Reticulons promote formation of ER-derived double-membrane vesicles that facilitate SARS-CoV-2 replication.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the etiologic agent for the global COVID-19 pandemic, triggers the formation of endoplasmic reticulum (ER)-derived replication organelles, including double-membrane vesicles (DMVs), in the host cell to support viral replication. Here, we clarify how SARS-CoV-2 hijacks host factors to construct the DMVs. We show that the ER morphogenic proteins reticulon-3 (RTN3) and RTN4 help drive DMV formation, enabling viral replication, which leads to productive infection. Different SARS-CoV-2 variants, including the delta variant, use the RTN-dependent pathway to promote infection. Mechanistically, our results reveal that the membrane-embedded reticulon homology domain (RHD) of the RTNs is sufficient to functionally support viral replication and physically engage NSP3 and NSP4, two viral non-structural membrane proteins known to induce DMV formation. Our findings thus identify the ER morphogenic RTN3 and RTN4 membrane proteins as host factors that help promote the biogenesis of SARS-CoV-2-induced DMVs, which can act as viral replication platforms.

NSP9 of SARS-CoV-2 attenuates nuclear transport by hampering nucleoporin 62 dynamics and functions in host cells.

Nuclear pore complexes (NPC) regulate molecular traffics on nuclear envelope, which plays crucial roles during cell fate specification and diseases. The viral accessory protein NSP9 of SARS-CoV-2 is reported to interact with nucleoporin 62 (NUP62), a structural component of the NPC, but its biological impact on the host cell remain obscure. Here, we established new cell line models with ectopic NSP9 expression and determined the subcellular destination and biological functions of NSP9. Confocal imaging identified NSP9 to be largely localized in close proximity to the endoplasmic reticulum. In agreement with the subcellular distribution of NSP9, association of NSP9 with NUP62 was observed in cytoplasm. Furthermore, the overexpression of NSP9 correlated with a reduction of NUP62 expression on the nuclear envelope, suggesting that attenuating NUP62 expression might have contributed to defective NPC formation. Importantly, the loss of NUP62 impaired translocation of p65, a subunit of NF-κB, upon TNF-α stimulation. Concordantly, NSP9 over-expression blocked p65 nuclear transport. Taken together, these data shed light on the molecular mechanisms underlying the modulation of host cells during SARS-CoV-2 infection.

Harnessing autophagy to fight SARS-CoV-2: An update in view of recent drug development efforts.

Drug repurposing is an attractive option for identifying new treatment strategies, in particular in extraordinary situations of urgent need such as the current coronavirus disease 2019 (Covid-19) pandemic. Recently, the World Health Organization announced testing of three drugs as potential Covid-19 therapeutics that are known for their dampening effect on the immune system. Thus, the underlying concept of selecting these drugs is to temper the potentially life-threatening overshooting of the immune system reacting to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. This viewpoint discusses the possibility that the impact of these and other drugs on autophagy contributes to their therapeutic effect by hampering the SARS-CoV-2 life cycle.

How Viruses Hijack and Modify the Secretory Transport Pathway.

Eukaryotic cells contain dynamic membrane-bound organelles that are constantly remodeled in response to physiological and environmental cues. Key organelles are the endoplasmic reticulum, the Golgi apparatus and the plasma membrane, which are interconnected by vesicular traffic through the secretory transport route. Numerous viruses, especially enveloped viruses, use and modify compartments of the secretory pathway to promote their replication, assembly and cell egression by hijacking the host cell machinery. In some cases, the subversion mechanism has been uncovered. In this review, we summarize our current understanding of how the secretory pathway is subverted and exploited by viruses belonging to Picornaviridae, Coronaviridae, Flaviviridae,Poxviridae, Parvoviridae and Herpesviridae families.

Disruption of the Golgi Apparatus and Contribution of the Endoplasmic Reticulum to the SARS-CoV-2 Replication Complex.

