Biology subjects

Elliott, G.

Publications and source records attributed to Elliott, G..

16 recordsLinked to original sources

bioRxiv2026

METTL3 promotes human coronavirus replication through an interferon-independent mechanism

N6-methyladenosine (m6A) is a pervasive mRNA modification that regulates RNA fate through effects on RNA-protein interactions, stability and translation. We previously showed that replication of human betacoronaviruses, OC43 (hCoV-OC43) and SARS-CoV-2, is sensitive to depletion or pharmacological inhibition of the m6A RNA methyltransferase METTL3, resulting in reduced viral RNA and protein accumulation. In other viral systems, such antiviral effects have been attributed to enhanced interferon (IFN) signalling and interferon-stimulated gene (ISG) induction. Here, using hCoV-OC43 we show a requirement for METTL3 that is independent of canonical IFN responses. Pharmacological inhibition of METTL3 with STM2457 failed to potentiate type I IFN signalling, global ISG expression, or the non-canonical inflammatory transcriptional programme associated with OC43 infection. Furthermore, pathogen-associated RNA sensing by RIG-I or MDA5 is not required for the antiviral effect of the STM2457. ISGs reported to be most potently antiviral against OC43 are either not significantly induced by METTL3 inhibition during infection or are not required for the antiviral activity. Nevertheless, defects in viral gene expression and progression through the viral life cycle are detectable within 6 h of STM2457 treatment and host cell transcription is dispensable for STM2457 antiviral activity. Lastly, a METTL3-directed Proteolysis Targeting Chimera (PROTAC) phenocopied STM2457, producing IFN-independent antiviral activity and ruling out off-target inhibition of viral RNA methyltransferases as a plausible explanation. Together, these findings define a direct, proviral role for METTL3 in coronavirus infection consistent with a model in which METTL3-catalysed m6A modification of viral RNA is required for efficient viral life cycle progression.

microbiology↗

bioRxiv2026

Integrating genome-wide neutral and putatively adaptive variation resolves population structure and differentiation in a recently diverged albatross complex

Understanding patterns of evolution and divergence in populations is important for defining conservation units and informing taxonomy. However, when populations have low genetic diversity, or are closely related, genetic differentiation can be difficult to detect, particularly when relying on small numbers of genetic markers. Whole-genome sequencing allows for genome-wide identification of both neutral and outlier variation, improving the resolution of subtle population structure and providing insight into evolutionary processes. Here, we investigated genomic differentiation in a recently diverged and highly threatened albatross complex, the Antipodean (D. antipodensis antipodensis) and Gibson's albatross (D. a. gibsoni), using genome-wide neutral and outlier datasets. Whole-genome resequencing of 86 individuals sampled across Antipodes Island (D. a. antipodensis) and the Auckland Islands (D. a. gibsoni) identified 381,176 neutrally evolving and 57 independently segregating outlier (putatively adaptive) SNPs. Analyses of both datasets revealed significant genetic differentiation between the Antipodean and Gibson's albatross, no evidence of contemporary gene flow and evidence of selective sweeps suggesting local adaptation. Within-population structure was also identified for the Gibson's albatross, with genetic differentiation among sample sites from different islands (Adams Island and Disappointment Island). Patterns of heterogenous differentiation across the genome suggest the taxa are on different evolutionary trajectories. Together with existing morphological and behavioural evidence, these genomic results support reassessment of their conservation and taxonomic status. More broadly, this study demonstrates the value of combining neutral and putatively adaptive genomic variation to resolve subtle population structure in recently diverged taxa.

evolutionary biology↗

bioRxiv2026

Loss of PKN2 drives fibroblast reprogramming and extracellular matrix remodelling in pulmonary fibrosis

