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Using lysophosphatidylcholine, a curvature-inducing lysolipid, we have isolated a reversible, "stalled pore" phenotype during syncytium formation induced by the p14 fusion-associated small transmembrane (FAST) protein and influenza virus hemagglutinin (HA) fusogens. This is the first evidence that lateral propagation of stable fusion pores leading to syncytiogenesis mediated by diverse viral fusogens is inhibited by promotion of positive membrane curvature in the outer leaflets of the lipid bilayer surrounding intercellular fusion pores.
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Células Gigantes/fisiologia , Células Gigantes/virologia , Hemaglutininas Virais/metabolismo , Lisofosfatidilcolinas/metabolismo , Fusão de Membrana/fisiologia , Modelos Biológicos , Proteínas Virais de Fusão/metabolismo , Animais , Chlorocebus aethiops , Microscopia de Fluorescência , Células VeroRESUMO
UNLABELLED: Orthoreovirus fusion-associated small transmembrane (FAST) proteins are dedicated cell-cell fusogens responsible for multinucleated syncytium formation and are virulence determinants of the fusogenic reoviruses. While numerous studies on the FAST proteins and enveloped-virus fusogens have delineated steps involved in membrane fusion and pore formation, little is known about the mechanics of pore expansion needed for syncytiogenesis. We now report that RNA interference (RNAi) knockdown of annexin A1 (AX1) expression dramatically reduced both reptilian reovirus p14 and measles virus F and H protein-mediated pore expansion during syncytiogenesis but had no effect on pore formation. A similar effect was obtained by chelating intracellular calcium, which dramatically decreased syncytiogenesis in the absence of detectable effects on p14-induced pore formation. Coimmunoprecipitation revealed calcium-dependent interaction between AX1 and p14 or measles virus F and H proteins, and fluorescence resonance energy transfer (FRET) demonstrated calcium-dependent p14-AX1 interactions in cellulo. Furthermore, antibody inhibition of extracellular AX1 had no effect on p14-induced syncytium formation but did impair cell-cell fusion mediated by the endogenous muscle cell fusion machinery in C2C12 mouse myoblasts. AX1 can therefore exert diverse, fusogen-specific effects on cell-cell fusion, functioning as an extracellular mediator of differentiation-dependent membrane fusion or as an intracellular promoter of postfusion pore expansion and syncytium formation following virus-mediated cell-cell fusion. IMPORTANCE: Numerous enveloped viruses and nonenveloped fusogenic orthoreoviruses encode membrane fusion proteins that induce syncytium formation, which has been linked to viral pathogenicity. Considerable insights into the mechanisms of membrane fusion have been obtained, but processes that drive postfusion expansion of fusion pores to generate syncytia are poorly understood. This study identifies intracellular calcium and annexin A1 (AX1) as key factors required for efficient pore expansion during syncytium formation mediated by the reptilian reovirus p14 and measles virus F and H fusion protein complexes. Involvement of intracellular AX1 in syncytiogenesis directly correlates with a requirement for intracellular calcium in p14-AX1 interactions and pore expansion but not membrane fusion and pore formation. This is the first demonstration that intracellular AX1 is involved in pore expansion, which suggests that the AX1 pathway may be a common host cell response needed to resolve virus-induced cell-cell fusion pores.
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Anexina A1/metabolismo , Cálcio/metabolismo , Regulação Viral da Expressão Gênica/genética , Células Gigantes/virologia , Vírus do Sarampo/metabolismo , Orthoreovirus/metabolismo , Proteínas Virais/metabolismo , Animais , Fusão Celular , Linhagem Celular , Chlorocebus aethiops , DNA Complementar/genética , Fibroblastos , Transferência Ressonante de Energia de Fluorescência , Regulação Viral da Expressão Gênica/fisiologia , Células Gigantes/fisiologia , Proteínas de Fluorescência Verde , Humanos , Camundongos , Orthoreovirus/patogenicidade , Plasmídeos/genética , Codorniz , Interferência de RNA , Células Vero , Proteínas Virais de Fusão/metabolismo , VirulênciaRESUMO
We previously found that enveloped virus binding and penetration are necessary to initiate an interferon-independent, IRF3-mediated antiviral response. To investigate whether membrane perturbations that accompany membrane fusion-dependent enveloped-virus entry are necessary and sufficient for antiviral-state induction, we utilized a reovirus fusion-associated small transmembrane (FAST) protein. Membrane disturbances during FAST protein-mediated fusion, in the absence of additional innate immune response triggers, are sufficient to elicit interferon-stimulated gene induction and establishment of an antiviral state. Using sensors of membrane disruption to activate an IRF3-dependent, interferon-independent antiviral state may provide cells with a rapid, broad-spectrum innate immune response to enveloped-virus infections.