A variety of immunolabeling procedures for both light and electron microscopy were used to examine the cellular origins of the host membranes supporting the SARS-CoV-2 replication complex. The endoplasmic reticulum has long been implicated as a source of membrane for the coronavirus replication organelle. Using dsRNA as a marker for sites of viral RNA synthesis, we provide additional evidence supporting ER as a prominent source of membrane. In addition, we observed a rapid fragmentation of the Golgi apparatus which is visible by 6 h and complete by 12 h post-infection. Golgi derived lipid appears to be incorporated into the replication organelle although protein markers are dispersed throughout the infected cell. The mechanism of Golgi disruption is undefined, but chemical disruption of the Golgi apparatus by brefeldin A is inhibitory to viral replication. A search for an individual SARS-CoV-2 protein responsible for this activity identified at least five viral proteins, M, S, E, Orf6, and nsp3, that induced Golgi fragmentation when expressed in eukaryotic cells. Each of these proteins, as well as nsp4, also caused visible changes to ER structure as shown by correlative light and electron microscopy (CLEM). Collectively, these results imply that specific disruption of the Golgi apparatus is a critical component of coronavirus replication.

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.

Increased susceptibility of human endothelial cells to infections by SARS-CoV-2 variants.

Coronavirus disease 2019 (COVID-19) spawned a global health crisis in late 2019 and is caused by the novel coronavirus SARS-CoV-2. SARS-CoV-2 infection can lead to elevated markers of endothelial dysfunction associated with higher risk of mortality. It is unclear whether endothelial dysfunction is caused by direct infection of endothelial cells or is mainly secondary to inflammation. Here, we investigate whether different types of endothelial cells are susceptible to SARS-CoV-2. Human endothelial cells from different vascular beds including umbilical vein endothelial cells, coronary artery endothelial cells (HCAEC), cardiac and lung microvascular endothelial cells, or pulmonary arterial cells were inoculated in vitro with SARS-CoV-2. Viral spike protein was only detected in HCAECs after SARS-CoV-2 infection but not in the other endothelial cells tested. Consistently, only HCAEC expressed the SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2), required for virus infection. Infection with the SARS-CoV-2 variants B.1.1.7, B.1.351, and P.2 resulted in significantly higher levels of viral spike protein. Despite this, no intracellular double-stranded viral RNA was detected and the supernatant did not contain infectious virus. Analysis of the cellular distribution of the spike protein revealed that it co-localized with endosomal calnexin. SARS-CoV-2 infection did induce the ER stress gene EDEM1, which is responsible for clearance of misfolded proteins from the ER. Whereas the wild type of SARS-CoV-2 did not induce cytotoxic or pro-inflammatory effects, the variant B.1.1.7 reduced the HCAEC cell number. Of the different tested endothelial cells, HCAECs showed highest viral uptake but did not promote virus replication. Effects on cell number were only observed after infection with the variant B.1.1.7, suggesting that endothelial protection may be particularly important in patients infected with this variant.

TMEM41B is a host factor required for the replication of diverse coronaviruses including SARS-CoV-2.

Antiviral therapeutics are a front-line defense against virally induced diseases. Because viruses frequently mutate to escape direct inhibition of viral proteins, there is interest in targeting the host proteins that the virus must co-opt to complete its replication cycle. However, a detailed understanding of the interactions between the virus and the host cell is necessary in order to facilitate development of host-directed therapeutics. As a first step, we performed a genome-wide loss of function screen using the alphacoronavirus HCoV-229E to better define the interactions between coronaviruses and host factors. We report the identification and validation of an ER-resident host protein, TMEM41B, as an essential host factor for not only HCoV-229E but also genetically distinct coronaviruses including the pandemic betacoronavirus SARS-CoV-2. We show that the protein is required at an early, but post-receptor engagement, stage of the viral lifecycle. Further, mechanistic studies revealed that although the protein was not enriched at replication complexes, it likely contributes to viral replication complex formation via mobilization of cholesterol and other lipids to facilitate host membrane expansion and curvature. Continued study of TMEM41B and the development of approaches to prevent its function may lead to broad spectrum anti-coronavirus therapeutics.

The chaperone GRP78 is a host auxiliary factor for SARS-CoV-2 and GRP78 depleting antibody blocks viral entry and infection.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the COVID-19 global pandemic, utilizes the host receptor angiotensin-converting enzyme 2 (ACE2) for viral entry. However, other host factors might also play important roles in SARS-CoV-2 infection, providing new directions for antiviral treatments. GRP78 is a stress-inducible chaperone important for entry and infectivity for many viruses. Recent molecular docking analyses revealed putative interaction between GRP78 and the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein (SARS-2-S). Here we report that GRP78 can form a complex with SARS-2-S and ACE2 on the surface and at the perinuclear region typical of the endoplasmic reticulum in VeroE6-ACE2 cells and that the substrate-binding domain of GRP78 is critical for this interaction. In vitro binding studies further confirmed that GRP78 can directly bind to the RBD of SARS-2-S and ACE2. To investigate the role of GRP78 in this complex, we knocked down GRP78 in VeroE6-ACE2 cells. Loss of GRP78 markedly reduced cell surface ACE2 expression and led to activation of markers of the unfolded protein response. Treatment of lung epithelial cells with a humanized monoclonal antibody (hMAb159) selected for its safe clinical profile in preclinical models depleted cell surface GRP78 and reduced cell surface ACE2 expression, as well as SARS-2-S-driven viral entry and SARS-CoV-2 infection in vitro. Our data suggest that GRP78 is an important host auxiliary factor for SARS-CoV-2 entry and infection and a potential target to combat this novel pathogen and other viruses that utilize GRP78 in combination therapy.