Introduction Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease characterised by aberrant fibroblast function, extracellular matrix (ECM) remodelling and defective tissue repair. Protein kinase N2 (PKN2) is associated with accelerated forced vital capacity decline in IPF, but its functional role in pulmonary fibrosis remains unknown. We hypothesised that PKN2 regulates fibroblast phenotype and tissue repair. Methods PKN2 expression was assessed in human lung tissue, induced sputum and primary airway and parenchymal fibroblasts from non-fibrotic controls and patients with interstitial lung disease (ILD). DNA methylation was profiled using the Illumina HumanMethylationEPIC array. PKN2 function was investigated by siRNA-mediated depletion in primary human lung fibroblasts using transcriptomic, proteomic and functional analyses. Tissue repair was assessed following pharmacological PKN inhibition in zebrafish. Results PKN2 expression was reduced in ILD lung tissue and primary airway and parenchymal fibroblasts and further suppressed by TGF-{beta}1. Differential methylation was identified across the PKN2 locus in both fibroblast populations. Integrated transcriptomic and proteomic profiling following PKN2 depletion revealed coordinated remodelling of ECM, cell adhesion, non-canonical WNT and VEGF pathways, including dysregulation of COL1A1, WNT, VEGF and MMP1. PKN2 loss increased VEGF and MMP-1 secretion and accelerated fibroblast wound closure. PKN inhibition altered epithelial organisation and collagen fibre alignment during zebrafish wound repair. Conclusion PKN2 loss drives fibroblast reprogramming and aberrant ECM remodelling, establishing PKN2 as an important regulator of pulmonary fibroblast homeostasis and tissue repair.

cell biology↗

bioRxiv2025

Foraging ecology drives viral community structure in New Zealand's aquatic birds

Wild migratory birds play a major role in the global spread of viruses, yet the diversity, host range and transmission patterns of viruses harboured by migratory species in Aotearoa/New Zealand remain largely unknown. This knowledge gap is critical given New Zealands position along major migratory flyways spanning Oceania, Antarctica and east Asia, where understanding viral diversity is key to assessing the risk of viral introductions such as highly pathogenic avian influenza virus and viral dispersal across these regions. To address this, we conducted the first large-scale metatranscriptomic survey of wild birds from New Zealand and its subantarctic islands, collecting 1,348 samples from 31 host species spanning four avian orders. We identified 118 avian viruses from 17 families, including 107 novel species, greatly expanding our knowledge of avian viral diversity. Viral communities differed significantly by host order and foraging behaviour, with scavenger birds harbouring more diverse viromes than non-scavengers. Although no HPAI subtypes were detected, we recovered a low-pathogenic avian influenza A/H1N9 virus from red knots (Calidris canutus) and a divergent tobanivirus from Auckland Island teal (Anas aucklandica), the first putative avian member of the Tobaniviridae. Notably, we detected 12 mammalian-associated viruses, primarily in scavenger birds, including Hedgehog hepatovirus, Rabbit haemorrhagic disease virus 2, and sea lion astroviruses, with mammalian host reads confirming their dietary origin. This study establishes the first virome baseline for New Zealands migratory birds, highlighting the ecological role of foraging in shaping viral communities and improving regional preparedness for HPAI and other emerging avian pathogens.

microbiology↗

bioRxiv2025

Differential time-restricted sensitivity of enveloped viruses to Sec61 translocon blockade

