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Purpose Rapid antigen-detecting tests (Ag-RDTs) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can transform pandemic control. Thus far, sensitivity (≤ 85%) of lateral-flow assays has limited scale-up. Conceivably, microfluidic immunofluorescence Ag-RDTs could increase sensitivity for SARS-CoV-2 detection. Methods This multi-centre diagnostic accuracy study investigated performance of the microfluidic immunofluorescence LumiraDx™ assay, enrolling symptomatic and asymptomatic participants with suspected SARS-CoV-2 infection. Participants collected a supervised nasal mid-turbinate (NMT) self-swab for Ag-RDT testing, in addition to a professionally collected nasopharyngeal (NP) swab for routine testing with reverse transcriptase polymerase chain reaction (RT-PCR). Results were compared to calculate sensitivity and specificity. Sub-analyses investigated the results by viral load, symptom presence and duration. An analytical study assessed exclusivity and limit-of-detection (LOD). In addition, we evaluated ease-of-use. Results The study was conducted between November 2nd 2020 and 4th of December 2020. 761 participants were enrolled, with 486 participants reporting symptoms on testing day. 120 out of 146 RT-PCR positive cases were detected positive by LumiraDx™, resulting in a sensitivity of 82.2% (95% CI 75.2–87.5%). Specificity was 99.3% (CI 98.3–99.7%). Sensitivity was increased in individuals with viral load ≥ 7 log10 SARS-CoV2 RNA copies/ml (93.8%; CI 86.2–97.3%). Testing against common respiratory commensals and pathogens showed no cross-reactivity and LOD was estimated to be 2–56 PFU/mL. The ease-of-use-assessment was favourable for lower throughput settings. Conclusion The LumiraDx™ assay showed excellent analytical sensitivity, exclusivity and clinical specificity with good clinical sensitivity using supervised NMT self-sampling. Trial registration number and registration date DRKS00021220 and 01.04.2020

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the COVID19 pandemic, is a highly pathogenic β-coronavirus. As other coronaviruses, SARS-CoV-2 is enveloped, replicates in the cytoplasm and assembles at intracellular membranes. Here, we structurally characterize the viral replication compartment and report critical insights into the budding mechanism of the virus, and the structure of extracellular virions close to their native state by in situ cryo-electron tomography and subtomogram averaging. We directly visualize RNA filaments inside the double membrane vesicles, compartments associated with viral replication. The RNA filaments show a diameter consistent with double-stranded RNA and frequent branching likely representing RNA secondary structures. We report that assembled S trimers in lumenal cisternae do not alone induce membrane bending but laterally reorganize on the envelope during virion assembly. The viral ribonucleoprotein complexes (vRNPs) are accumulated at the curved membrane characteristic for budding sites suggesting that vRNP recruitment is enhanced by membrane curvature. Subtomogram averaging shows that vRNPs are distinct cylindrical assemblies. We propose that the genome is packaged around multiple separate vRNP complexes, thereby allowing incorporation of the unusually large coronavirus genome into the virion while maintaining high steric flexibility between the vRNPs., Here the authors visualize SARS-CoV-2 infected cells by in situ cryo-electron tomography, delineating the structural organization and conformational changes that occur during virus replication and budding; and provide insight into vRNP architecture and RNA networks in double membrane vesicles.

Combinations of direct-acting antivirals are needed to minimize drug-resistance mutations and stably suppress replication of RNA viruses. Currently, there are limited therapeutic options against the Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) and testing of a number of drug regimens has led to conflicting results. Here we show that cobicistat, which is an-FDA approved drug-booster that blocks the activity of the drug metabolizing proteins Cytochrome P450-3As (CYP3As) and P-glycoprotein (P-gp), inhibits SARS-CoV-2 replication. Cell-to-cell membrane fusion assays indicated that the antiviral effect of cobicistat is exerted through inhibition of spike protein-mediated membrane fusion. In line with this, incubation with low micromolar concentrations of cobicistat decreased viral replication in three different cell lines including cells of lung and gut origin. When cobicistat was used in combination with the putative CYP3A target and nucleoside analog remdesivir, a synergistic effect on the inhibition of viral replication was observed in cell lines and in a primary human colon organoid. The cobicistat/remdesivir combination was able to potently abate viral replication to levels comparable to mock-infected cells leading to an almost complete rescue of infected cell viability. These data highlight cobicistat as a therapeutic candidate for treating SARS-CoV-2 infection and as a potential building block of combination therapies for COVID-19.

