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bioRxiv2024

Glutamic acid-lysine (EK) rich motif of RabD2 self-associates and regulates pathogenesis through multivesicular bodies pathway in E. histolytica

Entamoeba histolytica, an enteric pathogen, causes disease by adhering to and destroying the host tissues. The interactions between the parasite and host tissue enable rewiring of the gene expression and global membrane trafficking in the parasite. A fine balance between cargoes/receptors endocytosis and exocytosis is required to establish infection in the host. Multivesicular bodies (MVBs) act as sorting platforms, delivering cargoes/receptors to lysosomes for degradation or secreting their content through plasma membrane fusion. Some of the small GTPases are known to control MVB biogenesis in various organisms. However, the functional contribution of Rab GTPases in MVB biogenesis is poorly studied in E. histolytica. Here, we identified a novel atypical protein RabD2, with an N-terminal glutamic acid-lysine rich motif and a C-terminal conserved Rab domain. Our biochemical and cell biological assays provide evidence that RabD2 self-associates, and this interaction is controlled by the N-terminal EK-rich motif and the GTPase activity mutants (in a nucleotide-specific manner). RabD2 localizes on the surface of MVBs and controls their biogenesis. In line with these findings, overexpression of RabD2 upregulates global ubiquitination, directing the down regulation of the heavy chain of GalNAc lectin, ultimately leading to decreased adherence of E. histolytica trophozoites to host cells. Thus, amoebic RabD2 is a new class of Rab protein that forms high-ordered self-association variants and regulates the pathogenicity of E. histolytica through the biogenesis of MVBs. Author SummaryAmoebiasis is an enteral infection caused by Entamoeba histolytica that primarily remains asymptomatic but can eventually result in systemic complications like amoebic dysentery, liver abscess, and pulmonary effusions. Recent studies showed that Entamoeba ubiquitin is a robust antigen and linked with the invasive amoebiasis patient samples. Here, we identified a novel RabD2 that self-associates via its glutamic acid-lysine rich motif that causes high ubiquitination levels and biogenesis of multivesicular bodies. We uncovered for the first time that RabD2-mediated ubiquitin-dependent pathway is involved in the down regulation of the notable antigenic marker heavy chain of galactose-N-acetylgalactosamine lectin and thereby controls the parasite adherence to host cells. Further studies on crosstalk between parasite ubiquitination and antigenic receptors downregulation provide insights into how parasites use these strategies to establish the infection in the host intestine.

microbiology↗

bioRxiv2025

Inference and prediction for random walkmodels in biology

Parameter inference is a critical step in the process of interpreting biological data using mathematical models. Inference provides a means of deriving quantitative, mechanistic insights from sparse, noisy data. While methods for parameter inference, parameter identifiability, and model prediction are well-developed for deterministic continuum models, working with biological applications often requires stochastic modelling approaches to capture inherent variability and randomness that can be prominent in biological measurements and data. Random walk models are especially useful for capturing spatiotemporal processes, such as ecological population dynamics, molecular transport phenomena, and collective behaviour associated with multicellular phenomena. This review focuses on parameter inference, identifiability analysis, and model prediction for a suite of biologically-inspired, stochastic agent-based models relevant to animal dispersal and populations of biological cells. With a particular emphasis on model prediction, we highlight roles for numerical optimisation and automatic differentiation. Open-source Julia code is provided to support scientific reproducibility. We encourage readers to use this code directly or adapt it to suit their interests and applications.

systems biology↗

bioRxiv2017

Quantification of very low-abundant proteins in bacteria using the HaloTag and epi-fluorescence microscopy