The morphogenesis of enveloped viruses relies on the trafficking of transmembrane proteins through the secretory pathway to sites of virus envelopment. The first step in this pathway, their translocation into the endoplasmic reticulum, is therefore an attractive target for broad-spectrum intervention. Here, we tested if blockade of the Sec61 translocon by the Mycobacterium ulcerans exotoxin mycolactone, a potent inhibitor of Sec61, could block the production of virus glycoproteins and subsequent production of infectious virus from a range of human enveloped viruses: the DNA virus herpes simplex virus 1 (HSV1), and the RNA viruses, respiratory syncytial virus (RSV), influenza A virus (IAV), SARS coronavirus 2 (SARS CoV2) and Zika virus (ZIKV). In line with known translocation mechanisms, mycolactone blocked in vitro translocation and ectopic expression of type I transmembrane proteins but not type III, multipass or cytosolic proteins. Translocation of the type II protein RSV G was also blocked and although ectopically expressed G protein was detected, it was not glycosylated. Pretreatment of cells with mycolactone also blocked the synthesis of type I transmembrane proteins in infected cells and either the synthesis or glycosylation of type II transmembrane proteins, and the production of progeny from all viruses tested, while having no effect on virus entry or downstream synthesis of cytosolic proteins. While mycolactone treatment of HSV1 infected cells at various times after infection resulted in the immediate inhibition of virus production at the point of addition, IAV, RSV and ZIKV became resistant to the action of mycolactone surprisingly early in infection, and before virus glycoprotein synthesis was even detectable or virus production had begun. We therefore conclude that although inhibition of the translocation of virus transmembrane proteins through the Sec61 translocon can in principle block virus production, the morphogenesis of many enveloped RNA viruses requires only limited amounts of envelope proteins for successful propagation, providing novel insight into the biology of these viruses. Author SummaryMany circulating human pathogens are enveloped viruses that all use the cellular secretory pathway to target their envelope proteins to cellular sites of virus particle assembly. The potential to target this pathway could therefore offer a novel broad-spectrum therapy for existing, emerging and as yet unknown human pathogens. Here we have targeted the initial step in this pathway using a highly potent Sec61 inhibitor, mycolactone, to carry out the first comprehensive assessment of translocation disruption on a range of enveloped human viruses from different virus families, including herpes simplex virus, influenza A virus and SARS-CoV2. Our results have shown that Sec61 inhibition blocks the onward trafficking of many virus envelope proteins that are essential to produce infectious virus at assembly sites. However, unexpectedly, we found that several of the viruses were resistant to the effects of this toxin when it was added early in infection, indicating that the synthesis of these essential virus proteins occurs earlier in infection than previously recognised. Hence, while this approach may not be suitable as a broad intervention strategy, it has revealed new information on virus biology and provides us with a novel tool for exploring a wide range of enveloped viruses.

microbiology↗

bioRxiv2024

Spatial proteomics identifies a novel CRTC-dependent viral sensing pathway that stimulates production of Interleukin-11

Appropriate cellular recognition of viruses is essential for the generation of effective innate and adaptive antiviral immunity. Viral sensors and their signalling components thus provide a crucial first line of host defence. Many exhibit subcellular relocalisation upon activation, triggering expression of interferon and antiviral genes. To identify novel signalling factors we analysed protein relocalisation on a global scale during viral infection. CREB Regulated Transcription Coactivators-2 and 3 (CRTC2/3) exhibited early cytoplasmic-to-nuclear translocation upon a diversity of viral stimuli, in diverse cell types. This movement was depended on Mitochondrial Antiviral Signalling Protein (MAVS), cyclo-oxygenase proteins and protein kinase A. We identify a key effect of transcription stimulated by CRTC2/3 translocation as production of the pro-fibrogenic cytokine interleukin-11. This may be important clinically in viral infections associated with fibrosis, including SARS-CoV-2.

immunology↗

bioRxiv2024

Translational arrest and mRNA decay are independent activities of alphaherpesvirus virion host shutoff proteins

The herpes simplex virus 1 (HSV1) virion host shutoff (vhs) protein is an endoribonuclease that regulates the translational environment of the infected cell, by inducing the degradation of host mRNA via cellular exonuclease activity. To further understand the relationship between translational shutoff and mRNA decay, we have used ectopic expression to compare HSV1 vhs (vhsH) to its homologues from four other alphaherpesviruses - varicella zoster virus (vhsV), bovine herpesvirus 1 (vhsB), equine herpesvirus 1 (vhsE) and Mareks disease virus (vhsM). Only vhsH, vhsB and vhsE induced degradation of a reporter luciferase mRNA, with polyA+ in situ hybridisation indicating a global depletion of cytoplasmic polyA+ RNA and a concomitant increase in nuclear polyA+ RNA and the polyA tail binding protein PABPC1 in cells expressing these variants. By contrast, vhsV and vhsM failed to induce reporter mRNA decay and polyA+ depletion, but rather, induced cytoplasmic G3BP1 and polyA+ mRNA-containing granules and phosphorylation of the stress response proteins eIF2 and protein kinase R. Intriguingly, regardless of their apparent endoribonuclease activity, all vhs homologues induced an equivalent general blockade to translation as measured by single cell puromycin incorporation. Taken together, these data suggest that the activities of translational arrest and mRNA decay induced by vhs are separable and we propose that they represent sequential steps of the vhs host interaction pathway.