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Membrana Celular/fisiologia , Membrana Celular/virologia , Fator Regulador 3 de Interferon/metabolismo , Reoviridae/imunologia , Internalização do Vírus , Animais , Chlorocebus aethiops , Regulação da Expressão Gênica , Células VeroRESUMO
Genotypic and phenotypic adaptation is the consequence of ongoing natural selection in populations and is key to predicting and preventing drug resistance. Whereas classic antibiotic persistence is all-or-nothing, here we demonstrate that an antibiotic resistance gene displays linear dose-responsive selection for increased expression in proportion to rising antibiotic concentration in growing Escherichia coli populations. Furthermore, we report the potentially wide-spread nature of this form of emergent gene expression (EGE) by instantaneous phenotypic selection process under bactericidal and bacteriostatic antibiotic treatment, as well as an amino acid synthesis pathway enzyme under a range of auxotrophic conditions. We propose an analogy to Ohm's law in electricity (V = IR), where selection pressure acts similarly to voltage (V), gene expression to current (I), and resistance (R) to cellular machinery constraints and costs. Lastly, mathematical modeling using agent-based models of stochastic gene expression in growing populations and Bayesian model selection reveal that the EGE mechanism requires variability in gene expression within an isogenic population, and a cellular "memory" from positive feedbacks between growth and expression of any fitness-conferring gene. Finally, we discuss the connection of the observed phenomenon to a previously described general fluctuation-response relationship in biology.
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An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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The SARS-CoV-2 pandemic has shown how a rapid rise in demand for patient and community sample testing can quickly overwhelm testing capability globally. With most diagnostic infrastructure dependent on specialized instruments, their exclusive reagent supplies quickly become bottlenecks, creating an urgent need for approaches to boost testing capacity. We address this challenge by refocusing the London Biofoundry onto the development of alternative testing pipelines. Here, we present a reagent-agnostic automated SARS-CoV-2 testing platform that can be quickly deployed and scaled. Using an in-house-generated, open-source, MS2-virus-like particle (VLP) SARS-CoV-2 standard, we validate RNA extraction and RT-qPCR workflows as well as two detection assays based on CRISPR-Cas13a and RT-loop-mediated isothermal amplification (RT-LAMP). In collaboration with an NHS diagnostic testing lab, we report the performance of the overall workflow and detection of SARS-CoV-2 in patient samples using RT-qPCR, CRISPR-Cas13a, and RT-LAMP. The validated RNA extraction and RT-qPCR platform has been installed in NHS diagnostic labs, increasing testing capacity by 1000 samples per day.
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Betacoronavirus/genética , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Pneumonia Viral/diagnóstico , Pneumonia Viral/virologia , Betacoronavirus/isolamento & purificação , Bioensaio , COVID-19 , Teste para COVID-19 , Sistemas CRISPR-Cas , Técnicas de Laboratório Clínico/instrumentação , Técnicas de Laboratório Clínico/normas , Humanos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Pandemias , RNA Viral/análise , RNA Viral/genética , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , SARS-CoV-2 , Sensibilidade e EspecificidadeRESUMO
Synthetic biology aims to re-organise and control biological components to make functional devices. Along the way, the iterative process of designing and testing gene circuits has the potential to yield many insights into the functioning of the underlying chassis of cells. Thus, synthetic biology is converging with disciplines such as systems biology and even classical cell biology, to give a new level of predictability to gene expression, cell metabolism and cellular signalling networks. This review gives an overview of the contributions that synthetic biology has made in understanding gene expression, in terms of cell heterogeneity (noise), the coupling of growth and energy usage to expression, and spatiotemporal considerations. We mainly compare progress in bacterial and mammalian systems, which have some of the most-developed engineering frameworks. Overall, one view of synthetic biology can be neatly summarised as "creating in order to understand."