Cell biology is increasingly dependent on quantitative methods resulting in the need for microscopic labelling technologies that are highly sensitive and specific. Whilst the use of fluorescent proteins has led to major advances, they also suffer from their relatively low brightness and photo-stability, making the detection of very low abundance proteins using fluorescent protein-based methods challenging. Here, we characterize the use of the self-labelling protein tag called HaloTag, in conjunction with an organic fluorescent dye, to label and accurately count endogenous proteins present in very low numbers (<7) in individual Escherichia coli cells. This procedure can be used to detect single molecules in fixed cells with conventional epifluorescence illumination and a standard microscope. We show that the detection efficiency of proteins labelled with the HaloTag is [≥]80%, which is on par or better than previous techniques. Therefore, this method offers a simple and attractive alternative to current procedures to detect low abundance molecules.

systems biology↗

bioRxiv2023

Sinorhizobium meliloti FcrX coordinates cell cycle and division during free-living growth and symbiosis

Sinorhizobium meliloti is a soil bacterium that establishes a symbiosis within root nodules of legumes (Medicago sativa, for example) where it fixes atmospheric nitrogen into ammonia and obtains in return carbon sources and other nutrients. In this symbiosis, S. meliloti undergoes a drastic cellular change leading to a terminal differentiated form (called bacteroid) characterized by genome endoreduplication, increase of cell size and high membrane permeability. The bacterial cell cycle (mis)regulation is at the heart of this differentiation process. In free-living cells, the master regulator CtrA ensures the progression of cell cycle by activating cell division (controlled by the tubulin-like protein FtsZ) and simultaneously inhibiting supernumerary DNA replication, while on the other hand the downregulation of CtrA and FtsZ is essential for bacteroid differentiation during symbiosis, preventing endosymbiont division and permitting genome endoreduplication. Little is known in S. meliloti about regulators of CtrA and FtsZ, as well as the processes that control bacteroid development. Here, we combine cell biology, biochemistry and bacterial genetics approaches to understand the function(s) of FcrX, a new factor that controls both CtrA and FtsZ, in free-living growth and in symbiosis. Depletion of the essential gene fcrX led to abnormally high levels of FtsZ and CtrA and minicell formation. Using multiple complementary techniques, we showed that FcrX is able to interact physically with FtsZ and CtrA. Moreover, its transcription is controlled by CtrA itself and displays an oscillatory pattern in the cell cycle. We further showed that, despite a weak homology with FliJ-like proteins, only FcrX proteins from closely-related species are able to complement S. meliloti fcrX function. Finally, deregulation of FcrX showed abnormal symbiotic behaviors in plants suggesting a putative role of this factor during bacteroid differentiation. In conclusion, FcrX is the first known cell cycle regulator that acts directly on both, CtrA and FtsZ, thereby controlling cell cycle, division and symbiotic differentiation.

microbiology↗

bioRxiv2020

Stabilising selection causes grossly altered but stable karyotypes in metastatic colorectal cancer

Aneuploidy, the loss and gain of whole and part chromosomes, is near-ubiquitous in cancer genomes and likely defines cancer cell biology. However, the temporal evolutionary dynamics that select for aneuploidy remain uncharacterised. Here we perform longitudinal genomic analysis of 755 samples from a total of 167 patients with colorectal-derived neoplastic lesions that represent distinct stages of tumour evolution through metastasis and treatment. Adenomas typically had few copy number alterations (CNAs) and most were subclonal, whereas cancers had many clonal CNAs, suggesting that progression goes through a CNA bottleneck. Individual CRC glands from the same tumour typically had very similar karyotypes, despite evidence of ongoing instability at the cell level in patient tumours, cell lines and organoids. CNAs in metastatic lesions sampled from liver and other organs, after chemotherapy or targeted therapies, and in late recurrences were typically similar to the primary tumour. Mathematical modelling and statistical inference indicated that these data are consistent with the action of negative selection on CNAs that traps cancer cell genomes on a fitness peak defined by the specific pattern of chromosomal aberrations. These data suggest that the initial progression of colorectal cancer requires the traversal of a rugged fitness landscape and subsequent CNA evolution, including metastatic dissemination and therapeutic resistance, is constrained by negative selection.

cancer biology↗

bioRxiv2022

Computational design of ultrasensitive flexible peptide:receptor signaling complexes for enhanced chemotaxis

