Turnover of PPP1R15A mRNA encoding GADD34 controls responsiveness and adaptation to cellular stress.

The integrated stress response (ISR) is a key cellular signaling pathway activated by environmental alterations that represses protein synthesis to restore homeostasis. To prevent sustained damage, the ISR is counteracted by the upregulation of growth arrest and DNA damage-inducible 34 (GADD34), a stress-induced regulatory subunit of protein phosphatase 1 that mediates translation reactivation and stress recovery. Here, we uncover a novel ISR regulatory mechanism that post-transcriptionally controls the stability of PPP1R15A mRNA encoding GADD34. We establish that the 3' untranslated region of PPP1R15A mRNA contains an active AU-rich element (ARE) recognized by proteins of the ZFP36 family, promoting its rapid decay under normal conditions and stabilization for efficient expression of GADD34 in response to stress. We identify the tight temporal control of PPP1R15A mRNA turnover as a component of the transient ISR memory, which sets the threshold for cellular responsiveness and mediates adaptation to repeated stress conditions.

Bottom-up Assembled Synthetic SARS-CoV-2 Miniviruses Reveal Lipid Membrane Affinity of Omicron Variant Spike Glycoprotein.

The ongoing COVID-19 pandemic has been brought on by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The spike glycoprotein (S), which decorates the viral envelope forming a corona, is responsible for the binding to the angiotensin-converting enzyme 2 (ACE2) receptor and initiating the infection. In comparison to previous variants, Omicron S presents additional binding sites as well as a more positive surface charge. These changes hint at additional molecular targets for interactions between virus and cell, such as the cell membrane or proteoglycans on the cell surface. Herein, bottom-up assembled synthetic SARS-CoV-2 miniviruses (MiniVs), with a lipid composition similar to that of infectious particles, are implemented to study and compare the binding properties of Omicron and Alpha variants. Toward this end, a systematic functional screening is performed to study the binding ability of Omicron and Alpha S proteins to ACE2-functionalized and nonfunctionalized planar supported lipid bilayers. Moreover, giant unilamellar vesicles are used as a cell membrane model to perform competitive interaction assays of the two variants. Finally, two cell lines with and without presentation of the ACE2 receptor are used to confirm the binding properties of the Omicron and Alpha MiniVs to the cellular membrane. Altogether, the results reveal a significantly higher affinity of Omicron S toward both the lipid membrane and ACE2 receptor. The research presented here highlights the advantages of creating and using bottom-up assembled SARS-CoV-2 viruses to understand the impact of changes in the affinity of S for ACE2 in infection studies.

Extended methods for spatial cell classification with DBSCAN-CellX.

Local cell densities and positioning within cellular monolayers and stratified epithelia have important implications for cell interactions and the functionality of various biological processes. To analyze the relationship between cell localization and tissue physiology, density-based clustering algorithms, such as DBSCAN, allow for a detailed characterization of the spatial distribution and positioning of individual cells. However, these methods rely on predefined parameters that influence the outcome of the analysis. With varying cell densities in cell cultures or tissues impacting cell sizes and, thus, cellular proximities, these parameters need to be carefully chosen. In addition, standard DBSCAN approaches generally come short in appropriately identifying individual cell positions. We therefore developed three extensions to the standard DBSCAN-algorithm that provide: (i) an automated parameter identification to reliably identify cell clusters, (ii) an improved identification of cluster edges; and (iii) an improved characterization of the relative positioning of cells within clusters. We apply our novel methods, which are provided as a user-friendly OpenSource-software package (DBSCAN-CellX), to cellular monolayers of different cell lines. Thereby, we show the importance of the developed extensions for the appropriate analysis of cell culture experiments to determine the relationship between cell localization and tissue physiology.

Enhanced SARS-CoV-2 entry via UPR-dependent AMPK-related kinase NUAK2.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) remodels the endoplasmic reticulum (ER) to form replication organelles, leading to ER stress and unfolded protein response (UPR). However, the role of specific UPR pathways in infection remains unclear. Here, we found that SARS-CoV-2 infection causes marginal activation of signaling sensor IRE1α leading to its phosphorylation, clustering in the form of dense ER-membrane rearrangements with embedded membrane openings, and XBP1 splicing. By investigating the factors regulated by IRE1α-XBP1 during SARS-CoV-2 infection, we identified stress-activated kinase NUAK2 as a novel host-dependency factor for SARS-CoV-2, HCoV-229E, and MERS-CoV entry. Reducing NUAK2 abundance or kinase activity impaired SARS-CoV-2 particle binding and internalization by decreasing cell surface levels of viral receptors and viral trafficking likely by modulating the actin cytoskeleton. IRE1α-dependent NUAK2 levels were elevated in SARS-CoV-2-infected and bystander non-infected cells, promoting viral spread by maintaining ACE2 cell surface levels and facilitating virion binding to bystander cells.