BackgroundRapid antigen-detecting tests (Ag-RDTs) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can transform pandemic control. Thus far, sensitivity (≤85%) of lateral-flow assays has limited scale-up. Conceivably, microfluidic immunofluorescence Ag-RDTs could increase sensitivity for SARS-CoV-2 detection.Materials and MethodsThis multi-centre diagnostic accuracy study investigated performance of the microfluidic immunofluorescence LumiraDx™ assay, enrolling symptomatic and asymptomatic participants with suspected SARS-CoV-2 infection. Participants collected a supervised nasal mid-turbinate (NMT) self-swab for Ag-RDT testing, in addition to a professionally-collected nasopharyngeal (NP) swab for routine testing with reverse transcriptase polymerase chain reaction (RT-PCR). Results were compared to calculate sensitivity and specificity. Sub-analyses investigated the results by viral load, symptom presence and duration. An analytical study assessed exclusivity and limit-of-detection (LOD). In addition, we evaluated ease-of-use.ResultsStudy conduct was between November 2nd 2020 and January 21st 2021. 761 participants were enrolled, with 486 participants reporting symptoms on testing day. 120 out of 146 RT-PCR positive cases were detected positive by LumiraDx™, resulting in a sensitivity of 82.2% (95% CI: 75.2%-87.5%). Specificity was 99.3% (CI: 98.3-99.7%). Sensitivity was increased in individuals with viral load ≥ 7 log10 SARS-CoV2 RNA copies/ml (93.8%; CI: 86.2%-97.3%). Testing against common respiratory commensals and pathogens showed no cross-reactivity and LOD was estimated to be 2-56 PFU/mL. The ease-of-use-assessment was favourable for lower throughput settings.ConclusionThe LumiraDx™ assay showed excellent analytical sensitivity, exclusivity and clinical specificity with good clinical sensitivity using supervised NMT self-sampling.

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., 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.

Dengue virus (DENV) constitutes one of the most important arboviral pathogens affecting humans. The high prevalence of DENV infections, which cause more than 20,000 deaths annually, and the lack of effective vaccines or direct-acting antiviral drugs make it a global health concern. DENV genome replication occurs in close association with the host endomembrane system, which is remodeled to form the viral replication organelle that originates from endoplasmic reticulum (ER) membranes. To date, the viral and cellular determinants responsible for the biogenesis of DENV replication organelles are still poorly defined. The viral nonstructural protein 4A (NS4A) can remodel membranes and has been shown to associate with numerous host factors in DENV-replicating cells. In the present study, we used reverse and forward genetic screens and identified sites within NS4A required for DENV replication. We also mapped the determinants in NS4A required for interactions with other viral proteins. Moreover, taking advantage of our recently developed polyprotein expression system, we evaluated the role of NS4A in the formation of DENV replication organelles. Together, we report a detailed map of determinants within NS4A required for RNA replication, interaction with other viral proteins, and replication organelle formation. Our results suggest that NS4A might be an attractive target for antiviral therapy. IMPORTANCE DENV is the most prevalent mosquito-borne virus, causing around 390 million infections each year. There are no approved therapies to treat DENV infection, and the only available vaccine shows limited efficacy. The viral nonstructural proteins have emerged as attractive drug targets due to their pivotal role in RNA replication and establishment of virus-induced membranous compartments, designated replication organelles (ROs). The transmembrane protein NS4A, generated by cleavage of the NS4A-2K-4B precursor, contributes to DENV replication by unknown mechanisms. Here, we report a detailed genetic interaction map of NS4A and identify residues required for RNA replication and interaction between NS4A-2K-4B and NS2B-3 as well as NS1. Importantly, by means of an expression-based system, we demonstrate the essential role of NS4A in RO biogenesis and identify determinants in NS4A required for this process. Our data suggest that NS4A is an attractive target for antiviral therapy.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19. SARS-CoV-2 relies on cellular RNA-binding proteins (RBPs) to replicate and spread, although which RBPs control its life cycle remains largely unknown. Here, we employ a multi-omic approach to identify systematically and comprehensively the cellular and viral RBPs that are involved in SARS-CoV-2 infection. We reveal that SARS-CoV-2 infection profoundly remodels the cellular RNA-bound proteome, which includes wide-ranging effects on RNA metabolic pathways, non-canonical RBPs and antiviral factors. Moreover, we apply a new method to identify the proteins that directly interact with viral RNA, uncovering dozens of cellular RBPs and six viral proteins. Amongst them, several components of the tRNA ligase complex, which we show regulate SARS-CoV-2 infection. Furthermore, we discover that available drugs targeting host RBPs that interact with SARS-CoV-2 RNA inhibit infection. Collectively, our results uncover a new universe of host-virus interactions with potential for new antiviral therapies against COVID-19., Graphical Abstract, Kamel, Noerenberg, Cerikan and colleagues apply a multi-omic approach to identify the RNA-binding proteins that regulate SARS-CoV-2 infection. They discovered that the complement of RNA-binding proteins heavily remodels upon SARS-CoV-2 infection. They also show that the viral RNA interacts with dozens of cellular and six viral RNA-binding proteins. These host-virus interactions are fundamental for SARS-CoV-2 infection and have great potential for new therapeutic approaches against COVID-19.