Engineering protein biosensors that sensitively respond to specific biomolecules by triggering precise cellular responses is a major goal of diagnostics and synthetic cell biology. Previous biosensor designs have largely relied on binding structurally well-defined molecules. In contrast, approaches that couple the sensing of flexible compounds to intended cellular responses would greatly expand potential biosensor applications. Here, to address these challenges, we develop a computational strategy for designing signaling complexes between conformationally dynamic proteins and peptides. To demonstrate the power of the approach, we create ultrasensitive chemotactic receptor--peptide pairs capable of eliciting potent signaling responses and strong chemotaxis in primary human T cells. Unlike traditional approaches that engineer static binding complexes, our dynamic structure design strategy optimizes contacts with multiple binding and allosteric sites accessible through dynamic conformational ensembles to achieve unprecedented signaling efficacy and potency. Our study suggests that a conformationally adaptable binding interface coupled to a robust allosteric transmission region is a key evolutionary determinant of peptidergic GPCR signaling systems. The approach lays a foundation for designing peptide-sensing receptors and signaling peptide ligands for basic and therapeutic applications.

biophysics↗

bioRxiv2026

Adaptive evolution of Topoisomerase II triggers reproductive isolation in Drosophila

A fundamental driver of biological diversification is the evolution of reproductive barriers between species. Instability and mis-regulation of repetitive DNA underlie numerous post-zygotic reproductive barriers, yet the molecular mechanisms are unknown. A long-studied genetic incompatibility between Drosophila melanogaster and D. simulans arises from mis-segregation of the D. melanogaster-specific 359bp DNA satellite in hybrid embryos. Here we report that the D. simulans version of the essential enzyme Topoisomerase II/Top2 causes this lethal incompatibility. Combining interspecies gene swaps with cell biology and genetics revealed that D. simulans-specific adaptive divergence of Top2 DNA-interacting domains prevents the resolution of 359bp-induced topological stress. Our findings demonstrate that species-specific DNA satellite topology requires species-specific molecular machinery and that even vital housekeeping genes can underlie reproductive isolation between closely related species.

evolutionary biology↗

bioRxiv2020

Directed assembly and concentrating of micro/nanoparticles, cells, and vesicles via low-power near-infrared laser generated plasmonic microbubbles

Directed assembly and concentrating of micro- and nanoparticles via laser generated plasmonic microbubbles in a liquid environment is an emerging technology. For effective heating, visible light has been primarily employed in existing demonstrations. In this paper, we demonstrate a new plasmonic platform based on nanoporous gold disk (NPGD) array. Thanks to the highly tunable localized surface plasmon resonance of the NPGD array, microbubble of controlled size can be generated by near-infrared (NIR) light. Using NIR light provides several key advantages over visible light in less interference with standard microscopy and fluorescence imaging, preventing fluorescence photobleaching, less susceptible to absorption and scattering in turbid biological media, and much reduced photochemistry, phototoxicity and whatsoever. The large surface-to-volume ratio of NPGD further facilitates the heat transfer from these gold nanoheaters to the surroundings, achieving unprecedented low-power operation. While the microbubble is formed, the surrounding liquid circulates and direct microparticles randomly dispersed in the liquid to the bottom NPGD surface, yielding unique assemblies of microstructures. Such capability can also be employed in concentrating suspended colloidal nanoparticles at desirable sites and with preferred configuration, both enhancing the sensor performance. In addition to various micro- and nanoparticles, the plasmonic microbubbles are also shown to collect biological cells and nanovesicles. By using a spatial light modulator (SLM) to project the laser in arbitrary patterns, parallel assembly can be achieved to fabricate an array of clusters. These assemblies have been characterized using optical microscopy, scanning electron microscope, hyperspectral localized surface plasmon resonance imaging and hyperspectral Raman imaging.

bioengineering↗

bioRxiv2025

A genome-wide MAGIC kit for recombinase-independent mosaic analysis in Drosophila