The fifth element: a novel eIF2α kinase in microglia.

The cellular integrated stress response (ISR) is a central signaling pathway that tunes translation initiation in response to a wide range of cellular insults to promote cell survival. The critical node of this regulation involves the phosphorylation of the eukaryotic translation initiation factor 2α (eIF2α) by stress kinases. In this issue of EMBO reports, Wu et al (2023) report FAM69C as a novel eIF2α kinase that promotes ISR activation and stress granule (SG) assembly in microglia in response to oxidative stress. This work proposes a protective role for FAM69C and SGs in limiting damaging inflammatory responses commonly associated with neurodegenerative diseases.

Temporal control of the integrated stress response by a stochastic molecular switch.

Stress granules (SGs) are formed in the cytosol as an acute response to environmental cues and activation of the integrated stress response (ISR), a central signaling pathway controlling protein synthesis. Using chronic virus infection as stress model, we previously uncovered a unique temporal control of the ISR resulting in recurrent phases of SG assembly and disassembly. Here, we elucidate the molecular network generating this fluctuating stress response by integrating quantitative experiments with mathematical modeling and find that the ISR operates as a stochastic switch. Key elements controlling this switch are the cooperative activation of the stress-sensing kinase PKR, the ultrasensitive response of SG formation to the phosphorylation of the translation initiation factor eIF2α, and negative feedback via GADD34, a stress-induced subunit of protein phosphatase 1. We identify GADD34 messenger RNA levels as the molecular memory of the ISR that plays a central role in cell adaptation to acute and chronic stress.

SARS-CoV-2 variants of concern display enhanced intrinsic pathogenic properties and expanded organ tropism in mouse models.

SARS-CoV-2 variants of concern (VOCs) display enhanced transmissibility and resistance to antibody neutralization. Comparing the early 2020 isolate EU-1 to the VOCs Alpha, Beta, and Gamma in mice transgenic for human ACE2 reveals that VOCs induce a broadened scope of symptoms, expand systemic infection to the gastrointestinal tract, elicit the depletion of natural killer cells, and trigger variant-specific cytokine production patterns. Gamma infections result in accelerated disease progression associated with increased immune activation and inflammation. All four SARS-CoV-2 variants induce pDC depletion in the lungs, paralleled by reduced interferon responses. Remarkably, VOCs also use the murine ACE2 receptor for infection to replicate in the lungs of wild-type animals, which induce cellular and innate immune responses that apparently curtail the spread of overt disease. VOCs thus display distinct intrinsic pathogenic properties with broadened tissue and host range. The enhanced pathogenicity of VOCs and their potential for reverse zoonotic transmission pose challenges to clinical and pandemic management.

Synthetic virions reveal fatty acid-coupled adaptive immunogenicity of SARS-CoV-2 spike glycoprotein.

SARS-CoV-2 infection is a major global public health concern with incompletely understood pathogenesis. The SARS-CoV-2 spike (S) glycoprotein comprises a highly conserved free fatty acid binding pocket (FABP) with unknown function and evolutionary selection advantage1,2. Deciphering FABP impact on COVID-19 progression is challenged by the heterogenous nature and large molecular variability of live virus. Here we create synthetic minimal virions (MiniVs) of wild-type and mutant SARS-CoV-2 with precise molecular composition and programmable complexity by bottom-up assembly. MiniV-based systematic assessment of S free fatty acid (FFA) binding reveals that FABP functions as an allosteric regulatory site enabling adaptation of SARS-CoV-2 immunogenicity to inflammation states via binding of pro-inflammatory FFAs. This is achieved by regulation of the S open-to-close equilibrium and the exposure of both, the receptor binding domain (RBD) and the SARS-CoV-2 RGD motif that is responsible for integrin co-receptor engagement. We find that the FDA-approved drugs vitamin K and dexamethasone modulate S-based cell binding in an FABP-like manner. In inflammatory FFA environments, neutralizing immunoglobulins from human convalescent COVID-19 donors lose neutralization activity. Empowered by our MiniV technology, we suggest a conserved mechanism by which SARS-CoV-2 dynamically couples its immunogenicity to the host immune response.