Double membrane vesicles (DMVs) serve as replication organelles of plus-strand RNA viruses such as hepatitis C virus (HCV) and SARS-CoV-2. Viral DMVs are morphologically analogous to DMVs formed during autophagy, but lipids driving their biogenesis are largely unknown. Here we show that production of the lipid phosphatidic acid (PA) by acylglycerolphosphate acyltransferase (AGPAT) 1 and 2 in the ER is important for DMV biogenesis in viral replication and autophagy. Using DMVs in HCV-replicating cells as model, we found that AGPATs are recruited to and critically contribute to HCV and SARS-CoV-2 replication and proper DMV formation. An intracellular PA sensor accumulated at viral DMV formation sites, consistent with elevated levels of PA in fractions of purified DMVs analyzed by lipidomics. Apart from AGPATs, PA is generated by alternative pathways and their pharmacological inhibition also impaired HCV and SARS-CoV-2 replication as well as formation of autophagosome-like DMVs. These data identify PA as host cell lipid involved in proper replication organelle formation by HCV and SARS-CoV-2, two phylogenetically disparate viruses causing very different diseases, i.e. chronic liver disease and COVID-19, respectively. Host-targeting therapy aiming at PA synthesis pathways might be suitable to attenuate replication of these viruses., Double membrane vesicles (DMV) are used as replication organelles by several RNA viruses. Applying proteomics and lipidomics, Tabata and Prasad et al. find that two cellular acyltransferases (AGPAT1/2), responsible for synthesis of phosphatidic acid, play a role in the DMV-biogenesis of HCV and SARS-CoV-2, highlighting a common biogenesis mechanism for evolutionary distant positive-strand RNA viruses.

Two proteases produced by the SARS-CoV-2 virus, Mproand PLpro, are essential for viral replication and have become the focus of drug development programs for treatment of COVID-19. We screened a highly focused library of compounds containing covalent warheads designed to target cysteine proteases to identify new lead scaffolds for both Mproand PLproproteases. These efforts identified a small number of hits for the Mproprotease and no viable hits for the PLproprotease. Of the Mprohits identified as inhibitors of the purified recombinant protease, only two compounds inhibited viral infectivity in cellular infection assays. However, we observed a substantial drop in antiviral potency upon expression of TMPRSS2, a transmembrane serine protease that acts in an alternative viral entry pathway to the lysosomal cathepsins. This loss of potency is explained by the fact that our lead Mproinhibitors are also potent inhibitors of host cell cysteine cathepsins. To determine if this is a general property of Mproinhibitors, we evaluated several recently reported compounds and found that they are also effective inhibitors of purified human cathepsin L and B and showed similar loss in activity in cells expressing TMPRSS2. Our results highlight the challenges of targeting Mproand PLproproteases and demonstrate the need to carefully assess selectivity of SARS-CoV-2 protease inhibitors to prevent clinical advancement of compounds that function through inhibition of a redundant viral entry pathway.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19. SARS-CoV-2 relies on cellular RNA-binding proteins (RBPs) to replicate and spread, although which RBPs control SARS-CoV-2 infection remains largely unknown. Here, we employ a multi-omic approach to identify systematically and comprehensively which cellular and viral RBPs are involved in SARS-CoV-2 infection. We reveal that the cellular RNA-bound proteome is remodelled upon SARS-CoV-2 infection, having widespread effects on RNA metabolic pathways, non-canonical RBPs and antiviral factors. Moreover, we apply a new method to identify the proteins that directly interact with viral RNA, uncovering dozens of cellular RBPs and six viral proteins. Amongst them, several components of the tRNA ligase complex, which we show regulate SARS-CoV-2 infection. Furthermore, we discover that available drugs targeting host RBPs that interact with SARS-CoV-2 RNA inhibit infection. Collectively, our results uncover a new universe of host-virus interactions with potential for new antiviral therapies against COVID-19.

Summary Positive-strand RNA viruses replicate in distinct membranous structures called replication organelles (ROs). Mechanistic studies of RO formation have been difficult because perturbations affecting viral replication have an impact on viral protein amounts, thus affecting RO biogenesis. Here, we present a detailed guide on how to use a replication-independent expression system, designated pIRO (plasmid-induced replication organelle formation), inducing bona fide flavivirus ROs in transfected cells. This will be useful for mechanistic studies of viral and cellular factors driving flavivirus RO biogenesis. For complete details on the use and execution of this protocol, please refer to Cerikan et al. (2020)., Graphical Abstract, Highlights • A replication-independent system to form flaviviral replication organelles is described • The system is referred to as pIRO, an acronym for plasmid-induced replication organelle • The pIRO system has been established for dengue (pIRO-D) and Zika virus (pIRO-Z) • The pIRO system can be used under low biosafety conditions, Positive-strand RNA viruses replicate in distinct membranous structures called replication organelles (ROs). Mechanistic studies of RO formation have been difficult because perturbations affecting viral replication have an impact on viral protein amounts, thus affecting RO biogenesis. Here, we present a detailed guide on how to use a replication-independent expression system, designated pIRO (plasmid-induced replication organelle formation), inducing bona fide flavivirus ROs in transfected cells. This will be useful for mechanistic studies of viral and cellular factors driving flavivirus RO biogenesis.

Positive-strand RNA viruses have been the etiological agents in several major disease outbreaks over the last few decades. Examples of that are flaviviruses, such as dengue virus and Zika virus that cause millions of yearly infections and spread around the globe, and coronaviruses, such as SARS-CoV-2, which is the cause of the current pandemic. The severity of outbreaks caused by these viruses stresses the importance of virology research in determining mechanisms to limit virus spread and to curb disease severity. Such studies require molecular tools to decipher virus-host interactions and to develop effective interventions. Here, we describe the generation and characterization of a reporter system to visualize dengue virus and SARS-CoV-2 replication in live cells. The system is based on viral protease activity causing cleavage and nuclear translocation of an engineered fluorescent protein that is expressed in the infected cells. We show the suitability of the system for live cell imaging and visualization of single infected cells as well as for screening and testing of antiviral compounds. Given the modular building blocks, the system is easy to manipulate and can be adapted to any virus encoding a protease, thus offering a high degree of flexibility.IMPORTANCEReporter systems are useful tools for fast and quantitative visualization of viral replication and spread within a host cell population. Here we describe a reporter system that takes advantage of virus-encoded proteases that are expressed in infected cells to cleave an ER-anchored fluorescent protein fused to a nuclear localization sequence. Upon cleavage, the fluorescent protein 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.

