Defining basic rules for hardening influenza A virus liquid condensates

eLife, Год журнала: 2023, Номер 12

Опубликована: Апрель 4, 2023

In biological systems, liquid and solid-like biomolecular condensates may contain the same molecules but their behaviour, including movement, elasticity, viscosity, is different on account of distinct physicochemical properties. As such, it known that phase transitions affect function material properties can be tuned by several factors temperature, concentration, valency. It is, however, unclear if some are more efficient than others at regulating behaviour. Viral infections good systems to address this question as they form de novo part replication programmes. Here, we used influenza A virus (IAV) cytosolic condensates, AKA viral inclusions, provide a proof concept condensate hardening via changes in valency its components altering concentration or temperature cell. Liquid IAV inclusions hardened targeting vRNP (viral ribonucleoprotein) interactions NP (nucleoprotein) oligomerising molecule, nucleozin, both vitro vivo without affecting host proteome abundance nor solubility. This study starting point for understanding how pharmacologically modulate offer opportunities alternative antiviral strategies.

Язык: Английский

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Fluorescent protein tags affect the condensation properties of a phase-separating viral protein

Molecular Biology of the Cell, Год журнала: 2024, Номер 35(7)

Опубликована: Май 29, 2024

Fluorescent protein (FP) tags are extensively used to visualize and characterize the properties of biomolecular condensates despite a lack investigation into effects these on phase separation. Here, we characterized dynamic µNS, viral hypothesized undergo separation main component mammalian orthoreovirus factories. Our interest in sequence determinants nucleation process µNS led us compare size density formed by FP::µNS untagged protein. We found an FP-dependent increase droplet density, which suggests that FP can promote condensation. To further assess effect formation, fused mutants show could variably induce otherwise noncondensing proteins. By comparing fluorescent constructs with identified mNeonGreen as least artifactual tag minimally perturbed These results some more suitable for visualizing characterizing minimal experimental artifacts.

Язык: Английский

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RNA and condensates: Disease implications and therapeutic opportunities

Cell chemical biology, Год журнала: 2024, Номер 31(9), С. 1593 - 1609

Опубликована: Сен. 1, 2024

Язык: Английский

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Phosphorylation regulates viral biomolecular condensates to promote infectious progeny production

The EMBO Journal, Год журнала: 2024, Номер 43(2), С. 277 - 303

Опубликована: Янв. 2, 2024

Abstract Biomolecular condensates (BMCs) play important roles in diverse biological processes. Many viruses form BMCs which have been implicated various functions critical for the productive infection of host cells. The adenovirus L1-52/55 kilodalton protein (52K) was recently shown to viral that coordinate genome packaging and capsid assembly. Although packaging, we do not know how are regulated during infection. Here show phosphorylation serine residues 28 75 within N-terminal intrinsically disordered region 52K modulates vitro cells, promoting liquid-like properties. Furthermore, demonstrate promotes production infectious progeny particles. Collectively, our findings provide insights into condensate properties maintained a state conducive their function production. In addition, implications antiviral strategies aimed at targeting regulation limit multiplication.

Язык: Английский

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Viral amyloids: New opportunities for antiviral therapeutic strategies

Current Opinion in Structural Biology, Год журнала: 2023, Номер 83, С. 102706 - 102706

Опубликована: Сен. 30, 2023

Язык: Английский

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ATG9A regulates the dissociation of recycling endosomes from microtubules to form liquid influenza A virus inclusions

PLoS Biology, Год журнала: 2023, Номер 21(11), С. e3002290 - e3002290

Опубликована: Ноя. 20, 2023

It is now established that many viruses threaten public health establish condensates via phase transitions to complete their lifecycles, and knowledge on such processes may offer new strategies for antiviral therapy. In the case of influenza A virus (IAV), liquid known as viral inclusions, concentrate 8 distinct ribonucleoproteins (vRNPs) form IAV genome are viewed sites dedicated assembly 8-partite genomic complex. Despite not being delimited by host membranes, inclusions accumulate membranes inside a result vRNP binding recycling endocytic marker Rab11a, driver biogenesis these structures. We lack molecular understanding how Rab11a-recycling endosomes condensate specifically near endoplasmic reticulum (ER) exit upon infection. show here interact with ER fuse, divide, slide. uncover that, contrary previous indications, reported reduction in activity regulated process rather than competition cellular resources involving novel role factor ATG9A. infection, ATG9A mediates removal carrying vRNPs from microtubules. observe usage microtubules rescued when depleted, which prevents condensation Rab11a ER. The failure produce accumulates cytosol reduces release infectious virions. propose supports dynamics facilitating formation. This work advances our epidemic pandemic genomes formed. also reveals plasticity undergo response disclosing roles beyond its classical involvement autophagy.