Monitoring Virus-Induced Stress Granule Dynamics Using Long-Term Live-Cell Imaging.

The integrated stress response is a highly regulated signaling cascade that allows cells to react to a variety of external and internal stimuli. Activation of different stress-responsive kinases leads to the phosphorylation of their common downstream target, the eukaryotic translation initiation factor 2 alpha (eIF2α), which is a critical component of functional translation preinitiation complexes. As a consequence, stalled ribonucleoprotein complexes accumulate in the cytoplasm and condense into microscopically visible cytoplasmic stress granules (SGs). Over the past years, numerous microscopy approaches have been developed to study the spatiotemporal control of SG formation in response to a variety of stressors. Here, we apply long-term live-cell microscopy to monitor the dynamic cellular stress response triggered by infection with chronic hepatitis C virus (HCV) at single-cell level and study the behavior of infected cells that repeatedly switch between a stressed and unstressed state. We describe in detail the engineering of fluorescent SG-reporter cells expressing enhanced yellow fluorescent protein (YFP)-tagged T cell internal antigen 1 (TIA-1) using lentiviral delivery, as well as the production of mCherry-tagged HCV trans-complemented particles, which allow live tracking of SG assembly and disassembly, SG number and size in single infected cells over time.

Dengue virus is sensitive to inhibition prior to productive replication.

Uncovering vulnerable steps in the life cycle of viruses supports the rational design of antiviral treatments. However, information on viral replication dynamics obtained from traditional bulk assays with host cell populations is inherently limited as the data represent averages over a multitude of unsynchronized replication cycles. Here, we use time-lapse imaging of virus replication in thousands of single cells, combined with computational inference, to identify rate-limiting steps for dengue virus (DENV), a widespread human pathogen. Comparing wild-type DENV with a vaccine candidate mutant, we show that the viral spread in the mutant is greatly attenuated by delayed onset of productive replication, whereas wild-type and mutant virus have identical replication rates. Single-cell analysis done after applying the broad-spectrum antiviral drug, ribavirin, at clinically relevant concentrations revealed the same mechanism of attenuating viral spread. We conclude that the initial steps of infection, rather than the rate of established replication, are quantitatively limiting DENV spread.

SAR of novel benzothiazoles targeting an allosteric pocket of DENV and ZIKV NS2B/NS3 proteases.

In recent years, dengue virus (DENV) and Zika virus (ZIKV), both mosquito-borne members of the Flaviviridae family, have emerged as intercontinental health issues since their vectors have spread from their tropical origins to temperate climate zones due to climate change and increasing globalization. DENV and ZIKV are positive-sense, single-stranded RNA viruses, whose genomes consist of three structural (capsid, membrane precursor, envelope) and seven non-structural (NS) proteins, all of which are initially expressed as a single precursor polyprotein. For virus maturation, the polyprotein processing is accomplished by host proteases and the viral NS2B/NS3 protease complex, whose inhibitors have been shown to be effective antiviral agents with loss of viral pathogenicity. In this work, we elucidate new structure-activity relationships of benzo[d]thiazole-based allosteric NS2B/NS3 inhibitors. We developed a new series of Y-shaped inhibitors, which, with its larger hydrophobic contact surface, should bind to previously unaddressed regions of the allosteric NS2B/NS3 binding pocket. By scaffold-hopping, we varied the benzo[d]thiazole core and identified benzofuran as a new lead scaffold shifting the selectivity of initially ZIKV-targeting inhibitors to higher activities towards the DENV protease. In addition, we were able to increase the ligand efficiency from 0.27 to 0.41 by subsequent inhibitor truncation and identified N-(5,6-dihydroxybenzo[d]thiazol-2-yl)-4-iodobenzamide as a novel sub-micromolar NS2B/NS3 inhibitor. Utilizing cell-based assays, we could prove the antiviral activity in cellulo. Overall, we report new series of sub-micromolar allosteric DENV and ZIKV inhibitors with good efficacy profile in terms of cytotoxicity and protease inhibition selectivity.

An epigenetic 'extreme makeover': the methylation of flaviviral RNA (and beyond).