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., Graphical Abstract, Highlights • Integrative imaging approaches reveal SARS-CoV-2-induced cellular alterations • SARS-CoV-2 extensively remodels the cellular endomembrane system • Pharmacological inhibition of cytoskeleton remodeling restricts viral replication • We provide a comprehensive repository of virus-induced ultrastructural cell changes, Cortese et al. use integrative imaging techniques to generate a publicly available repository of morphological alterations induced by SARS-CoV-2 in lung cells. Accumulation of ER-derived double-membrane vesicles, the viral replication organelle, occurs concomitantly with cytoskeleton remodeling and Golgi fragmentation. Pharmacological alteration of cytoskeleton dynamics restricts viral replication and spread.

SARS-CoV-2 is a novel virus that has rapidly spread, causing a global pandemic. In the majority of infected patients, SARS-CoV-2 leads to mild disease; however, in a significant proportion of infections, individuals develop severe symptoms that can lead to permanent lung damage or death. These severe cases are often associated with high levels of pro-inflammatory cytokines and low antiviral responses which can lead to systemic complications. We have evaluated transcriptional and cytokine secretion profiles from infected cell cultures and detected a distinct upregulation of inflammatory cytokines that parallels samples taken from infected patients. Building on these observations, we found a specific activation of NF-κB and a block of IRF3 nuclear translocation in SARS-CoV-2 infected cells. This NF-κB response is mediated by cGAS-STING activation and could be attenuated through STING targeting drugs. Our results show that SARS-CoV-2 curates a cGAS-STING mediated NF-κB driven inflammatory immune response in epithelial cells that likely contributes to inflammatory responses seen in patients and might be a target to suppress severe disease symptoms.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virions are surrounded by a lipid bilayer from which spike (S) protein trimers protrude. Heavily glycosylated S trimers bind the ACE2 receptor and mediate entry of virions into target cells. S exhibits extensive conformational flexibility: it modulates the exposure of its receptor binding site and later undergoes complete structural rearrangement to drive fusion of viral and cellular membranes. The structures and conformations of soluble, overexpressed, purified S proteins have been studied in detail using cryo-electron microscopy. The structure and distribution of S on the virion surface, however, has not been characterised. Here we applied cryo-electron microscopy and tomography to image intact SARS-CoV-2 virions, determining the high-resolution structure, conformational flexibility and distributions of S trimers in situ on the virion surface. These results provide a basis for understanding the conformations of S present on the virion, and for studying their interactions with neutralizing antibodies.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the COVID19 pandemic, is a highly pathogenic β-coronavirus. As other coronaviruses, SARS-CoV-2 is enveloped, replicates in the cytoplasm and assembles at intracellular membranes. Here, we structurally characterize the viral replication compartment and report critical insights into the budding mechanism of the virus, and the structure of extracellular virions close to their native state byin situcryo-electron tomography and subtomogram averaging. We directly visualized RNA filaments inside the double membrane vesicles, compartments associated with viral replication. The RNA filaments show a diameter consistent with double-stranded RNA and frequent branching likely representing RNA secondary structures. We found that assembled S trimers in lumenal cisternae do not alone induce membrane bending but laterally reorganize on the envelope during virion assembly. The viral ribonucleoprotein complexes (vRNPs) are accumulated at the curved membrane characteristic for budding sites suggesting that vRNP recruitment is enhanced by membrane curvature. Subtomogram averaging shows that vRNPs are distinct cylindrical assemblies. We propose that the genome is packaged around multiple separate vRNP complexes, thereby allowing incorporation of the unusually large coronavirus genome into the virion while maintaining high steric flexibility between the vRNPs.

