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Virus infection is a process that requires combined contributions from both virus and host factors. For this process to be efficient within the crowded host environment, viruses have evolved ways to manipulate and reorganize host structures to produce cellular microenvironments. Positive-strand RNA virus replication and assembly occurs in association with cytoplasmic membranes, causing a reorganization of these membranes to create microenvironments that support viral processes. Similarities between virus-induced membrane domains and cellular organelles have led to the description of these structures as virus replication organelles (vRO). Electron microscopy analysis of vROs in positive-strand RNA virus infected cells has revealed surprising morphological similarities between genetically diverse virus species. For all positive-strand RNA viruses, vROs can be categorized into two groups: those that make invaginations into the cellular membranes (In-vRO), and those that cause the production of protrusions from cellular membranes (Pr-vRO), most often in the form of double membrane vesicles (DMVs). In this review, we will discuss the current knowledge on the structure and biogenesis of these two different vRO classes as well as comparing morphology and function of vROs between various positive-strand RNA viruses. Finally, we will discuss recent studies describing pharmaceutical intervention in vRO formation as an avenue to control virus infection.

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.

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 long-lasting lung damage or death. These severe cases are often associated with high levels of pro-inflammatory cytokines and low antiviral responses, which can cause systemic complications. Here, we have evaluated transcriptional and cytokine secretion profiles and detected a distinct upregulation of inflammatory cytokines in infected cell cultures and 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 was mediated by cGAS-STING activation and could be attenuated through several STING-targeting drugs. Our results show that SARS-CoV-2 directs a cGAS-STING mediated, NF-κB-driven inflammatory immune response in human epithelial cells that likely contributes to inflammatory responses seen in patients and could be therapeutically targeted to suppress severe disease symptoms., Neufeldt et al. evaluate transcriptional and cytokine secretion profiles of cells and patient sera following SARS-CoV-2 infection and detect distinct upregulation of inflammatory cytokines. They also demonstrate that this upregulation is mediated by cGAS-STING and NF-κB signalling, which could provide a potential avenue for the development of future therapies against SARS-CoV-2.

The lack of effective antiviral treatments against SARS-CoV-2 is a significant limitation in the fight against the COVID-19 pandemic. Single-drug regimens have so far yielded limited results, indicating that combinations of antivirals might be required, as previously seen for other RNA viruses.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 coronavirus 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. Two independent cell-to-cell membrane fusion assays showed 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 remdesivir, a synergistic effect on the inhibition of viral replication was observed in cell lines and in a primary human colon organoid. This was consistent with the effects of cobicistat on two of its known targets, CYP3A4 and P-gp, the silencing of which boosted the in vitro antiviral activity of remdesivir in a cobicistat-like manner. When administered in vivo to Syrian hamsters at a high dose, cobicistat decreased viral load and mitigated clinical progression. 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. IMPORTANCE The lack of effective antiviral treatments against SARS-CoV-2 is a significant limitation in the fight against the COVID-19 pandemic. Single-drug regimens have so far yielded limited results, indicating that combinations of antivirals might be required, as previously seen for other RNA viruses. Our work introduces the drug booster cobicistat, which is approved by the FDA and typically used to potentiate the effect of anti-HIV protease inhibitors, as a candidate inhibitor of SARS-CoV-2 replication. Beyond its direct activity as an antiviral, we show that cobicistat can enhance the effect of remdesivir, which was one of the first drugs proposed for treatment of SARS-CoV-2. Overall, the dual action of cobicistat as a direct antiviral and a drug booster can provide a new approach to design combination therapies and rescue the activity of compounds that are only partially effective in monotherapy.

Targeting sarbecoviruses As we continue to battle the COVID-19 pandemic, we must confront the possibility of new pathogenic coronaviruses emerging in humans in the future. With this in mind, Rappazzo et al. isolated antibodies from a survivor of the 2003 severe acute respiratory syndrome coronavirus (SARS-CoV), used yeast display libraries to introduce diversity into these antibodies, and then screened for binding to SARS-CoV-2. One of the affinity-matured progeny strongly neutralized SARS-CoV-2, SARS-CoV, and two SARS-related viruses from bats. In addition, this antibody bound to the receptor-binding domains from a panel of sarbecoviruses, suggesting broader activity, and provided protection against SARS-CoV and SARS-CoV-2 in mouse models. Science, this issue p. 823, An affinity-optimized human monoclonal antibody displays broad in vivo efficacy in murine models of SARS and COVID-19., The recurrent zoonotic spillover of coronaviruses (CoVs) into the human population underscores the need for broadly active countermeasures. We employed a directed evolution approach to engineer three severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies for enhanced neutralization breadth and potency. One of the affinity-matured variants, ADG-2, displays strong binding activity to a large panel of sarbecovirus receptor binding domains and neutralizes representative epidemic sarbecoviruses with high potency. Structural and biochemical studies demonstrate that ADG-2 employs a distinct angle of approach to recognize a highly conserved epitope that overlaps the receptor binding site. In immunocompetent mouse models of SARS and COVID-19, prophylactic administration of ADG-2 provided complete protection against respiratory burden, viral replication in the lungs, and lung pathology. Altogether, ADG-2 represents a promising broad-spectrum therapeutic candidate against clade 1 sarbecoviruses.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.