Positive-strand RNA virus replication organelles at a glance.

Membrane-bound replication organelles (ROs) are a unifying feature among diverse positive-strand RNA viruses. These compartments, formed as alterations of various host organelles, provide a protective niche for viral genome replication. Some ROs are characterised by a membrane-spanning pore formed by viral proteins. The RO membrane separates the interior from immune sensors in the cytoplasm. Recent advances in imaging techniques have revealed striking diversity in RO morphology and origin across virus families. Nevertheless, ROs share core features such as interactions with host proteins for their biogenesis and for lipid and energy transfer. The restructuring of host membranes for RO biogenesis and maintenance requires coordinated action of viral and host factors, including membrane-bending proteins, lipid-modifying enzymes and tethers for interorganellar contacts. In this Cell Science at a Glance article and the accompanying poster, we highlight ROs as a universal feature of positive-strand RNA viruses reliant on virus-host interplay, and we discuss ROs in the context of extensive research focusing on their potential as promising targets for antiviral therapies and their role as models for understanding fundamental principles of cell biology.

Dynamic conformational changes of a tardigrade group-3 late embryogenesis abundant protein modulate membrane biophysical properties.

A number of intrinsically disordered proteins (IDPs) encoded in stress-tolerant organisms, such as tardigrade, can confer fitness advantage and abiotic stress tolerance when heterologously expressed. Tardigrade-specific disordered proteins including the cytosolic-abundant heat-soluble proteins are proposed to confer stress tolerance through vitrification or gelation, whereas evolutionarily conserved IDPs in tardigrades may contribute to stress tolerance through other biophysical mechanisms. In this study, we characterized the mechanism of action of an evolutionarily conserved, tardigrade IDP, HeLEA1, which belongs to the group-3 late embryogenesis abundant (LEA) protein family. HeLEA1 homologs are found across different kingdoms of life. HeLEA1 is intrinsically disordered in solution but shows a propensity for helical structure across its entire sequence. HeLEA1 interacts with negatively charged membranes via dynamic disorder-to-helical transition, mainly driven by electrostatic interactions. Membrane interaction of HeLEA1 is shown to ameliorate excess surface tension and lipid packing defects. HeLEA1 localizes to the mitochondrial matrix when expressed in yeast and interacts with model membranes mimicking inner mitochondrial membrane. Yeast expressing HeLEA1 shows enhanced tolerance to hyperosmotic stress under nonfermentative growth and increased mitochondrial membrane potential. Evolutionary analysis suggests that although HeLEA1 homologs have diverged their sequences to localize to different subcellular organelles, all homologs maintain a weak hydrophobic moment that is characteristic of weak and reversible membrane interaction. We suggest that such dynamic and weak protein-membrane interaction buffering alterations in lipid packing could be a conserved strategy for regulating membrane properties and represent a general biophysical solution for stress tolerance across the domains of life.

Usp25-Erlin1/2 activity limits cholesterol flux to restrict virus infection.

Reprogramming lipid metabolic pathways is a critical feature of activating immune responses to infection. However, how these reconfigurations occur is poorly understood. Our previous screen to identify cellular deubiquitylases (DUBs) activated during influenza virus infection revealed Usp25 as a prominent hit. Here, we show that Usp25-deleted human lung epithelial A549 cells display a >10-fold increase in pathogenic influenza virus production, which was rescued upon reconstitution with the wild type but not the catalytically deficient (C178S) variant. Proteomic analysis of Usp25 interactors revealed a strong association with Erlin1/2, which we confirmed as its substrate. Newly synthesized Erlin1/2 were degraded in Usp25-/- or Usp25C178S cells, activating Srebp2, with increased cholesterol flux and attenuated TLR3-dependent responses. Our study therefore defines the function of a deubiquitylase that serves to restrict a range of viruses by reprogramming lipid biosynthetic flux to install appropriate inflammatory responses.

Yeast Svf1 binds ceramides and contributes to sphingolipid metabolism at the ER cis-Golgi interface.