Key Points Flaviviruses and hepaciviruses share similarities in their fundamental replication mechanisms and strategies to manipulate the host cell, yet important differences exist, likely reflecting the use of distinct host cell pathways.RNA replication of Flaviviridae family members occurs in tight association with endoplasmic reticulum-derived membranes, which are reorganized into viral replication organelles. Whereas the morphology and the architecture of these replication organelles are well defined, relatively little is known about the viral and cellular factors orchestrating their biogenesis.Protein folding, modification and degradation are essential, tightly regulated cellular processes, and a number of common host factors and pathways that are involved in these processes appear to be exploited by both flaviviruses and hepaciviruses at different steps of their replication cycle. These include heat shock protein 70 (HSP70) network components, the unfolded protein response, the ubiquitin-dependent proteasome system and autophagy.Accumulating evidence indicates that lipids and lipid metabolism fulfil essential roles in the life cycle of Flaviviridae viruses. They alter the lipid composition of cellular membranes, serving as scaffold for the assembly of the viral replicase by changing their biophysical properties, such as curvature, permeability and fluidity.The identification of host cell pathways and factors commonly used by members of the Flaviviridae family might help in the development of broad-spectrum antiviral drugs that target multiple members of this family and/or other virus families.As exemplified by members of the Flaviviridae family, the use of host cell pathways does not follow conventional phylogeny but, rather, reveals unexpected commonalities with distantly related viruses, raising the question of evolutionary relationships between these viruses. Supplementary information The online version of this article (doi:10.1038/nrmicro.2017.170) contains supplementary material, which is available to authorized users., In this Review, Bartenschlager and colleagues discuss howFlaviviridaeviruses rewire cellular pathways and co-opt organelles. They compare strategies employed by flaviviruses with those employed by hepaciviruses and discuss the importance of these virus–host interactions in the context of viral replication and antiviral therapies. Supplementary information The online version of this article (doi:10.1038/nrmicro.2017.170) contains supplementary material, which is available to authorized users., Members of the Flaviviridae virus family comprise a large group of enveloped viruses with a single-strand RNA genome of positive polarity. Several genera belong to this family, including the Hepacivirus genus, of which hepatitis C virus (HCV) is the prototype member, and the Flavivirus genus, which contains both dengue virus and Zika virus. Viruses of these genera differ in many respects, such as the mode of transmission or the course of infection, which is either predominantly persistent in the case of HCV or acutely self-limiting in the case of flaviviruses. Although the fundamental replication strategy of Flaviviridae members is similar, during the past few years, important differences have been discovered, including the way in which these viruses exploit cellular resources to facilitate viral propagation. These differences might be responsible, at least in part, for the various biological properties of these viruses, thus offering the possibility to learn from comparisons. In this Review, we discuss the current understanding of how Flaviviridae viruses manipulate and usurp cellular pathways in infected cells. Specifically, we focus on comparing strategies employed by flaviviruses with those employed by hepaciviruses, and we discuss the importance of these interactions in the context of viral replication and antiviral therapies. Supplementary information The online version of this article (doi:10.1038/nrmicro.2017.170) contains supplementary material, which is available to authorized users.

Replication and amplification of the viral genome is a key process for all viruses. For hepatitis C virus (HCV), a positive-strand RNA virus, amplification of the viral genome requires the synthesis of a negative-sense RNA template, which is in turn used for the production of new genomic RNA. This process is governed by numerous proteins, both host and viral, as well as distinct lipids and specific RNA elements within the positive- and negative-strand RNAs. Moreover, this process requires specific changes to host cell ultrastructure to create microenvironments conducive to viral replication. This review will focus on describing the processes and factors involved in facilitating or regulating HCV genome replication.

Summary Dengue virus (DENV) and Zika virus (ZIKV), members of the Flavivirus genus, rearrange endoplasmic reticulum membranes to induce invaginations known as vesicle packets (VPs), which are the assumed sites for viral RNA replication. Mechanistic information on VP biogenesis has so far been difficult to attain due to the necessity of studying their formation under conditions of viral replication, where perturbations reducing replication will inevitably impact VP formation. Here, we report a replication-independent expression system, designated pIRO (plasmid-induced replication organelle formation) that induces bona fide DENV and ZIKV VPs that are morphologically indistinguishable from those in infected cells. Using this system, we demonstrate that sequences in the 3′ terminal RNA region of the DENV, but not the ZIKV genome, contribute to VP formation in a non-replicative manner. These results validate the pIRO system that opens avenues for mechanistically dissecting virus replication from membrane reorganization., Graphical Abstract, Highlights • A replication-independent system to study DENV/ZIKV replication organelle formation • This system is called pIRO (i.e., plasmid-induced replication organelle formation) • Replication organelles induced with pIRO system analogous to those in infected cell • Non-replicative role of 3′ terminal RNA elements in organelle formation, Cerikan et al. devise an RNA replication-independent expression system designated pIRO (plasmid-induced replication organelle formation) phenocopying DENV/ZIKV-induced vesicle packets (VPs), the viral replication organelle. The authors find that RNA elements residing in the 3′ untranslated region of either virus genome are required for VP generation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virions are surrounded by a lipid bilayer from which spike (S) protein trimers protrude1. Heavily glycosylated S trimers bind to the angiotensin-converting enzyme 2 receptor and mediate entry of virions into target cells2–6. S exhibits extensive conformational flexibility: it modulates exposure of its receptor-binding site and subsequently undergoes complete structural rearrangement to drive fusion of viral and cellular membranes2,7,8. The structures and conformations of soluble, overexpressed, purified S proteins have been studied in detail using cryo-electron microscopy2,7,9–12, but the structure and distribution of S on the virion surface remain unknown. Here we applied cryo-electron microscopy and tomography to image intact SARS-CoV-2 virions and determine the high-resolution structure, conformational flexibility and distribution of S trimers in situ on the virion surface. These results reveal the conformations of S on the virion, and provide a basis from which to understand interactions between S and neutralizing antibodies during infection or vaccination. Cryo-electron microscopy and tomography studies reveal the structures, conformations and distributions of spike protein trimers on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virions and provide a basis for understanding the interactions of the spike protein with neutralizing antibodies.