microbiology↗

bioRxiv2023

Pharmacologic inhibition of BAF chromatin remodeling complexes as a therapeutic approach to transcription factor-dependent cancers

The BRG/Brahma-associated factors (BAF or mSWI/SNF) family of chromatin remodeling complexes are critical regulators of gene expression and are major determinants of cancer and other diseases. Two paralog ATPases, SMARCA4 and SMARCA2 (BRG1 and BRM, respectively), provide the enzymatic activity required for chromatin remodeling. Here, we discover and characterize a novel series of compounds that potently and selectively inhibit SMARCA4/SMARCA2. Mutational and biochemical studies demonstrate that these inhibitors act through a unique mode of inhibition, distinct from reported SMARCA4/SMARCA2 inhibitors. Across a range of cancer cell lines, SMARCA4/SMARCA2 inhibition resulted in lineage-specific changes in chromatin accessibility at binding sites for key transcription factors (TFs). In uveal melanoma (UM), BAF inhibition resulted in loss of enhancer occupancy of SOX10 and MITF, two essential TFs, leading to down-regulation of the melanocytic gene expression program. In a mouse xenograft model of UM, SMARCA4/SMARCA2 inhibition was well tolerated and resulted in dose-dependent tumor regression correlating with pharmacodynamic modulation of BAF-target gene expression. These data provide the foundation for first-in-human studies of BAF ATPase inhibition as a novel therapeutic to treat TF-dependent cancers.

cancer biology↗

bioRxiv2022

Dysregulated nuclear export of the late herpes simplex virus 1 transcriptome through the vhs-VP22 axis uncouples virus cytopathic effect and virus production

Herpes simplex virus 1 (HSV1) expresses its genes in a classical cascade culminating in the production of large amounts of structural proteins to facilitate virus assembly. HSV1 lacking the virus protein VP22 ({Delta}22) exhibits late translational shutoff, a phenotype that has been attributed to the unrestrained activity of the virion host shutoff (vhs) protein, a virus-encoded endoribonuclease which induces mRNA degradation during infection. We have previously shown that vhs is also involved in regulating the nuclear-cytoplasmic compartmentalisation of the virus transcriptome, and in the absence of VP22 many virus transcripts are sequestered in the nucleus late in infection. Here we show that despite expressing minimal amounts of structural proteins and failing to plaque on human fibroblasts, the strain 17 {Delta}22 virus replicates and spreads as efficiently as Wt virus, but without causing cytopathic effect (CPE). Nonetheless, CPE-causing virus spontaneously appeared on {Delta}22-infected human fibroblasts, and four viruses isolated in this way had all acquired point mutations in vhs which rescued viral mRNA export and late protein translation. However, unlike a virus deleted for vhs, these viruses still induced the degradation of cellular mRNA, suggesting that vhs mutation in the absence of VP22 is necessary to overcome a disturbance in mRNA export rather than mRNA degradation. The ultimate outcome of secondary mutations in vhs is therefore the rescue of virus-induced CPE caused by late protein synthesis, and while there is a clear selective pressure on HSV1 to mutate vhs for optimal production of late structural proteins, the purpose of this is over and above that of virus production. Author SummaryHSV is a human pathogen that lytically infects cells of the epidermis. Following viral genome replication, structural proteins are produced in abundance to enable the rapid assembly and release of large quantities of infectious progeny. Infected cells also exhibit cytopathic effect (CPE), morphological changes that are exemplified by cell rounding and the breakage of cell-to-cell contacts, facilitating virus dissemination. Here we show that HSV1 with a mutation that results in the nuclear retention of viral mRNA and concomitant shutdown of late protein synthesis, also fails to cause CPE. However, unexpectedly, we found that this virus is still able to release large numbers of infectious virus which can spread between cells without any evidence of cell damage. Nonetheless, despite efficient virus productivity, this virus spontaneously mutates to rescue late protein production and CPE, with mutations mapping to the process of mRNA export. There is therefore a clear selective pressure on HSV1 to optimize the synthesis of late structural proteins, but the purpose of this is over and above that of virus production, a result that has implications for why viruses in general express such large amounts of structural proteins.