Beyond their high clinical relevance worldwide, flaviviruses (comprising dengue and Zika viruses) are of particular interest to understand the spatiotemporal control of RNA metabolism. Indeed, their positive single-stranded viral RNA genome (vRNA) undergoes in the cytoplasm replication, translation and encapsidation, three steps of the flavivirus life cycle that are coordinated through a fine-tuned equilibrium. Over the last years, RNA methylation has emerged as a powerful mechanism to regulate messenger RNA metabolism at the posttranscriptional level. Not surprisingly, flaviviruses exploit RNA epigenetic strategies to control crucial steps of their replication cycle as well as to evade sensing by the innate immune system. This review summarizes the current knowledge about vRNA methylation events and their impacts on flavivirus replication and pathogenesis. We also address the important challenges that the field of epitranscriptomics faces in reliably and accurately identifying RNA methylation sites, which should be considered in future studies on viral RNA modifications.

A Versatile Reporter System To Monitor Virus-Infected Cells and Its Application to Dengue Virus and SARS-CoV-2.

Positive-strand RNA viruses have been the etiological agents in several major disease outbreaks over the last few decades. Examples of this include flaviviruses, such as dengue virus and Zika virus, which cause millions of yearly infections around the globe, and coronaviruses, such as SARS-CoV-2, the source of the current pandemic. The severity of outbreaks caused by these viruses stresses the importance of research aimed at determining methods to limit virus spread and to curb disease severity. Such studies require molecular tools to decipher virus-host interactions and to develop effective treatments. Here, we describe the generation and characterization of a reporter system that can be used to visualize and identify cells infected with dengue virus or SARS-CoV-2. This system is based on viral protease activity that mediates cleavage and nuclear translocation of an engineered fluorescent protein stably expressed in cells. We show the suitability of this system for live cell imaging, for visualization of single infected cells, and for screening and testing of antiviral compounds. With the integrated modular building blocks, this system is easy to manipulate and can be adapted to any virus encoding a protease, thus offering a high degree of flexibility.IMPORTANCE Reporter systems are useful tools for fast and quantitative visualization of virus-infected cells within a host cell population. Here, we describe a reporter system that takes advantage of virus-encoded proteases expressed in infected cells to cleave an ER-anchored fluorescent protein fused to a nuclear localization sequence. Upon cleavage, the GFP moiety translocates to the nucleus, allowing for rapid detection of the infected cells. Using this system, we demonstrate reliable reporting activity for two major human pathogens from the Flaviviridae and the Coronaviridae families: dengue virus and SARS-CoV-2. We apply this reporter system to live cell imaging and use it for proof-of-concept to validate antiviral activity of a nucleoside analogue. This reporter system is not only an invaluable tool for the characterization of viral replication, but also for the discovery and development of antivirals that are urgently needed to halt the spread of these viruses.

Integrative Imaging Reveals SARS-CoV-2-Induced Reshaping of Subcellular Morphologies.

Pathogenesis induced by SARS-CoV-2 is thought to result from both an inflammation-dominated cytokine response and virus-induced cell perturbation causing cell death. Here, we employ an integrative imaging analysis to determine morphological organelle alterations induced in SARS-CoV-2-infected human lung epithelial cells. We report 3D electron microscopy reconstructions of whole cells and subcellular compartments, revealing extensive fragmentation of the Golgi apparatus, alteration of the mitochondrial network and recruitment of peroxisomes to viral replication organelles formed by clusters of double-membrane vesicles (DMVs). These are tethered to the endoplasmic reticulum, providing insights into DMV biogenesis and spatial coordination of SARS-CoV-2 replication. Live cell imaging combined with an infection sensor reveals profound remodeling of cytoskeleton elements. Pharmacological inhibition of their dynamics suppresses SARS-CoV-2 replication. We thus report insights into virus-induced cytopathic effects and provide alongside a comprehensive publicly available repository of 3D datasets of SARS-CoV-2-infected cells for download and smooth online visualization.

Chemical targeting of NEET proteins reveals their function in mitochondrial morphodynamics.

Several human pathologies including neurological, cardiac, infectious, cancerous, and metabolic diseases have been associated with altered mitochondria morphodynamics. Here, we identify a small organic molecule, which we named Mito-C. Mito-C is targeted to mitochondria and rapidly provokes mitochondrial network fragmentation. Biochemical analyses reveal that Mito-C is a member of a new class of heterocyclic compounds that target the NEET protein family, previously reported to regulate mitochondrial iron and ROS homeostasis. One of the NEET proteins, NAF-1, is identified as an important regulator of mitochondria morphodynamics that facilitates recruitment of DRP1 to the ER-mitochondria interface. Consistent with the observation that certain viruses modulate mitochondrial morphogenesis as a necessary part of their replication cycle, Mito-C counteracts dengue virus-induced mitochondrial network hyperfusion and represses viral replication. The newly identified chemical class including Mito-C is of therapeutic relevance for pathologies where altered mitochondria dynamics is part of disease etiology and NEET proteins are highlighted as important therapeutic targets in anti-viral research.