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 employed an integrative light and electron microscopy based imaging analysis to determine morphological organelle alterations induced in SARS-CoV-2 infected human lung epithelial cells. We report 3D 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 remodelling of cytoskeleton elements; pharmacological inhibition of their dynamics strongly suppresses SARS-CoV-2 replication. Taken together, we provide critical insights into virus-induced cytopathic effects, while alongside presenting a comprehensive and publicly available repository of 3D data-sets of SARS-CoV-2 infected cells. Funding: This work was supported in part by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – Project number 240245660 – SFB 1129 (TP11, TP13, TP14 and project Z2) to R.B., A.R., S.B. and Y.S. R.B. was supported in addition by the German Center for Infection Research (DZIF), project numbers 8029801806 and 8029705705, and the DFG, project number 272983813 – TRR 179.SB and MS received additional financial support from the DFG, project number 415089553 (Heisenberg) and 272983813 (TRR179) to S.B. and project number 416072091 to MS. Conflict of Interest: The authors declare no competing interests.

With no antiviral drugs or widely available vaccines, Dengue virus (DENV) constitutes a public health concern. DENV replicates at ER-derived cytoplasmic structures that include substructures called convoluted membranes (CMs); however, the purpose of these membrane alterations remains unclear. We determine that DENV nonstructural protein (NS)4B, a promising drug target with unknown function, associates with mitochondrial proteins and alters mitochondria morphology to promote infection. During infection, NS4B induces elongation of mitochondria, which physically contact CMs. This restructuring compromises the integrity of mitochondria-associated membranes, sites of ER-mitochondria interface critical for innate immune signaling. The spatio-temporal parameters of CM biogenesis and mitochondria elongation are linked to loss of activation of the fission factor Dynamin-Related Protein-1. Mitochondria elongation promotes DENV replication and alleviates RIG-I-dependent activation of interferon responses. As Zika virus infection induces similar mitochondria elongation, this perturbation may protect DENV and related viruses from innate immunity and create a favorable replicative environment.

Dengue virus (DENV) has emerged as major human pathogen. Despite the serious socio-economic impact of DENV-associated diseases, antiviral therapy is missing. DENV replicates in the cytoplasm of infected cells and induces a membranous replication organelle, formed by invaginations of the endoplasmic reticulum membrane and designated vesicle packets (VPs). Nonstructural protein 1 (NS1) of DENV is a multifunctional protein. It is secreted from cells to counteract antiviral immune responses, but also critically contributes to the severe clinical manifestations of dengue. In addition, NS1 is indispensable for viral RNA replication, but the underlying molecular mechanism remains elusive. In this study, we employed a combination of genetic, biochemical and imaging approaches to dissect the determinants in NS1 contributing to its various functions in the viral replication cycle. Several important observations were made. First, we identified a cluster of amino acid residues in the exposed region of the β-ladder domain of NS1 that are essential for NS1 secretion. Second, we revealed a novel interaction of NS1 with the NS4A-2K-4B cleavage intermediate, but not with mature NS4A or NS4B. This interaction is required for RNA replication, with two residues within the connector region of the NS1 “Wing” domain being crucial for binding of the NS4A-2K-4B precursor. By using a polyprotein expression system allowing the formation of VPs in the absence of viral RNA replication, we show that the NS1 –NS4A-2K-4B interaction is not required for VP formation, arguing that the association between these two proteins plays a more direct role in the RNA amplification process. Third, through analysis of polyproteins containing deletions in NS1, and employing a trans-complementation assay, we show that both cis and trans acting elements within NS1 contribute to VP formation, with the capability of NS1 mutants to form VPs correlating with their capability to support RNA replication. In conclusion, these results reveal a direct role of NS1 in VP formation that is independent from RNA replication, and argue for a critical function of a previously unrecognized NS4A-2K-NS4B precursor specifically interacting with NS1 and promoting viral RNA replication., Author summary Dengue virus (DENV) is one of the most prevalent mosquito-transmitted human pathogens. The only licensed vaccine has limited efficacy and an antiviral therapy is not available. The multifunctional non-structural protein 1 (NS1) of DENV is secreted from infected cells, counteracts antiviral immune response and contributes to the pathogenesis of DENV infection. In addition, NS1 is essential for the viral replication cycle but the underlying mechanism is unknown. Here we determined the viral interactome of NS1 and identified a novel interaction of NS1 with the NS4A-2K-4B cleavage intermediate, but not with NS4A and NS4B. This interaction is required for RNA replication. Additionally, we identified a domain in NS1 important for efficient secretion of this protein. Finally, we demonstrate that NS1 is required for the biogenesis of the membranous DENV replication organelle. This function does not require RNA replication and is independent from NS1 interaction with NS4A-2K-4B. Our results provide new insights into the role of NS1 in DENV RNA replication and establish a genetic map of residues in NS1 required for the diverse functions of this protein. These results should aid in the design of antiviral strategies targeting NS1, with the aim to suppress viral replication as well as severe disease manifestations.

