Biology subjects

Mercer, J.

Publications and source records attributed to Mercer, J..

At least 19 recordsLinked to original sources

bioRxiv2026

Agentic-AI-ready genome-wide poxvirus-host interaction screen refined by a protein language model

Recent mpox outbreaks highlight the necessity to understand the interactions between poxviruses and the human host. These can be discovered systematically through screening for host genes involved in infection at the single-cell level using RNA interference. However, off-target effects and assay noise obscure true gene-phenotype relationships, hampering the discovery of therapeutically relevant targets. Here, we show that integrating protein-protein interaction information derived from a protein language model boosts the discovery of vaccinia virus-host interactions. We propose ICARus - a positive-unlabelled read-out refinement framework to achieve this. Our approach enhances the identification of human genes with potential antiviral function. We provide the raw and refined read-outs of a genome-wide screen for vaccinia virus host factors as an agentic-AI-enabled community resource. Our findings provide a generalisable strategy for robust hit prioritisation in functional screens, accelerating discovery across complex biological systems.

systems biology↗

bioRxiv2026

Critical period plasticity enables credit assignment

Synaptic plasticity is often guided by instructive inputs to neural circuits, but learning only succeeds when these instructions reach neurons that mediate relevant outputs. This creates the credit assignment problem: how does a neuron receive instructions suited to its own behavioral function? Here we report a developmental mechanism that enables credit assignment by using instructive inputs to organize the downstream architecture through which learning is expressed. In the olivocerebellar learning system, the inferior olive provides instructive inputs that guide plasticity within the cerebellum. During circuit assembly in zebrafish, we find these same inputs regulate long-range cerebellar projections during a highly plastic, two-day critical period. Developmental experience caused specific maturation of projections to targets that were coactivated with olivary inputs. Mathematical theory and computational modeling show how the resulting architecture constrains later learning, such that a single input can sculpt downstream connectivity and then leverage that circuit to assign credit. Simulated learning became paradoxically more robust if we reduced plasticity after a developmental critical period, protecting key architecture from corruption during learning. Thus, instructive inputs can first build the circuits they later teach, coordinating development and learning to enable effective credit assignment.

neuroscience↗

bioRxiv2026

Granularity screening identifies candidate genes involved in vaccinia virus induced LC3 lipidation

Autophagy is a catabolic process used for the degradation of organelles and proteins. Macroautophagy involves the formation of autophagosomes and subsequent fusion with lysosomes to mediate cargo degradation. It also functions as a cellular defence mechanism, known as xenophagy, during infection. Previous studies show that different viruses manipulate the autophagy pathway of the host cell to assure successful replication and/or virion assembly. Vaccinia virus (VACV), the prototypic poxvirus, replicates exclusively in the cytoplasm of host cells. It is known that VACV infection causes LC3 lipidation and prevents autophagosome formation, yet the double membrane vesicles formed during autophagy do not serve as the source of the mature VACV membrane. To date the viral protein(s) causing increased LC3 lipidation have not been identified. Here we developed an image-based screening approach based on LC3 granularity to identify candidate VACV genes affecting its lipidation. We identify several candidate viral membrane proteins as effectors of LC3 lipidation, suggesting that the interplay between VACV and autophagy is more directed than previously thought.

cell biology↗

bioRxiv2023

Alternative cell entry mechanisms for SARS-CoV-2 and multiple animal viruses

The cell entry mechanism of SARS-CoV-2, the causative agent of the COVID-19 pandemic, is not fully understood. Most animal viruses hijack cellular endocytic pathways as an entry route into the cell. Here, we show that in cells that do not express serine proteases such as TMPRSS2, genetic depletion of all dynamin isoforms blocked the uptake and strongly reduced infection with SARS-CoV-2 and its variant Delta. However, increasing the viral loads partially and dose-dependently restored infection via a thus far uncharacterized entry mechanism. Ultrastructural analysis by electron microscopy showed that this dynamin-independent endocytic processes appeared as 150-200 nm non-coated invaginations and was efficiently used by numerous mammalian viruses, including alphaviruses, influenza, vesicular stomatitis, bunya, adeno, vaccinia, and rhinovirus. Both the dynamin-dependent and dynamin-independent infection of SARS-CoV-2 required a functional actin cytoskeleton. In contrast, the alphavirus Semliki Forest virus, which is smaller in diameter, required actin only for the dynamin-independent entry. The presence of TMPRSS2 protease rescued SARS-CoV-2 infection in the absence of dynamins. Collectively, these results indicate that some viruses such as canine parvovirus and SARS-CoV-2 mainly rely on dynamin for endocytosis-dependent infection, while other viruses can efficiently bypass this requirement harnessing an alternative infection entry route dependent on actin.