microbiology↗

bioRxiv2022

Herpes simplex virus 1 expressing GFP-tagged virion host shutoff (vhs) protein uncouples the activities of degradation and nuclear retention of the infected cell transcriptome

Virion host shutoff (vhs) protein is an endoribonuclease encoded by herpes simplex virus 1 (HSV1). Vhs causes a number of changes to the infected cell environment that favour translation of late (L) virus proteins: cellular mRNAs are degraded, immediate-early (IE) and early (E) viral transcripts are sequestered in the nucleus with polyA binding protein (PABPC1), and dsRNA is degraded to help dampen the PKR-dependent stress response. To further our understanding of the cell biology of vhs, we constructed a virus expressing vhs tagged at its C-terminus with GFP. When first expressed, vhs-GFP localised to juxtanuclear clusters, and later it colocalised and interacted with its binding partner VP16, and was packaged into virions. Despite vhs-GFP maintaining activity when expressed in isolation, it failed to degrade mRNA or relocalise PABPC1 during infection, while viral transcript levels were similar to those seen for a vhs knockout virus. PKR phosphorylation was also enhanced in vhs-GFP infected cells, in line with a failure to degrade dsRNA. Nonetheless, mRNA FISH revealed that as in Wt but not {Delta}vhs infection, IE and E, but not L transcripts were retained in the nucleus of vhs-GFP infected cells at late times. Moreover, a representative cellular transcript which is ordinarily highly susceptible to vhs degradation, was also retained in the nucleus. These results reveal that the vhs-induced nuclear retention of the infected cell transcriptome is dependent on vhs expression but not on its endoribonuclease activity, uncoupling these two functions of vhs. ImportanceLike many viruses, herpes simplex virus 1 (HSV1) expresses an endoribonuclease, the virion host shutoff (vhs) protein, which regulates the RNA environment of the infected cell and facilitates the classical cascade of virus protein translation. It does this by causing the degradation of some mRNA molecules and the nuclear retention of others. Here we describe a virus expressing vhs tagged at its C-terminus with green fluorescent protein (GFP) and show that the vhs-GFP fusion protein retains the physical properties of native vhs, but does not induce the degradation of mRNA. Nonetheless, vhs-GFP maintains the ability to trap the infected cell transcriptome in the nucleus, proving for the first time that mRNA degradation is not a prerequisite for vhs effects on the nuclear transcriptome. This virus has therefore uncoupled the nuclear retention and degradation activities of vhs, providing new understanding of vhs during infection.

microbiology↗

bioRxiv2021

Cell-to-cell transmission of HSV1 in human keratinocytes in the absence of the major entry receptor, nectin1