Members of the Flavivirus genus rely extensively on the host cell endomembrane network to generate complex membranous replication organelles (ROs) that facilitate viral genome replication and the production of virus particles. For dengue virus and Zika virus, these ROs included vesicles which are formed through membrane invagination into the endoplasmic reticulum (ER) lumen, termed invaginated vesicles or vesicle packets (VPs), as well as large areas of bundled smooth ER, termed convoluted membranes. Though the morphology of these virus-induced membrane structures has been well characterized, the viral and host constituents that make up flaviviral ROs are still poorly understood. Here, we identified a subset of ER resident proteins (atlastins), normally required for maintaining ER tubule networks, as critical host factors for flavivirus infection. Specific changes in atlastin (ATL) levels had dichotomous effects on flaviviruses with ATL2 depletion, leading to replication organelle defects and ATL3 depletion to changes in viral assembly/release pathways. These different depletion phenotypes allowed us to exploit virus infection to characterize non-conserved functional domains between the three atlastin paralogues. Additionally, we established the ATL interactome and show how it is reprogrammed upon viral infection. Screening of specific ATL interactors confirmed non-redundant ATL functions and identified a role for ATL3 in vesicle trafficking. Our data demonstrate that ATLs are central host factors that coordinate the ER network and shape the ER during flavivirus infection.

Tissue culture medium routinely contains fetal bovine serum (FBS). Here we show that culturing human hepatoma cells in their native, adult serum (human serum, HS) results in the restoration of key morphological and metabolic features of normal liver cells. When moved to HS, these cells show differential transcription of 22-32% of the genes, stop proliferating, and assume a hepatocyte-like morphology. Metabolic analysis shows that the Warburg-like metabolic profile, typical for FBS-cultured cells, is replaced by a diverse metabolic profile consistent within vivohepatocytes. We demonstrate the formation of large lipid and glycogen stores, increased glycogenesis, increased β-oxidation, increased ketogenesis, and decreased glycolysis. Finally, organ-specific functions are restored, including xenobiotics degradation and secretion of bile, very low density lipoprotein, and albumin. Thus, organ-specific functions are not necessarily lost in cell cultures, but might be merely suppressed in FBS. Together, we showed that cells that are representative of normal physiology can be produced from cancer cells simply by replacing FBS by HS in culture media. The effect of serum is often overseen in cell culture and we provide a detailed study in the changes that occur, provide insight in some of the serum components that may play a role in the establishment of the different phenotypes, and discuss how these finding might be beneficial to a variety of research fields.

Summary A global concern has emerged with the pandemic spread of Zika virus (ZIKV) infections that can cause severe neurological symptoms in adults and newborns. ZIKV is a positive-strand RNA virus replicating in virus-induced membranous replication factories (RFs). Here we used various imaging techniques to investigate the ultrastructural details of ZIKV RFs and their relationship with host cell organelles. Analyses of human hepatic cells and neural progenitor cells infected with ZIKV revealed endoplasmic reticulum (ER) membrane invaginations containing pore-like openings toward the cytosol, reminiscent to RFs in Dengue virus-infected cells. Both the MR766 African strain and the H/PF/2013 Asian strain, the latter linked to neurological diseases, induce RFs of similar architecture. Importantly, ZIKV infection causes a drastic reorganization of microtubules and intermediate filaments forming cage-like structures surrounding the viral RF. Consistently, ZIKV replication is suppressed by cytoskeleton-targeting drugs. Thus, ZIKV RFs are tightly linked to rearrangements of the host cell cytoskeleton., Graphical Abstract, Highlights • ZIKV induces ER membrane invaginations similar to Dengue virus • ZIKV induces profound alterations of the cytoskeleton • Microtubules and intermediate filaments surround the ZIKV replication factory • ZIKV replication is sensitive to cytoskeleton-targeting drugs, Cortese et al. show that ZIKV infection in both human hepatoma and neuronal progenitor cells induces drastic structural modification of the cellular architecture. Microtubules and intermediate filaments surround the viral replication factory composed of vesicles corresponding to ER membrane invagination toward the ER lumen. Importantly, alteration of microtubule flexibility impairs ZIKV replication.

The Zika virus (ZIKV) has recently attracted major research interest as infection was unexpectedly associated with neurological manifestations in developing foetuses and with Guillain-Barré syndrome in infected adults. Understanding the underlying molecular mechanisms requires reverse genetic systems, which allow manipulation of infectious cDNA clones at will. In the case of flaviviruses, to which ZIKV belongs, several reports have indicated that the construction of full-length cDNA clones is difficult due to toxicity during plasmid amplification in Escherichia coli. Toxicity of flaviviral cDNAs has been linked to the activity of cryptic prokaryotic promoters within the region encoding the structural proteins leading to spurious transcription and expression of toxic viral proteins. Here, we employ an approach based on in silico prediction and mutational silencing of putative promoters to generate full-length cDNA clones of the historical MR766 strain and the contemporary French Polynesian strain H/PF/2013 of ZIKV. While for both strains construction of full-length cDNA clones has failed in the past, we show that our approach generates cDNA clones that are stable on single bacterial plasmids and give rise to infectious viruses with properties similar to those generated by other more complex assembly strategies. Further, we generate luciferase and fluorescent reporter viruses as well as sub-genomic replicons that are fully functional and suitable for various research and drug screening applications. Taken together, this study confirms that in silico prediction and silencing of cryptic prokaryotic promoters is an efficient strategy to generate full-length cDNA clones of flaviviruses and reports novel tools that will facilitate research on ZIKV biology and development of antiviral strategies.