microbiology↗

bioRxiv2023

56Fe ion exposure increases the incidence of lung and brain tumors at a similar rate in male and female mice

The main deterrent to long-term space travel is the risk of Radiation Exposure Induced Death (REID). The National Aeronautics and Space Administration (NASA) has adopted Permissible Exposure Levels (PELs) to limit the probability of REID to 3% for the risk of death due to radiation-induced carcinogenesis. The most significant contributor to current REID estimates for astronauts is the risk of lung cancer. Recently updated lung cancer estimates from Japans atomic bomb survivors showed that the excess relative risk of lung cancer by age 70 is roughly four-fold higher in females compared to males. However, whether sex differences may impact the risk of lung cancer due to exposure to high charge and energy (HZE) radiation is not well studied. Thus, to evaluate the impact of sex differences on the risk of solid cancer development post-HZE radiation exposure, we irradiated Rbfl/fl; Trp53fl/+ male and female mice infected with Adeno-Cre with various doses of 320 kVp X-rays or 600 MeV/n 56Fe ions and monitored them for any radiation-induced malignancies. We observed that lung adenomas/carcinomas and esthesioneuroblastomas (ENBs) were the most common primary malignancies in X-ray and 56Fe ion-exposed mice, respectively. In addition, 1 Gy 56Fe ion exposure compared to X-rays led to a significantly higher incidence of lung adenomas/carcinomas (p=0.02) and ENBs (p<0.0001). However, we did not find a significantly higher incidence of any solid malignancies in female mice as compared to male mice, regardless of radiation quality. Furthermore, gene expression analysis of ENBs suggested a distinct gene expression pattern with similar hallmark pathways altered, such as MYC targets and MTORC1 signaling, in X-ray and 56Fe ion-induced ENBs. Thus, our data revealed that 56Fe ion exposure significantly accelerated the development of lung adenomas/carcinomas and ENBs compared to X-rays, but the rate of solid malignancies was similar between male and female mice, regardless of radiation quality.

cancer biology↗

bioRxiv2022

The Vaccinia virus chondroitin sulfate binding protein drives host membrane curvature to facilitate fusion

Virus binding serves to define virus tropism and species specificity1. Virus binding proteins are classically considered as facilitators of cell surface attachment prior to receptor engagement and virus internalization. For efficient entry vaccinia virus (VACV) - the prototypic poxvirus - relies on four binding proteins and an eleven-protein entry fusion complex (EFC)2. We recently demonstrated that VACV binding and fusion proteins are organized into distinct functional domains, with localization of EFC proteins to virion tips directly influencing membrane fusion activity3. However, the relationship between virus binding protein distribution, virion binding orientation and subsequent membrane fusion remain unexplored. Here, we show that virus binding proteins guide side-on virion binding and promote curvature of the host membrane towards EFC-containing virion tips to facilitate virus fusion. Using a cell-derived membrane-bleb model system together with super-resolution and electron microscopy we found that side-bound VACV virions induce membrane invagination in the presence of low pH. Repression or deletion of individual binding proteins revealed that three of four contribute to binding orientation, amongst which the chondroitin sulphate binding protein, D8, is required for host membrane bending. Consistent with low-pH dependent macropinocytic entry of vaccinia virus4,5, loss of D8 prevents virion-associated macropinosome membrane bending, disrupts fusion pore formation and infection kinetics. Our results extend the role of viral binding proteins from mere attachment factors to active participants in successful viral membrane fusion and further illustrate the influence of virus protein architecture on successful infection.

microbiology↗

bioRxiv2021

Programmable large DNA deletion, replacement, integration, and inversion with twin prime editing and site-specific recombinases