Mosaic analysis has been instrumental in advancing developmental and cell biology. Most current mosaic techniques rely on exogenous site-specific recombination sequences that need to be introduced into the genome, limiting their application. Mosaic analysis by gRNA-induced crossing-over (MAGIC) was recently developed in Drosophila to eliminate this requirement by inducing somatic recombination through CRISPR/Cas9-generated DNA double-strand breaks. However, MAGIC has not been widely adopted because gRNA-markers, a required component for this technique, are not yet available for most chromosomes. Here, we present a complete, genome-wide gRNA-marker kit that incorporates optimized designs for enhanced clone induction and more effective clone labeling in both positive MAGIC (pMAGIC) and negative MAGIC (nMAGIC). With this kit, we demonstrate clonal analysis in a broad range of Drosophila tissues, including cell types that have been difficult to analyze using recombinase-based systems. Notably, MAGIC enables clonal analysis of pericentromeric genes and deficiency chromosomes and in interspecific hybrid animals, opening new avenues for gene function study, rapid gene discovery, and understanding cellular basis of speciation. This MAGIC kit complements existing systems and makes mosaic analysis accessible to address a wider range of biological questions. IMPACT STATEMENTA comprehensive toolkit enables genome-wide, recombinase-independent mosaic analysis in Drosophila, permitting clonal analysis of pericentromeric genes, deficiency chromosomes, and interspecific hybrids previously inaccessible to standard methods.

genetics↗

bioRxiv2025

Deletion of the Envelope gene attenuates SARS-CoV-2 infection by altered Spike localization and increased cell-to-cell transmission

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19, a highly transmissible acute respiratory infection that can result in severe pneumonia and death. Many details of SARS-CoV-2 infection are not fully understood, including the cell biology and host-virus interactions involved in coronavirus assembly and release, in which the Envelope (E) structural protein is instrumental. Deletion of E in other coronaviruses has been shown previously to either attenuate or abrogate infection. To determine the role of E on SARS-CoV-2 virus production and infectivity, we produced reporter SARS-CoV-2 with or without the E gene deleted using a bacterial artificial chromosome. Replication of {delta}E SARS-CoV-2 was attenuated in Vero E6 cells expressing human ACE2 and TMPRSS2 and in human epithelial cell lines. Electron and immunofluorescence microscopy and virology assays showed that {delta}E SARS-CoV-2 increased cell surface expression of Spike (S) glycoprotein, leading to reduced S incorporation into {delta}E SARS-CoV-2 particles and promotion of increased cell-to-cell transmission that evades neutralizing antibody inhibition. Trans-complementation of E partially rescued {delta}E SARS-CoV-2 S incorporation and restored cell-free transmission. In addition to validating the role of E in retention of S in the ER-Golgi intermediate complex (ERGIC), our results showed that a lack of E led to reorganization of the ERGIC and Golgi during SARS-CoV-2 infection. Improved understanding of E in SARS-CoV-2 replication and host pathogenesis may help development of novel therapeutics.

microbiology↗

bioRxiv2020

Human SYNGAP1 Regulates the Development of Neuronal Activity by Controlling Dendritic and Synaptic Maturation

SYNGAP1 is a major genetic risk factor for global developmental delay, autism spectrum disorder, and epileptic encephalopathy. De novo loss-of-function variants in this gene cause a neurodevelopmental disorder defined by cognitive impairment, social-communication disorder, and early-onset seizures. Cell biological studies in mouse and rat neurons have shown that Syngap1 regulates developing excitatory synapse structure and function, with loss-of-function variants driving formation of larger dendritic spines and stronger glutamatergic transmission. However, studies to date have been limited to mouse and rat neurons. Therefore, it remains unknown how SYNGAP1 loss-of-function impacts the development and function of human neurons. To address this, we employed CRISPR/Cas9 technology to ablate SYNGAP1 protein expression in neurons derived from a human induced pluripotent stem cell line (hiPSC). Reducing SynGAP protein expression in developing hiPSC-derived neurons enhanced dendritic morphogenesis, leading to larger neurons compared to those derived from isogenic controls. Consistent with larger dendritic fields, we also observed a greater number of morphologically defined excitatory synapses in cultures containing these neurons. Moreover, neurons with reduced SynGAP protein had stronger excitatory synapses and expressed synaptic activity earlier in development. Finally, distributed network spiking activity appeared earlier, was substantially elevated, and exhibited greater bursting behavior in SYNGAP1 null neurons. We conclude that SYNGAP1 regulates the postmitotic maturation of human neurons made from hiPSCs, which influences how activity develops within nascent neural networks. Alterations to this fundamental neurodevelopmental process may contribute to the etiology of SYNGAP1-related disorders.