The hepatitis C virus (HCV) is a positive strand RNA virus of the Flavivirus family that replicates in the cytoplasm of infected hepatocytes. Previously, several nuclear localization signals (NLS) and nuclear export signals (NES) have been identified in HCV proteins, however, there is little evidence that these proteins travel into the nucleus during infection. We have recently shown that nuclear pore complex (NPC) proteins (termed nucleoporins or Nups) are present in the membranous web and are required during HCV infection. In this study, we identify a total of 11 NLS and NES sequences in various HCV proteins. We show direct interactions between HCV proteins and importin α5 (IPOA5/kapα1), importin β3 (IPO5/kap β3), and exportin 1 (XPO1/CRM1) both in-vitro and in cell culture. These interactions can be disrupted using peptides containing the specific NLS or NES sequences of HCV proteins. Moreover, using a synchronized infection system, we show that these peptides inhibit HCV infection during distinct phases of the HCV life cycle. The inhibitory effects of these peptides place them in two groups. The first group binds IPOA5 and inhibits infection during the replication stage of HCV life cycle. The second group binds IPO5 and is active during both early replication and early assembly. This work delineates the entire life cycle of HCV and the active involvement of NLS sequences during HCV replication and assembly. Given the abundance of NLS sequences within HCV proteins, our previous finding that Nups play a role in HCV infection, and the relocation of the NLS double-GFP reporter in HCV infected cells, this work supports our previous hypothesis that NPC-like structures and nuclear transport factors function in the membranous web to create an environment conducive to viral replication.

Hepatitis C virus (HCV) is a positive-strand RNA virus of the Flaviviridae family and a major cause of liver disease worldwide. HCV replicates in the cytoplasm, and the synthesis of viral proteins induces extensive rearrangements of host cell membranes producing structures, collectively termed the membranous web (MW). The MW contains the sites of viral replication and assembly, and we have identified distinct membrane fractions derived from HCV-infected cells that contain replication and assembly complexes enriched for viral RNA and infectious virus, respectively. The complex membrane structure of the MW is thought to protect the viral genome limiting its interactions with cytoplasmic pattern recognition receptors (PRRs) and thereby preventing activation of cellular innate immune responses. Here we show that PRRs, including RIG-I and MDA5, and ribosomes are excluded from viral replication and assembly centers within the MW. Furthermore, we present evidence that components of the nuclear transport machinery regulate access of proteins to MW compartments. We show that the restricted assess of RIG-I to the MW can be overcome by the addition of a nuclear localization signal sequence, and that expression of a NLS-RIG-I construct leads to increased immune activation and the inhibition of viral replication., Author Summary Hepatitis C virus (HCV) is a positive-strand RNA virus and it is a major cause of liver disease worldwide affecting more than 170 million individuals. Infection of cells with HCV leads to rearrangement of cytoplasmic host cell membranes and the formation of the membranous web (MW) containing viral replication and assembly complexes. The MW is thought to function in concentrating viral components, regulating virus replication, and immune evasion. Our analysis has provided new insight into the organization of the MW and the mechanisms that contribute to the formation and maintenance of distinct compartments within the MW. We show that the MW limits access of host cell innate immune receptors to sites of viral replication and assembly. Moreover, we show that components of the nuclear transport machinery, normally involved in regulating traffic between the cytoplasm and the nucleus, have a role in limiting immune receptor access to compartments within the MW. These findings provide important insights in how HCV, and likely other positive-strand RNA viruses, organize their replication factories and evaded recognition by host cell immune receptors.

Hepatitis C virus (HCV) infection induces formation of a membranous web structure in the host cell cytoplasm where the viral genome replicates and virions assemble. The membranous web is thought to concentrate viral components and hide viral RNA from pattern recognition receptors. We have uncovered a role for nuclear pore complex proteins (Nups) and nuclear transport factors (NTFs) in the membranous web. We show that HCV infection leads to increased levels of cytoplasmic Nups that accumulate at sites enriched for HCV proteins. Moreover, we detected interactions between specific HCV proteins and both Nups and NTFs. We hypothesize that cytoplasmically positioned Nups facilitate formation of the membranous web and contribute to the compartmentalization of viral replication. Accordingly, we show that transport cargo proteins normally targeted to the nucleus are capable of entering regions of the membranous web, and that depletion of specific Nups or Kaps inhibits HCV replication and assembly., Author Summary Hepatitis C virus (HCV) is a positive strand RNA virus and is a major cause of liver disease worldwide, affecting more than 170 million individuals. Infection of cells with HCV leads to rearrangement of cytoplasmic host cell membranes into viral replication and assembly complexes collectively known as the membranous web. This membranous web is thought to be involved in concentrating viral components and immune evasion, though the mechanisms by which these functions are achieved remains an important question in the field. Here, we report that nuclear envelope structures that transport macromolecules into and out of the nucleus, termed nuclear pore complexes (NPCs), are also present in the membranous web of cells infected with HCV and other positive strand RNA viruses. Our results suggest that these NPCs function to regulate access of proteins into the interior of the membranous web, thus contributing to the establishment of an environment conducive to viral replication and viral immune evasion. Consistent with this idea, we show that NPC proteins are required for HCV assembly. Our discovery that nuclear transport proteins play a role in HCV replication, and potentially other viral infections, may lead to the discovery of new targets for antiviral therapies.