Herpes simplex virus 1 (HSV1) infects the stratified epithelia of the epidermis, oral or genital mucosa, where the main cell type is the keratinocyte. Here we have used nTERT human keratinocytes to generate a CRISPR-Cas9 knockout (KO) of the primary candidate HSV1 receptor, nectin1, resulting in a cell line that is refractory to HSV1 entry. Nonetheless, a small population of KO cells was able to support infection, and strikingly at later times, those cells originally resistant to HSV1 infection had also been infected. Appearance of this later population was blocked by inhibition of virus genome replication, or infection with a{Delta} UL34 virus defective in capsid export to the cytoplasm, while newly formed GFP-tagged capsids were detected in cells surrounding the initial infected cell, suggesting that virus was spreading following replication in the original susceptible cells. Neutralizing human serum failed to block virus transmission in KO cells, which was dependent on the cell-to-cell spread glycoproteins gI and gE, indicating that virus was spreading by direct cell-to-cell transmission. In line with these results, both HSV1 and HSV2 formed plaques on nectin1 KO cells, albeit at a reduced titre, confirming that once the original cell population was infected, the virus could spread into all other cells in the monolayer. Additional siRNA depletion of the second major HSV1 receptor HVEM, or PTP1B, a cellular factor shown elsewhere to be involved in cell-to-cell transmission, had no effect on virus spread in the absence of nectin1. We conclude that although nectin1 is required for extracellular entry in to the majority of human keratinocytes, it is dispensable for direct cell-to-cell transmission. Author SummaryHerpes simplex virus 1 (HSV1) infects the epithelia of the epidermis, oral or genital mucosa to cause cold sores, genital herpes, or more serious outcomes such as keratitis and neonatal herpes. Like many viruses, HSV1 can spread through the extracellular environment or by direct cell-to-cell transmission, with the latter mechanism being important for avoiding antibody responses in the host. Here we have studied HSV1 entry and transmission in the human keratinocyte, the main cell type in the target epithelia, by generating a CRISPR-Cas9 knockout of the primary candidate virus receptor, nectin1. While HSV1 was unable to infect the majority of nectin1 knockout keratinocytes, a small population of these nectin1 KO cells remained susceptible to virus entry, and once infected, the virus was able to spread unhindered into the rest of the monolayer. This spread continued in the presence of neutralising serum which blocks extracellular virus, and required the glycoproteins gE and gI which are known to be involved in cell-to-cell spread. Hence, while nectin1 is required for virus entry into the majority of human keratinocyte cells, it is dispensable for cell-to-cell transmission of the virus. These data have implications for the mechanism of HSV1 epithelial spread and pathogenesis.

microbiology↗

bioRxiv2021

Exquisite Sensitivity to Dual BRG1/BRM ATPase Inhibitors Reveals Broad SWI/SNF Dependencies in Acute Myeloid Leukemia

Various subunits of mammalian SWI/SNF chromatin remodeling complexes display loss-of-function mutations characteristic of tumor suppressors in different cancers, but an additional role for SWI/SNF supporting cell survival in distinct cancer contexts is emerging. In particular, genetic dependence on the catalytic subunit BRG1/SMARCA4 has been observed in acute myeloid leukemia (AML), yet the feasibility of direct therapeutic targeting of SWI/SNF catalytic activity in leukemia remains unknown. Here, we evaluated the activity of dual BRG1/BRM ATPase inhibitors across a genetically diverse panel of cancer cell lines and observed that hematopoietic cancer cell lines were among the most sensitive compared to other lineages. This result was striking in comparison to data from pooled short hairpin RNA screens, which showed that only a subset of leukemia cell lines display sensitivity to BRG1 knockdown. We demonstrate that combined genetic knockdown of BRG1 and BRM is required to recapitulate the effects of dual inhibitors, suggesting that SWI/SNF dependency in human leukemia extends beyond a predominantly BRG1-driven mechanism. Through gene expression and chromatin accessibility studies, we show that the dual inhibitors act at genomic loci associated with oncogenic transcription factors, and observe a downregulation of leukemic pathway genes including MYC, a well-established target of BRG1 activity in AML. Overall, small molecule inhibition of BRG1/BRM induced common transcriptional responses across leukemia models resulting in a spectrum of cellular phenotypes. Our studies reveal the breadth of SWI/SNF dependency and support targeting SWI/SNF catalytic function as a potential therapeutic strategy in AML.

cancer biology↗

bioRxiv2020

Novel role for ESCRT-III component CHMP4C in the integrity of the endocytic network utilized for herpes simplex virus envelopment