neuroscience↗

bioRxiv2023

Vimentin intermediate filaments structure and mechanically support microtubules in cells

The eukaryotic cytoskeleton is an intricate network of three types of mechanically distinct biopolymers - actin filaments, microtubules and intermediate filaments (IFs). These filamentous networks determine essential cellular functions and properties. Among them, microtubules are important for intracellular transport and establishing cell polarity during migration. Despite their intrinsic stiffness, they exhibit characteristic bending and buckling in cells due to non thermal forces acting on them. Interactions between cytoskeletal filaments have been found but are complex and diverse with respect to their effect on the mechanical behavior of the filaments and the architecture of networks. We systematically study how actin and vimentin IFs influence the network structure and local bending of microtubules by analyzing fluorescence microscopy images of mouse fibroblasts on protein micropatterns. Our automated analysis averages over large amounts of data to mitigate the effect of the considerable natural variance in biological cell data. We find that the radial orientation of microtubules in circular cells is robust and is established independently of vimentin and actin networks. Observing the local curvature of microtubules, we find highly similar average bending of microtubules in the entire cell regardless of the cytoskeletal surrounding. Small systematic differences cannot be attributed directly to vimentin and actin densities. Our results suggest that, on average, microtubules in unpolarized mouse fibroblasts are unexpectedly independent of the rest of the cytoskeleton in their global network structure and their local response to forces.

biophysics↗

bioRxiv2024

Maturation and conformational switching of a de novo designed phase-separating polypeptide

Cellular compartments formed by biomolecular condensation are a widespread feature of cell biology. These organelle-like assemblies compartmentalize macromolecules dynamically within the crowded intracellular environment. However, the intermolecular interactions that produce condensed droplets may also create arrested states and potentially pathological assemblies such as fibers, aggregates, and gels, through droplet maturation. Protein liquid-liquid phase separation is a metastable process, so maturation may be an intrinsic property of phase-separating proteins, where nucleation of different phases or states arise in supersaturated condensates. Here, we describe the formation of both phase-separated droplets and proteinaceous fibers driven by a de novo designed polypeptide. We characterize the formation of supramolecular fibers in vitro and in bacterial cells. We show that client proteins can be targeted to the fibers in cells using the droplet-forming construct. Finally, we explore the interplay between phase separation and fiber formation of the de novo polypeptide, showing that the droplets mature with a post-translational switch to largely {beta} conformations, analogous to models of pathological phase separation.

synthetic biology↗

bioRxiv2020

Population dynamics with threshold effects give rise to a diverse family of Allee effects

The Allee effect describes populations that deviate from logistic growth models and arises in applications including ecology and cell biology. A common justification for incorporating Allee effects into population models is that the population in question has altered growth mechanisms at some critical density, often referred to as a threshold effect. Despite the ubiquitous nature of threshold effects arising in various biological applications, the explicit link between local threshold effects and global Allee effects has not been considered. In this work, we examine a continuum population model that incorporates threshold effects in the local growth mechanisms. We show that this model gives rise to a diverse family of Allee effects and we provide a comprehensive analysis of which choices of local growth mechanisms give rise to specific Allee effects. Calibrating this model to a recent set of experimental data describing the growth of a population of cancer cells provides an interpretation of the threshold population density and growth mechanisms associated with the population.

