two members of the receptor tyrosine phosphatase family suggest that homophilic specificity is driven by a combination of shape complementarity and specific but limited sequence differences. SIGNIFICANCE STATEMENTCell-cell contacts are dynamically regulated, in part, by the actions of tyrosine kinases and phosphatases. The R2B family of receptor tyrosine phosphatases combine an adhesive extracellular domain with intracellular catalytic domains that bind and dephosphorylate key cell adhesion and polarity proteins. Previous work demonstrated that the extracellular domains form head-to-tail homodimers but, as the interface was composed of residues conserved across the family, homophilic specificity determinants remained unclear. We have used a range of structural techniques including X-ray crystallography, small angle X-ray scattering and AlphaFold modelling to demonstrate that, despite their similarity, two members of the R2B family possess significant differences in their overall shape. Our results support that a combination of subtle shape and sequence variations may determine homophilic binding.

biochemistry↗

bioRxiv2022

Herpes simplex virus 1 protein pUL21 stimulates cellular ceramide transport by activating CERT

Herpes simplex virus (HSV)-1 dramatically alters the architecture and protein composition of cellular membranes during infection, but its effects upon membrane lipid composition remain unclear. HSV-1 pUL21 is a virus-encoded protein phosphatase adaptor that promotes dephosphorylation of multiple cellular and virus proteins, including the cellular ceramide transport protein CERT. CERT mediates non- vesicular transport of ceramide from the ER to the trans-Golgi network, whereupon ceramide is converted to sphingomyelin and other sphingolipids that play important roles in cell proliferation, cell signalling and membrane trafficking. Using click chemistry to profile the kinetics of sphingolipid metabolism in cultured cells, we show that pUL21-mediated dephosphorylation activates CERT and increases the rate of ceramide to sphingomyelin conversion. Purified pUL21 and full-length CERT interact with sub-micromolar affinity and we map the domains responsible for the interaction. Solving the solution structure of the pUL21 C-terminal domain in complex with the CERT PH and START domains using small-angle X-ray scattering allows us to identify a single amino acid mutation on the surface of pUL21 that disrupts CERT binding in vitro and in cultured cells. Sphingolipid profiling demonstrates that ceramide to sphingomyelin conversion is severely diminished in the context of HSV- 1 infection, a defect that is compounded when infecting with a virus encoding the mutated form of pUL21 that lacks the ability to activate CERT. However, virus replication and spread are not significantly altered when pUL21-mediated CERT dephosphorylation is abolished, highlighting that dephosphorylation of other cellular and/or viral targets underpins the important role of pUL21 in HSV-1 biology. SignificanceHerpes simplex virus (HSV)-1 causes a life-long dormant infection of neurons, sporadically reactivating to manifest as cold-sores or genital herpes. While the impact of HSV-1 upon the protein content of infected cells has been well studied, we know relatively little about its impact upon cellular lipids. Using bioorthogonal labelling in cultured cells we show that HSV-1 protein pUL21 activates the key cellular lipid transport protein CERT to accelerate the conversion of ceramide to sphingomyelin. HSV-1 infection dramatically alters the kinetics of ceramide metabolism, leading to ceramide accumulation. Mutation of HSV-1 pUL21 to prevent CERT activation further enhances ceramide accumulation but this does not alter the replication or spread of HSV-1, highlighting that other cellular and/or viral proteins represent the critical targets of pUL21-mediated dephosphorylation in cultured cells.

microbiology↗

bioRxiv2022

Molecular mechanism of Afadin substrate recruitment to the receptor phosphatase PTPRK via its pseudophosphatase domain