The targeted deletion, replacement, integration, or inversion of DNA sequences at specified locations in the genome could be used to study or treat many human genetic diseases. Here, we describe twin prime editing (twinPE), a method for the programmable replacement or excision of DNA sequence at endogenous human genomic sites without requiring double-strand DNA breaks. TwinPE uses a prime editor (PE) protein and two prime editing guide RNAs (pegRNAs) that template the synthesis of complementary DNA flaps on opposing strands of genomic DNA, resulting in the replacement of endogenous DNA sequence between the PE-induced nick sites with pegRNA-encoded sequences. We show that twinPE in human cells can perform precise deletions of at least 780 bp and precise replacements of genomic DNA sequence with new sequences of at least 108 bp. By combining single or multiplexed twinPE with site-specific serine recombinases either in separate steps or in a single step, we demonstrate targeted integration of gene-sized DNA plasmids (>5,000 bp) into safe-harbor loci including AAVS1, CCR5, and ALB in human cells. To our knowledge, these results represent the first RNA-programmable insertion of gene-sized DNA sequences into targeted genomic sites of unmodified human cells without requiring double-strand breaks or homology-directed repair. Twin PE combined with recombinases also mediated a 40,167-bp inversion at IDS that corrects a common Hunter syndrome allele. TwinPE expands the capabilities of precision gene editing without requiring double-strand DNA breaks and synergizes with other tools to enable the correction or complementation of large or complex pathogenic alleles in human cells.

genomics↗

bioRxiv2021

Vaccinia Virus Arrests and Shifts the Cell Cycle

Modulation of the host cell cycle is a common strategy used by viruses to create a pro-replicative environment. To facilitate viral genome replication, vaccinia virus (VACV) has been reported to alter cell cycle regulation and trigger the host cell DNA damage response. However, the cellular factors and viral effectors that mediate these changes remain unknown. Here, we set out to investigate the effect of VACV infection on cell proliferation and host cell cycle progression. Using a subset of VACV mutants we characterize the stage of infection required for inhibition of cell proliferation and define the viral effectors required to dysregulate the host cell cycle. Consistent with previous studies, we show that VACV inhibits, and subsequently shifts the host cell cycle. We demonstrate that these two phenomena are independent of one another, with viral early genes being responsible for cell cycle inhibition, and post-replicative viral gene(s) responsible for the cell cycle shift. Extending previous findings, we show that the viral kinase F10 is required to activate the DNA damage checkpoint and that the viral B1/B12 (pseudo) kinases mediate degradation of checkpoint effectors p53 and p21 during infection. We conclude that VACV modulates host cell proliferation and host cell cycle progression through temporal expression of multiple VACV effector proteins.

microbiology↗

bioRxiv2021

Vaccinia virus subverts xenophagy through phosphorylation and nuclear targeting of p62

Autophagy is an essential degradation program required to maintain cell homeostasis. Amongst its many functions is the engulfment and destruction of cytosolic pathogens, termed Xenophagy. Not surprisingly, many pathogens have adapted various strategies to circumvent or co-opt autophagic degradation during infection. For poxviruses, it is known that infection activates autophagy, which however is not required for successful replication. Despite the fact that these complex viruses replicate exclusively in the cytoplasm, autophagy-mediated control of poxvirus infection has not been explored. Using the prototypic poxvirus, vaccinia virus (VACV), we show that over-expression of the xenophagy receptors p62, NDP52 and Tax1Bp1 restricts poxvirus infection. While NDP52 and Tax1Bp1 were degraded, p62 was found to initially target cytoplasmic virions before being shunted to the nucleus. Nuclear translocation of p62 during infection was dependent upon p62 NLS2, and VACV kinase mediated phosphorylation of associated p62 residues T269/S272. These results indicate that VACV actively targets the xenophagy receptor p62 during the early stages of infection to avoid destruction, and further suggest that poxviruses exhibit a unique multi-layered control of autophagy in order to facilitate cytoplasmic replication.

microbiology↗

bioRxiv2020

Poxviruses package viral redox proteins in lateral bodies and modulate the host oxidative response

All poxviruses contain a set of proteinaceous structures termed lateral bodies (LB) that deliver viral effector proteins into the host cytosol during virus entry. To date, the spatial proteotype of LBs remains unknown. Using the prototypic poxvirus, vaccinia virus (VACV), we employed a quantitative comparative mass spectrometry strategy to determine the poxvirus LB proteome. We identified a large population of cellular proteins, the majority being mitochondrial, and 15 viral LB proteins. Strikingly, one-third of these comprise the full set of VACV redox proteins whose LB residency could be confirmed using super-resolution microscopy. We further show that VACV infection exerts an anti-oxidative effect on host cells and that artificial induction of oxidative stress impacts early gene expression and virion production. In addition to defining the spatial proteotype of these enigmatic viral structures, these findings implicate poxvirus redox proteins as modulators of host oxidative anti-viral responses and provide a solid starting point for future investigations into the role of LB resident proteins in host immunomodulation.