Dance with the Devil: Stress Granules and Signaling in Antiviral Responses.

Cells have evolved highly specialized sentinels that detect viral infection and elicit an antiviral response. Among these, the stress-sensing protein kinase R, which is activated by double-stranded RNA, mediates suppression of the host translation machinery as a strategy to limit viral replication. Non-translating mRNAs rapidly condensate by phase separation into cytosolic stress granules, together with numerous RNA-binding proteins and components of signal transduction pathways. Growing evidence suggests that the integrated stress response, and stress granules in particular, contribute to antiviral defense. This review summarizes the current understanding of how stress and innate immune signaling act in concert to mount an effective response against virus infection, with a particular focus on the potential role of stress granules in the coordination of antiviral signaling cascades.

A Coupled Mathematical Model of the Intracellular Replication of Dengue Virus and the Host Cell Immune Response to Infection.

Dengue virus (DV) is a positive-strand RNA virus of the Flavivirus genus. It is one of the most prevalent mosquito-borne viruses, infecting globally 390 million individuals per year. The clinical spectrum of DV infection ranges from an asymptomatic course to severe complications such as dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), the latter because of severe plasma leakage. Given that the outcome of infection is likely determined by the kinetics of viral replication and the antiviral host cell immune response (HIR) it is of importance to understand the interaction between these two parameters. In this study, we use mathematical modeling to characterize and understand the complex interplay between intracellular DV replication and the host cells' defense mechanisms. We first measured viral RNA, viral protein, and virus particle production in Huh7 cells, which exhibit a notoriously weak intrinsic antiviral response. Based on these measurements, we developed a detailed intracellular DV replication model. We then measured replication in IFN competent A549 cells and used this data to couple the replication model with a model describing IFN activation and production of IFN stimulated genes (ISGs), as well as their interplay with DV replication. By comparing the cell line specific DV replication, we found that host factors involved in replication complex formation and virus particle production are crucial for replication efficiency. Regarding possible modes of action of the HIR, our model fits suggest that the HIR mainly affects DV RNA translation initiation, cytosolic DV RNA degradation, and naïve cell infection. We further analyzed the potential of direct acting antiviral drugs targeting different processes of the DV lifecycle in silico and found that targeting RNA synthesis and virus assembly and release are the most promising anti-DV drug targets.

CDK1 couples proliferation with protein synthesis.

Cell proliferation exerts a high demand on protein synthesis, yet the mechanisms coupling the two processes are not fully understood. A kinase and phosphatase screen for activators of translation, based on the formation of stress granules in human cells, revealed cell cycle-associated kinases as major candidates. CDK1 was identified as a positive regulator of global translation, and cell synchronization experiments showed that this is an extramitotic function of CDK1. Different pathways including eIF2α, 4EBP, and S6K1 signaling contribute to controlling global translation downstream of CDK1. Moreover, Ribo-Seq analysis uncovered that CDK1 exerts a particularly strong effect on the translation of 5'TOP mRNAs, which includes mRNAs encoding ribosomal proteins and several translation factors. This effect requires the 5'TOP mRNA-binding protein LARP1, concurrent to our finding that LARP1 phosphorylation is strongly dependent on CDK1. Thus, CDK1 provides a direct means to couple cell proliferation with biosynthesis of the translation machinery and the rate of protein synthesis.

Norovirus infection results in eIF2α independent host translation shut-off and remodels the G3BP1 interactome evading stress granule formation.

Viral infections impose major stress on the host cell. In response, stress pathways can rapidly deploy defence mechanisms by shutting off the protein synthesis machinery and triggering the accumulation of mRNAs into stress granules to limit the use of energy and nutrients. Because this threatens viral gene expression, viruses need to evade these pathways to propagate. Human norovirus is responsible for gastroenteritis outbreaks worldwide. Here we examined how norovirus interacts with the eIF2α signaling axis controlling translation and stress granules. While norovirus infection represses host cell translation, our mechanistic analyses revealed that eIF2α signaling mediated by the stress kinase GCN2 is uncoupled from translational stalling. Moreover, infection results in a redistribution of the RNA-binding protein G3BP1 to replication complexes and remodelling of its interacting partners, allowing the avoidance from canonical stress granules. These results define novel strategies by which norovirus undergo efficient replication whilst avoiding the host stress response and manipulating the G3BP1 interactome.