Ceramides are essential precursors of complex sphingolipids and act as potent signaling molecules. Ceramides are synthesized in the endoplasmic reticulum (ER) and receive their head-groups in the Golgi apparatus, yielding complex sphingolipids (SPs). Transport of ceramides between the ER and the Golgi is executed by the essential ceramide transport protein (CERT) in mammalian cells. However, yeast cells lack a CERT homolog, and the mechanism of ER to Golgi ceramide transport remains largely elusive. Here, we identified a role for yeast Svf1 in ceramide transport between the ER and the Golgi. Svf1 is dynamically targeted to membranes via an N-terminal amphipathic helix (AH). Svf1 binds ceramide via a hydrophobic binding pocket that is located in between two lipocalin domains. We showed that Svf1 membrane-targeting is important to maintain flux of ceramides into complex SPs. Together, our results show that Svf1 is a ceramide binding protein that contributes to sphingolipid metabolism at Golgi compartments.

Computed structures of core eukaryotic protein complexes.

Protein-protein interactions play critical roles in biology, but the structures of many eukaryotic protein complexes are unknown, and there are likely many interactions not yet identified. We take advantage of advances in proteome-wide amino acid coevolution analysis and deep-learning­based structure modeling to systematically identify and build accurate models of core eukaryotic protein complexes within the Saccharomyces cerevisiae proteome. We use a combination of RoseTTAFold and AlphaFold to screen through paired multiple sequence alignments for 8.3 million pairs of yeast proteins, identify 1505 likely to interact, and build structure models for 106 previously unidentified assemblies and 806 that have not been structurally characterized. These complexes, which have as many as five subunits, play roles in almost all key processes in eukaryotic cells and provide broad insights into biological function.

Structure of the complete, membrane-assembled COPII coat reveals a complex interaction network.

COPII mediates Endoplasmic Reticulum to Golgi trafficking of thousands of cargoes. Five essential proteins assemble into a two-layer architecture, with the inner layer thought to regulate coat assembly and cargo recruitment, and the outer coat forming cages assumed to scaffold membrane curvature. Here we visualise the complete, membrane-assembled COPII coat by cryo-electron tomography and subtomogram averaging, revealing the full network of interactions within and between coat layers. We demonstrate the physiological importance of these interactions using genetic and biochemical approaches. Mutagenesis reveals that the inner coat alone can provide membrane remodelling function, with organisational input from the outer coat. These functional roles for the inner and outer coats significantly move away from the current paradigm, which posits membrane curvature derives primarily from the outer coat. We suggest these interactions collectively contribute to coat organisation and membrane curvature, providing a structural framework to understand regulatory mechanisms of COPII trafficking and secretion.

Combinatorial multivalent interactions drive cooperative assembly of the COPII coat.

Protein secretion is initiated at the endoplasmic reticulum by the COPII coat, which self-assembles to form vesicles. Here, we examine the mechanisms by which a cargo-bound inner coat layer recruits and is organized by an outer scaffolding layer to drive local assembly of a stable structure rigid enough to enforce membrane curvature. An intrinsically disordered region in the outer coat protein, Sec31, drives binding with an inner coat layer via multiple distinct interfaces, including a newly defined charge-based interaction. These interfaces combinatorially reinforce each other, suggesting coat oligomerization is driven by the cumulative effects of multivalent interactions. The Sec31 disordered region could be replaced by evolutionarily distant sequences, suggesting plasticity in the binding interfaces. Such a multimodal assembly platform provides an explanation for how cells build a powerful yet transient scaffold to direct vesicle traffic.

Subtomogram averaging of COPII assemblies reveals how coat organization dictates membrane shape.

Eukaryotic cells employ membrane-bound carriers to transport cargo between compartments in a process essential to cell functionality. Carriers are generated by coat complexes that couple cargo capture to membrane deformation. The COPII coat mediates export from the endoplasmic reticulum by assembling in inner and outer layers, yielding carriers of variable shape and size that allow secretion of thousands of diverse cargo. Despite detailed understanding of COPII subunits, the molecular mechanisms of coat assembly and membrane deformation are unclear. Here we present a 4.9 Å cryo-tomography subtomogram averaging structure of in vitro-reconstituted membrane-bound inner coat. We show that the outer coat (Sec13-Sec31) bridges inner coat subunits (Sar1-Sec23-Sec24), promoting their assembly into a tight lattice. We directly visualize the membrane-embedded Sar1 amphipathic helix, revealing that lattice formation induces parallel helix insertions, yielding tubular curvature. We propose that regulators like the procollagen receptor TANGO1 modulate this mechanism to determine vesicle shape and size.