Enveloped viruses exploit cellular trafficking pathways for their morphogenesis, providing potential scope for the development of new antiviral therapies. We have previously shown that herpes simplex virus 1 (HSV1) utilises recycling endocytic membranes as the source of its envelope, in a process involving four Rab GTPases. To identify novel factors involved in HSV1 envelopment, we have screened an siRNA library targeting over eighty human trafficking proteins including coat proteins, adaptor proteins, fusion factors, fission factors and Rab effectors. Depletion of eleven factors reduced virus yield by 20- to 100-fold, including three early secretory pathway proteins; four late secretory pathway proteins; and four endocytic pathway proteins, three of which are membrane fission factors. Five of the eleven targets were chosen for further analysis in virus infection where it was found that the absence of only one, the fission factor CHMP4C, known for its role in the cytokinesis checkpoint, specifically reduced virus production at the final stage of morphogenesis. Ultrastructural and confocal microscopy of CHMP4C-depleted, HSV1-infected cells, showed an accumulation of endocytic membranes; extensive tubulation of recycling, transferrin receptor-positive endosomes indicative of aberrant fission; and a failure in virus envelopment. No effect on the late endocytic pathway was detected, while exogenous CHMP4C was shown to localise to recycling endosomes. Taken together, these data reveal a novel role for the CHMP4C fission factor in the integrity of the recycling endosomal network, which has been unveiled through the dependence of HSV1 on these membranes for the acquisition of their envelopes. ImportanceCellular transport pathways play a fundamental role in secretion and membrane biogenesis. Enveloped viruses exploit these pathways to direct their membrane proteins to sites of envelopment, and as such, are powerful tools for unravelling subtle activities of trafficking factors, potentially pinpointing therapeutic targets. Using the sensitive biological readout of virus production, over eighty trafficking factors involved in diverse and poorly defined cellular processes have been screened for involvement in the complex process of HSV1 envelopment. Out of eleven potential targets, CHMP4C - a key component in the cell-cycle abscission checkpoint - stood out as being required for the physical process of virus wrapping in endocytic tubules, where it was shown to localise. In the absence of CHMP4C, recycling endocytic membranes failed to undergo scission, causing transient tubulation and accumulation of membranes and unwrapped virus. These data reveal a new role for this important cellular factor in the biogenesis of recycling endocytic membranes.

cell biology↗

bioRxiv2020

Biomarker-guided treatment strategies for ovarian cancer identified from a heterogeneous panel of patient-derived tumor xenografts

Advanced ovarian cancers are a leading cause of cancer-related death in women. Such cancers are currently treated with surgery and chemotherapy which is often temporarily successful but exhibits a high rate of relapse after which treatment options are few. Here we assess the responses of a panel of patient-derived ovarian cancer xenografts (PDXs) to 19 mono and combination therapies, including small molecules and antibody-drug conjugates. The PDX panel aimed to mimic the heterogeneity of disease observed in patients, and exhibited a distribution of responsiveness to standard of care chemotherapy similar to human clinical data. Three monotherapies and one drug combination were found to be active in different subsets of PDXs. By analyzing gene expression data we identified gene expression biomarkers predictive of responsiveness to each of three novel targeted therapy regimens. While no single treatment had as high a response rate as chemotherapy, nearly 90% of PDXs were eligible for and responded to at least one biomarker-guided treatment, including tumors resistant to standard chemotherapy. Biomarker frequency was similar in human patients, suggesting the possibility of a new therapeutic approach to ovarian cancer and demonstrating the potential power of PDX-based trials in broadening the reach of precision cancer medicine.

cancer biology↗

bioRxiv2019

In-Depth Characterization and Validation in BRG1-Mutant Lung Cancers Define Novel Catalytic Inhibitors of SWI/SNF Chromatin Remodeling