biophysics↗

bioRxiv2022

The dual character of the inhibitory functions of CD6

T-cell membrane scaffold proteins play important roles in T cell biology, functioning as multi-functional signaling hubs. CD6 assembles a large intracellular signalosome but, unlike typical membrane-attached scaffolds like LAT or PAG, it has a sizeable ectodomain that binds a well-characterized ligand, CD166. It is unclear whether CD6 has net inhibitory or costimulatory functions or how its ectodomain influences these activities. To explore these questions, we dissected the signaling functions of the extracellular and cytoplasmic regions of CD6. We found that CD6 was delivered to the immunological synapse and suppressed T cell responsiveness in vitro wholly dependently of its cytoplasmic domain, indicating that CD6 very potently imposes tonic inhibition, acting as a structural and signaling inhibitory hub. However, the cell-intrinsic suppression of autoimmunity by CD6 in vivo was also impacted by extracellular interactions, demonstrated by the increased susceptibility of mice to experimental autoimmune encephalomyelitis after removal of the ligand binding region of the ectodomain of CD6. Our work identifies CD6 as a new class of on/off switching scaffold-receptor that constrains immune responsiveness at two speeds. First, it sets signaling thresholds via tonic inhibition, functioning as a cytoplasmic membrane-bound scaffold and, second, by cycling between signaling-enabling and signalinginhibiting ectodomain isoforms it functions as an immune checkpoint.

immunology↗

bioRxiv2025

Fluid-Squid: DIY Multiplexed Imaging of Cells and Tissues

Recent advances in multiplexed single-cell characterization have revolutionized our insight into cell biology, but many available technologies remain limited by high costs or a lack of customizability. To address these challenges, we developed Fluid-Squid, a cost-effective, quantitative imaging platform that integrates automated fluidics to support customizable, do-it-yourself multiplexed imaging workflows. Using Fluid-Squid, we successfully imaged fresh frozen human intestinal tissues with a 36-plex oligonucleotide-barcoded antibody panel and further demonstrated the feasibility of lyophilizing such multiplexed panels. We also adapted existing multiplexed imaging workflows to characterize individual cells to identify immune cell populations, phenotype, and antigen-specific cells from mouse splenocytes and human peripheral blood mononuclear cells (PBMCs) with a 39-antibody panel. To further expand its utility, we developed a barcoding strategy that allows for the pooling and simultaneous staining of multiple samples, reducing time, costs, and batch effects in single-cell experiments. This approach facilitated rapid titration to optimize antibody concentrations and assess the impact of various blood preparation methods on cell type retention. Overall, our work provides a new open-source framework for automated fluidics and microscopy in a flexible, cost-effective platform, empowering adaptable multiplexed characterization of both single cells and tissues.

bioengineering↗

bioRxiv2026

A molecular description of plant cellulose biosynthesis inhibition

Dictating cell growth and morphology, cellulose biosynthesis is intrinsic to plant cell biology. Accordingly, cellulose biosynthesis inhibitors (CBIs) are important herbicides, toxins, and experimental tools. We currently lack mechanistic understanding of CBI activity, preventing engineering of herbicide selectivity and disease immunity. Contrasting classical inhibitors, we unexpectedly identify the unusual Streptomyces phytotoxin thaxtomin A as the only in vitro-active CBI, with unprecedentedly broad-spectrum activity against various cellulose synthase enzymes. High-resolution cryo-electron microscopy reveals that thaxtomin A leverages exotic nitroaromatic chemistry to target a strictly conserved site in cellulose synthases polysaccharide secretion channel. Strikingly, in vitro biosynthesis and biophysical assays demonstrate thaxtomin As near-picomolar efficacy. Plant and algal systems reveal that its global arrest of cellulose biosynthesis produces an osmotically driven crisis in expanding cells. Finally, site-directed mutagenesis generates the first toxin-resistant cellulose synthase. Our results underscore celluloses critical function in plant lifeforms and inform efforts to inhibit related enzymes across kingdoms.

plant biology↗

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↗