microbiology↗

bioRxiv2020

Modulation of early host innate immune response by a Fowlpox virus (FWPV) lateral body protein

The avian pathogen, fowlpox virus (FWPV) has been successfully used as vaccine vector in poultry and humans but relatively little is known about its ability to modulate host antiviral immune responses in these hosts, which are replication permissive and non-permissive, respectively. FWPV is highly resistant to avian type I interferon (IFN) and able to completely block the host IFN-response. Microarray screening of host IFN-regulated gene expression in cells infected with 59 different, non-essential FWPV gene knock-out mutants revealed that FPV184 confers immunomodulatory capacity. We report that FPV184-knockout virus (FWPV{Delta}184) induces the cellular IFN response as early as 2 hours post-infection. The wild-type, uninduced phenotype can be rescued by transient expression of FPV184 in FWPV{Delta}184-infected cells. Ectopic expression of FPV184 inhibited polyI:C activation of the chicken IFN-{beta} promoter and IFN- activation of the chicken Mx promoter. Confocal and correlative super-resolution light and electron microscopy demonstrated that FPV184 has a functional nuclear localisation signal domain and is packaged in the lateral bodies of the virions. Taken together, these results provide a paradigm for a late poxvirus structural protein packaged in the lateral bodies and capable of supressing IFN induction early during the next round of infection.

microbiology↗

bioRxiv2020

Acrylamide Inhibits Vaccinia Virus Through Vimentin-independent Anti-Viral Granule Formation

The replication and assembly of vaccinia virus (VACV), the prototypic poxvirus, occurs exclusively in the cytoplasm of host cells. While the role of cellular cytoskeletal components in these processes remains poorly understood, vimentin - a type III intermediate filament - has been shown to associate with viral replication sites and to be incorporated into mature VACV virions. Here we employed chemical and genetic approaches to further investigate the role of vimentin during the VACV lifecycle. The collapse of vimentin filaments, using acrylamide, was found to inhibit VACV infection at the level of genome replication, intermediate- and late- gene expression. However, we found that CRISPR-mediated knockout of vimentin did not impact VACV replication. Combining these tools, we demonstrate that acrylamide treatment results in the formation of antiviral granules (AVGs) known to mediate translational inhibition of many viruses. We conclude that vimentin is dispensable for poxvirus replication and assembly and that acrylamide, as a potent inducer of AVGs during VACV infection, serves to bolster cells antiviral response to poxvirus infection. Summary StatementAcrylamide inhibits poxvirus replication by inducing anti-viral granules and blocking translation. This inhibition is independent of the effect of acrylamide on vimentin filaments which were found to be dispensable for viral replication and assembly.

microbiology↗

bioRxiv2020

Vaccinia virus hijacks ESCRT-mediated multivesicular body formation for virus egress

Unlike most enveloped viruses, poxvirus egress is a complex process whereby cytoplasmic single membrane-bound virions are wrapped in a cell-derived double membrane. These triple membrane-bound particles, termed intracellular enveloped virions (IEVs), are then released from infected cells by fusion. While the wrapping double membrane is thought to be derived from virus-modified trans-Golgi or early endosomal cisternae, the cellular factors that regulate virus wrapping remain largely undefined. To identify novel cell factors required for this process the prototypic poxvirus, vaccinia virus (VACV), was subjected to a high-throughput RNAi screen directed against cellular membrane trafficking proteins. Focusing on the endosomal sorting complexes required for transport (ESCRT), we demonstrate that ESCRT-III and VPS4 are required for packaging of virus into multivesicular bodies (MVBs). EM-based characterization of these MVB-IEVs showed that they account for half of IEV production indicating that MVBs serve as a second major source of VACV wrapping membrane. These data support a model whereby, in addition to cisternae-based wrapping, VACV hijacks ESCRT-mediated MVB formation to facilitate virus egress and spread.