Protein tyrosine phosphatase receptor-type kappa (PTPRK) is a transmembrane receptor that links extracellular homophilic interactions to intracellular catalytic activity. Previously we showed that PTPRK promotes cell-cell adhesion by selectively dephosphorylating several cell junction regulators including the protein Afadin (Fearnley et al., 2019). Here we demonstrate that Afadin is recruited for dephosphorylation by directly binding to the PTPRK D2 pseudophosphatase domain. We mapped this interaction to a putative coiled coil (CC) domain in Afadin that is separated by more than 100 amino acids from the substrate pTyr residue. We identify the residues that define PTP specificity, explaining how Afadin is selectively dephosphorylated by PTPRK yet not by the closely-related receptor tyrosine phosphatase PTPRM. Our work demonstrates that PTP substrate specificity can be determined by protein-protein interactions distal to the active site. This explains how PTPRK and other PTPs achieve substrate specificity despite a lack of specific sequence context at the substrate pTyr. Furthermore, by demonstrating that these interactions are phosphorylation-independent and mediated via binding to a non-catalytic domain, we highlight how receptor PTPs could function as intracellular scaffolds in addition to catalyzing protein dephosphorylation.

molecular biology↗

bioRxiv2021

Contour, a semi-automated segmentation and quantitation tool for cryo-soft-X-ray tomography

Cryo-soft-X-ray tomography is being increasingly used in biological research to study the morphology of cellular compartments and how they change in response to different stimuli, such as viral infections. Segmentation of these compartments is limited by time-consuming manual tools or machine learning algorithms that require extensive time and effort to train. Here we describe Contour, a new, easy-to-use, highly automated segmentation tool that enables accelerated segmentation of tomograms to delineate distinct cellular compartments. Using Contour, cellular structures can be segmented based on their projection intensity and geometrical width by applying a threshold range to the image and excluding noise smaller in width than the cellular compartments of interest. This method is less laborious and less prone to errors from human judgement than current tools that require features to be manually traced, and does not require training datasets as would machine-learning driven segmentation. We show that high-contrast compartments such as mitochondria, lipid droplets, and features at the cell surface can be easily segmented with this technique in the context of investigating herpes simplex virus 1 infection. Contour can extract geometric measurements from 3D segmented volumes, providing a new method to quantitate cryo-soft-X-ray tomography data. Contour can be freely downloaded at github.com/kamallouisnahas/Contour. Impact StatementMore research groups are using cryo-soft-X-ray tomography as a correlative imaging tool to study the ultrastructure of cells and tissues but very few tomograms are segmented with existing segmentation programs. Segmentation is usually a prerequisite for measuring the geometry of features in tomograms but the time- and labour-intensive nature of current segmentation techniques means that such measurements are rarely across a large number of tomograms, as is required for robust statistical analysis. Contour has been designed to facilitate the automation of segmentation and, as a result, reduce manual effort and increase the number of tomograms that can be segmented. Because it requires minimal manual intervention, Contour is not as prone to human error as programs that require the users to trace the edges of cellular features. Geometry measurements of the segmented volumes can be calculated using this program, providing a new platform to quantitate cryoSXT data. Contour also supports quantitation of volumes imported from other segmentation programs. The generation of a large sample of segmented volumes with Contour that can be used as a representative training dataset for machine learning applications is a long-term aspiration of this technique.

cell biology↗

bioRxiv2021

The crystal structure of vaccinia virus protein E2 and perspectives on the prediction of novel viral protein folds

The morphogenesis of vaccinia virus (VACV, family Poxviridae), the smallpox vaccine, is a complex process involving multiple distinct cellular membranes and resulting in multiple different forms of infectious virion. Efficient release of enveloped virions, which promote systemic spread of infection within hosts, requires the VACV protein E2 but the molecular basis of E2 function remains unclear and E2 lacks sequence homology to any well-characterised family of proteins. We solved the crystal structure of VACV E2 to 2.3 [A] resolution, revealing that it comprises two domains with novel folds: an N-terminal annular (ring) domain and a C-terminal head domain. The C-terminal head domain displays weak structural homology with cellular (pseudo)kinases but lacks conserved surface residues or kinase features, suggesting that it is not enzymatically active, and possesses a large surface basic patch that might interact with phosphoinositide lipid headgroups. Recent deep learning methods have revolutionised our ability to predict the three-dimensional structures of proteins from primary sequence alone. VACV E2 is an exemplar difficult viral protein target for structure prediction, being comprised of multiple novel domains and lacking sequence homologues outside Poxviridae. AlphaFold2 nonetheless succeeds in predicting the structures of the head and ring domains with high and moderate accuracy, respectively, allowing accurate inference of multiple structural properties. The advent of highly accurate virus structure prediction marks a step-change in structural virology and beckons a new era of structurally-informed molecular virology.