Язык: Английский

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Localising enzymes to biomolecular condensates increases their accumulation and benefits engineered metabolic pathway performance in Nicotiana benthamiana

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2025, Номер unknown

Опубликована: Янв. 29, 2025

Summary Establishment of N. benthamiana as a robust biofactory is complicated by issues such product toxicity and proteolytic degradation target proteins / introduced enzymes. Here we investigate whether biomolecular condensates can be used to address these problems. We engineered in leaves using transient expression synthetic modular scaffolds. The vivo properties the that resulted were consistent with them being liquid-like bodies thermodynamic features typical multicomponent phase-separating systems. show recruitment enzymes led several fold yield increases one- three-step metabolic pathways (citramalate biosynthesis poly-3-hydroxybutyrate (PHB) biosynthesis, respectively). This enhanced could for reasons including improved enzyme kinetics, metabolite channelling or avoidance cytotoxicity retention pathway within condensate, which was demonstrated PHB. However, also observed several-fold increase amount accumulated when they targeted condensates. suggests more stable localised condensate than freely diffusing cytosol. hypothesise this stability likely main driver increased production. Our findings provide foundation leveraging plant engineering advance versatile industrial applications.

Язык: Английский

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Fluorescence Loss After Photoactivation (FLAPh): A Pulse-Chase Cellular Assay for Understanding Kinetics and Dynamics of Viral Inclusions

Methods in molecular biology, Год журнала: 2025, Номер unknown, С. 125 - 140

Опубликована: Янв. 1, 2025

Язык: Английский

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Localising enzymes to biomolecular condensates increase their accumulation and benefits engineered metabolic pathway performance in Nicotiana benthamiana

Plant Biotechnology Journal, Год журнала: 2025, Номер unknown

Опубликована: Апрель 9, 2025

Summary The establishment of Nicotiana benthamiana as a robust biofactory is complicated by issues such product toxicity and proteolytic degradation target proteins/introduced enzymes. Here we investigate whether biomolecular condensates can be used to address these problems. We engineered in N. leaves using transient expression synthetic modular scaffolds. vivo properties the that resulted were consistent with them being liquid‐like bodies thermodynamic features typical multicomponent phase‐separating systems. show recruitment enzymes led several‐fold yield increases one‐ three‐step metabolic pathways (citramalate biosynthesis poly‐3‐hydroxybutyrate (PHB) biosynthesis, respectively). This enhanced could for several reasons including improved enzyme kinetics, metabolite channelling or avoidance cytotoxicity retention pathway within condensate, which was demonstrated PHB. However, also observed increase amount accumulated when they targeted condensates. suggests more stable localised condensate than freely diffusing cytosol. hypothesise this stability likely main driver increased production. Our findings provide foundation leveraging plant engineering advance versatile industrial applications.

Язык: Английский

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O-GlcNAcylation reduces proteome solubility and regulates the formation of biomolecular condensates in human cells

Nature Communications, Год журнала: 2025, Номер 16(1)

Опубликована: Апрель 30, 2025

O-GlcNAcylation plays critical roles in the regulation of protein functions and cellular activities, including interactions with other macromolecules. While formation biomolecular condensates (or biocondensates) regulated by a few individual proteins has been reported, systematic investigation on biocondensate remains to be explored. Here we systematically study regulating solubility its impacts RNA-protein using mass spectrometry-based chemoproteomics. Unexpectedly, observe system-wide decrease modified O-GlcNAcylation, glycoproteins involved focal adhesion actin binding exhibiting most significant decrease. Furthermore, sites located disordered regions fewer acidic aromatic residues nearby are related greater drop solubility. Additionally, discover that specific group events promotes dissociation under heat stress, while some enhance during recovery phase. Using site mutagenesis, inhibition O-GlcNAc transferase, fluorescence microscopy, validate regulates biocondensates for YTHDF3 NUFIP2. This work advances our understanding condensates.

Язык: Английский

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