cell biology↗

bioRxiv2019

In vivo control of Toxoplasma gondii by zebrafish macrophages

Toxoplasma gondii is an obligate intracellular parasite capable of invading any nucleated cell. Three main clonal lineages (type I, II, III) exist and murine models have driven the understanding of general and strain-specific immune mechanisms underlying Toxoplasma infection. However, murine models are limited for studying parasite-leukocyte interactions in vivo, and discrepancies exist between cellular immune responses observed in mouse versus human cells. Here, we develop a zebrafish infection model to study the innate immune response to Toxoplasma in vivo. By infecting the zebrafish hindbrain ventricle, and using high-resolution microscopy techniques coupled with computer vision driven automated image analysis, we reveal that Toxoplasma invades and replicates inside a parasitophorous vacuole to which type I and III parasites recruit host cell mitochondria. We show that type II and III strains maintain a higher infectious burden than type I strains. To understand how parasites are being cleared in vivo, we analyzed Toxoplasma-macrophage interactions using time-lapse and correlative light and electron microscopy. Strikingly, macrophages are recruited to the infection site and play a key role in Toxoplasma control. These results highlight in vivo control of Toxoplasma by macrophages, and illuminate the possibility to exploit zebrafish for discoveries within the field of parasite immunity.

microbiology↗

bioRxiv2019

Mimicry embedding for advanced neuralnetwork training of 3D biomedical micrographs

The use of deep neural networks (DNNs) for analysis of complex biomedical images shows great promise but is hampered by a lack of large verified datasets for rapid network evolution. Here we present a novel \"mimicry embedding\" strategy for rapid application of neural network architecture-based analysis of biomedical imaging datasets. Embedding of a novel biological dataset, such that it mimics a verified dataset, enables efficient deep learning and seamless architecture switching. We apply this strategy across various microbiological phenotypes; from super-resolved viruses to in vivo parasitic infections. We demonstrate that mimicry embedding enables efficient and accurate analysis of three-dimensional microscopy datasets. The results suggest that transfer learning from pre-trained network data may be a powerful general strategy for analysis of heterogeneous biomedical imaging datasets.

bioinformatics↗

bioRxiv2018

Fix your membrane receptor imaging: Actin cytoskeleton and CD4 membrane organization disruption by chemical fixation

Single-molecule localization microscopy (SMLM) techniques allow near molecular scale resolution (~ 20nm) as well as precise and robust analysis of protein organization at different scales. SMLM hardware, analytics and probes have been the focus of a variety of studies and are now commonly used in laboratories across the world. Protocol reliability and artefact identification are increasingly seen as important aspects of super-resolution microscopy. The reliability of these approaches thus requires in-depth evaluation so that biological findings are based on solid foundations. Here we explore how different fixation approaches that disrupt or preserve the actin cytoskeleton affect membrane protein organization. Using CD4 as a model, we show that fixation-mediated disruption of the actin cytoskeleton correlates with changes in CD4 membrane organization. We highlight how these artefacts are easy to overlook and how careful sample preparation is essential for extracting meaningful results from super-resolution microscopy.

cell biology↗

bioRxiv2018

NanoJ: a high-performance open-source super-resolution microscopy toolbox

Super-resolution microscopy has become essential for the study of nanoscale biological processes. This type of imaging often requires the use of specialised image analysis tools to process a large volume of recorded data and extract quantitative information. In recent years, our team has built an open-source image analysis framework for super-resolution microscopy designed to combine high performance and ease of use. We named it NanoJ - a reference to the popular ImageJ software it was de-veloped for. In this paper, we highlight the current capabilities of NanoJ for several essential processing steps: spatio-temporal alignment of raw data (NanoJ-Core), super-resolution image re-construction (NanoJ-SRRF), image quality assessment (NanoJ-SQUIRREL), structural modelling (NanoJ-VirusMapper) and control of the sample environment (NanoJ-Fluidics). We expect to expand NanoJ in the future through the development of new tools designed to improve quantitative data analysis and measure the reliability of fluorescent microscopy studies.

bioinformatics↗

bioRxiv2018

An Artificial Intelligence Workflow for Defining Host-Pathogen Interactions

For image-based infection biology, accurate unbiased quantification of host-pathogen interactions is essential, yet often performed manually or using limited enumeration employing simple image analysis algorithms based on image segmentation. Host protein recruitment to pathogens is often refractory to accurate automated assessment due to its heterogeneous nature. An intuitive intelligent image analysis program to assess host protein recruitment within general cellular pathogen defense is lacking. We present HRMAn (Host Response to Microbe Analysis), an open-source image analysis platform based on machine learning algorithms and deep learning. We show that HRMAn has the capability to learn phenotypes from the data, without relying on researcher-based assumptions. Using Toxoplasma gondii and Salmonella typhimurium we demonstrate HRMAns capacity to recognize, classify and quantify pathogen killing, replication and cellular defense responses.

microbiology↗