microbiology↗

bioRxiv2021

Near-native state imaging by cryo-soft-X-ray tomography reveals remodelling of cytoplasmic vesicles and mitochondria during HSV-1 infection

Herpes simplex virus-1 (HSV-1) is a large, enveloped DNA virus and its assembly in the cell is a complex multi-step process during which viral particles interact with numerous cellular compartments such as the nucleus and organelles of the secretory pathway. Transmission electron microscopy and fluorescence microscopy are commonly used to study HSV-1 infection. However, 2D imaging limits our understanding of the 3D geometric changes to cellular compartments that accompany infection and sample processing can introduce morphological artefacts that complicate interpretation. In this study, we used soft X-ray tomography to observe differences in whole-cell architecture between HSV-1 infected and uninfected cells. To protect the near-native structure of cellular compartments we used a non-disruptive sample preparation technique involving rapid cryopreservation, and a fluorescent reporter virus was used to facilitate correlation of structural changes with the stage of infection in individual cells. We observed viral capsids and assembly intermediates interacting with nuclear and cytoplasmic membranes. Additionally, we observed differences in the morphology of specific organelles between uninfected and infected cells. The local concentration of cytoplasmic vesicles at the juxtanuclear compartment increased and their mean width decreased as infection proceeded, and lipid droplets transiently increased in size. Furthermore, mitochondria in infected cells were elongated and highly branched, suggesting that HSV-1 infection alters the dynamics of mitochondrial fission/fusion. Our results demonstrate that high-resolution 3D images of cellular compartments can be captured in a near-native state using soft X-ray tomography and have revealed that infection causes striking changes to the morphology of intracellular organelles. ImportanceUltrastructural changes to the morphology and organization of cellular compartments during herpes simplex virus-1 (HSV-1) infection have not previously been studied under near-physiological conditions. In this study, soft X-ray tomography was used to image the ultrastructure of vitrified cells during HSV-1 infection, identifying striking changes to the abundance and organization of cytoplasmic vesicles and mitochondria. The concentration of vesicles in the juxtanuclear region increased with time post infection, which could represent an increasing supply of vesicles to support capsid envelopment, and there is a transient increase in the size of lipid droplets in infected cells. Furthermore, we show that mitochondria elongate and form highly-branched networks as infection progresses. These findings offer insight into stages of virion morphogenesis and the cellular response to infection, highlighting the utility of cryo-soft-X-ray tomography for monitoring the near-native state ultrastructure of infected cells.

microbiology↗

bioRxiv2021

A tetrameric assembly of saposin A: increasing structural diversity in lipid transfer proteins

Saposins are lipid transfer proteins required for the degradation of sphingolipids in the lysosome. These small proteins bind lipids by transitioning from a closed, monomeric state to an open conformation exposing a hydrophobic surface that binds and shields hydrophobic lipid tails from the aqueous environment. Saposins form a range of multimeric assemblies to encompass these bound lipids and present them to hydrolases in the lysosome. This lipid-binding property of human saposin A has been exploited to form lipoprotein nanodiscs suitable for structural studies of membrane proteins. Here we present the crystal structure of a unique tetrameric assembly of murine saposin A produced serendipitously, following modifications of published protocols for making lipoprotein nanodiscs. The structure of this new saposin oligomer highlights the diversity of tertiary arrangement that can be adopted by these important lipid transfer proteins.

biochemistry↗