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Nucléolo Celular/metabolismo , Biologia Sintética/métodos , Biologia de Sistemas/métodos , Animais , Bactérias/metabolismo , Simulação por Computador , Expressão Gênica , Redes Reguladoras de Genes , Engenharia Genética , Humanos , Transdução de SinaisRESUMO
Reovirus fusion-associated small transmembrane (FAST) proteins are the only known nonenveloped virus fusogens and are dedicated to inducing cell-to-cell, not virus-cell, membrane fusion. Numerous structural and functional attributes distinguish this novel family of viral fusogens from all enveloped virus membrane fusion proteins. Both families of viral fusogens play key roles in virus dissemination and pathogenicity, but employ different mechanisms to mediate membrane apposition and merger. However, convergence of these distinct families of viral membrane fusion proteins on common pathways needed for pore expansion and syncytium formation suggests syncytiogenesis represents a cellular response to the presence of cell-cell fusion pores. Together, FAST proteins and enveloped virus fusion proteins provide exceptional insights into the ubiquitous process of cell-cell membrane fusion and syncytium formation.
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Células Gigantes/fisiologia , Fusão de Membrana , Reoviridae/metabolismo , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/metabolismo , Sequência de Aminoácidos , Dados de Sequência Molecular , Orthoreovirus/metabolismo , Filogenia , Alinhamento de Sequência , Internalização do VírusRESUMO
The reovirus fusion-associated small transmembrane (FAST) proteins evolved to induce cell-cell, rather than virus-cell, membrane fusion. It is unclear whether the FAST protein fusion reaction proceeds in the same manner as the enveloped virus fusion proteins. We now show that fluorescence-based cell-cell and cell-RBC hemifusion assays are unsuited for detecting lipid mixing in the absence of content mixing during FAST protein-mediated membrane fusion. Furthermore, membrane curvature agents that inhibit hemifusion or promote pore formation mediated by influenza hemagglutinin had no effect on p14-induced cell-cell fusion, even under conditions of limiting p14 concentrations. Standard assays used to detect fusion intermediates induced by enveloped virus fusion proteins are therefore not applicable to the FAST proteins. These results suggest the possibility that the nature of the fusion intermediates or the mechanisms used to transit through the various stages of the fusion reaction may differ between these distinct classes of viral fusogens.
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Proteínas de Membrana/metabolismo , Reoviridae/fisiologia , Reoviridae/patogenicidade , Proteínas Virais/metabolismo , Internalização do Vírus , Fusão Celular , Membrana Celular/efeitos dos fármacos , HumanosRESUMO
The completion of chromosome segregation during anaphase requires the hypercondensation of the approximately 1-Mb rDNA array, a reaction dependent on condensin and Cdc14 phosphatase. Using systematic genetic screens, we identified 29 novel genetic interactions with budding yeast condensin. Of these, FOB1, CSM1, LRS4, and TOF2 were required for the mitotic condensation of the tandem rDNA array localized on chromosome XII. Interestingly, whereas Fob1 and the monopolin subunits Csm1 and Lrs4 function in rDNA condensation throughout M phase, Tof2 was only required during anaphase. We show that Tof2, which shares homology with the Cdc14 inhibitor Net1/Cfi1, interacts with Cdc14 phosphatase and its deletion suppresses defects in mitotic exit network (MEN) components. Consistent with these genetic data, the onset of Cdc14 release from the nucleolus was similar in TOF2 and tof2Delta cells; however, the magnitude of the release was dramatically increased in the absence of Tof2, even when the MEN pathway was compromised. These data support a model whereby Tof2 coordinates the biphasic release of Cdc14 during anaphase by restraining a population of Cdc14 in the nucleolus after activation of the Cdc14 early anaphase release (FEAR) network, for subsequent release by the MEN.