Members of the ATP-dependent SWI/SNF chromatin remodeling complexes are among the most frequently mutated genes in cancer, suggesting their dysregulation plays a critical role. The synthetic lethality between SWI/SNF catalytic subunits BRM/SMARCA2 and BRG1/SMARCA4 has instigated great interest in targeting BRM. Here we have performed a critical and in-depth investigation of novel dual inhibitors (BRM011 and BRM014) of BRM and BRG1 in order to validate their utility as chemical probes of SWI/SNF catalytic function, while obtaining insights into the therapeutic potential of SWI/SNF inhibition. In corroboration of on-target activity, we discovered compound resistant mutations through pooled screening of BRM variants in BRG1-mut cancer cells. Strikingly, genome-wide transcriptional and chromatin profiling (ATAC-Seq) provided further evidence of pharmacological perturbation of SWI/SNF chromatin remodeling as BRM011 treatment induced specific changes in chromatin accessibility and gene expression similar to genetic depletion of BRM. Finally, these compounds have the capacity to inhibit the growth of tumor-xenografts, yielding important insights into the feasibility of developing BRM/BRG1 ATPase inhibitors for the treatment of BRG1-mut lung cancers. Overall, our studies not only establish the feasibility of inhibiting SWI/SNF catalytic function, providing a framework for SWI/SNF therapeutic targeting, but have also yielded successful elucidation of small-molecule inhibitors that will be of importance in probing SWI/SNF function in various disease contexts.

cancer biology↗

bioRxiv2018

Nuclear-cytoplasmic compartmentalization of the herpes simplex virus 1 infected cell transcriptome is co-ordinated by the viral endoribonuclease vhs and cofactors to facilitate the translation of late proteins

HSV1 encodes an endoribonuclease termed virion host shutoff (vhs) that is produced late in infection and packaged into virions. Paradoxically, vhs is active against not only host but also virus transcripts, and is involved in host shutoff and the temporal expression of the virus transcriptome. Two other virus proteins - VP22 and VP16 - are proposed to regulate vhs to prevent uncontrolled and lethal mRNA degradation but their mechanism of action is unknown. We have performed dual transcriptomic analysis and single-cell mRNA FISH of human fibroblasts, a cell type where in the absence of VP22, HSV1 infection results in extreme translational shutoff. In Wt infection, host mRNAs exhibited a wide range of susceptibility to vhs ranging from resistance to 1000-fold reduction, a variation that was independent of their relative abundance or transcription rate. However, vhs endoribonuclease activity was not found to be overactive against any of the cell transcriptome in {Delta}22-infected cells but rather was delayed, while its activity against the virus transcriptome and in particular late mRNA was minimally enhanced. Intriguingly, immediate-early and early transcripts exhibited vhs-dependent nuclear retention later in Wt infection but late transcripts were cytoplasmic. However, in the absence of VP22, not only early but also late transcripts were retained in the nucleus, a characteristic that extended to cellular transcripts that were not efficiently degraded by vhs. Moreover, the ability of VP22 to bind VP16 enhanced but was not fundamental to the rescue of vhs-induced nuclear retention of late transcripts. Hence, translational shutoff in HSV1 infection is primarily a result of vhs-induced nuclear retention and not degradation of infected cell mRNA. We have therefore revealed a new mechanism whereby vhs and its co-factors including VP22 elicit a temporal and spatial regulation of the infected cell transcriptome, thus co-ordinating efficient late protein production.\n\nAuthor SummaryHerpesviruses are large DNA viruses that replicate in the nucleus and express their genes by exploiting host cell mRNA biogenesis mechanisms including transcription, nuclear export, translation and turnover. As such, these viruses express multiple factors that enable the appropriation of cellular pathways for optimal virus production, and work in concert to shut off host gene expression and to overexpress virus genes in a well-described cascade that occurs in a temporal pattern of immediate-early, early and late proteins. We have analysed global and single cell changes in the host and virus transcriptome to uncover a novel mechanism by which the viral endoribonuclease, termed vhs, turns off early virus gene expression. This is achieved through the vhs-induced nuclear retention of the entire infected cell transcriptome at the onset of late gene expression. To enable the switch from early to late protein production the virus then requires a second factor called VP22 to specifically inhibit the nuclear retention of late transcripts allowing their translation in the cytoplasm. In this way, HSV1 elicits a temporal and spatial regulation of the infected cell transcriptome to co-ordinate efficient late protein production, a process that may be relevant to herpesviruses in general.

microbiology↗