Functional landscape of SARS-CoV-2 cellular restriction.

A deficient interferon (IFN) response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been implicated as a determinant of severe coronavirus disease 2019 (COVID-19). To identify the molecular effectors that govern IFN control of SARS-CoV-2 infection, we conducted a large-scale gain-of-function analysis that evaluated the impact of human IFN-stimulated genes (ISGs) on viral replication. A limited subset of ISGs were found to control viral infection, including endosomal factors inhibiting viral entry, RNA binding proteins suppressing viral RNA synthesis, and a highly enriched cluster of endoplasmic reticulum (ER)/Golgi-resident ISGs inhibiting viral assembly/egress. These included broad-acting antiviral ISGs and eight ISGs that specifically inhibited SARS-CoV-2 and SARS-CoV-1 replication. Among the broad-acting ISGs was BST2/tetherin, which impeded viral release and is antagonized by SARS-CoV-2 Orf7a protein. Overall, these data illuminate a set of ISGs that underlie innate immune control of SARS-CoV-2/SARS-CoV-1 infection, which will facilitate the understanding of host determinants that impact disease severity and offer potential therapeutic strategies for COVID-19.

Annulate lamellae and intracellular pathogens.

Annulate lamellae (AL) have been observed many times over the years on electron micrographs of rapidly dividing cells, but little is known about these unusual organelles consisting of stacked sheets of endoplasmic reticulum-derived membranes with nuclear pore complexes (NPCs). Evidence is growing for a role of AL in viral infection. AL have been observed early in the life cycles of the hepatitis C virus (HCV) and, more recently, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), suggesting a specific induction of mechanisms potentially useful to these pathogens. Like other positive-strand RNA viruses, these viruses induce host cells membranes rearrangements. The NPCs of AL could potentially mediate exchanges between these partially sealed compartments and the cytoplasm. AL may also be involved in regulating Ca2+ homeostasis or cell cycle control. They were recently observed in cells infected with Theileria annulata, an intracellular protozoan parasite inducing cell proliferation. Further studies are required to clarify their role in intracellular pathogen/host-cell interactions.

Assembly and Cellular Exit of Coronaviruses: Hijacking an Unconventional Secretory Pathway from the Pre-Golgi Intermediate Compartment via the Golgi Ribbon to the Extracellular Space.

Coronaviruses (CoVs) assemble by budding into the lumen of the intermediate compartment (IC) at the endoplasmic reticulum (ER)-Golgi interface. However, why CoVs have chosen the IC as their intracellular site of assembly and how progeny viruses are delivered from this compartment to the extracellular space has remained unclear. Here we address these enigmatic late events of the CoV life cycle in light of recently described properties of the IC. Of particular interest are the emerging spatial and functional connections between IC elements and recycling endosomes (REs), defined by the GTPases Rab1 and Rab11, respectively. The establishment of IC-RE links at the cell periphery, around the centrosome and evidently also at the noncompact zones of the Golgi ribbon indicates that-besides traditional ER-Golgi communication-the IC also promotes a secretory process that bypasses the Golgi stacks, but involves its direct connection with the endocytic recycling system. The initial confinement of CoVs to the lumen of IC-derived large transport carriers and their preferential absence from Golgi stacks is consistent with the idea that they exit cells following such an unconventional route. In fact, CoVs may share this pathway with other intracellularly budding viruses, lipoproteins, procollagen, and/or protein aggregates experimentally introduced into the IC lumen.

Intracellular host cell membrane remodelling induced by SARS-CoV-2 infection in vitro.

BACKGROUND INFORMATION: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection induces an alteration in the endomembrane system of the mammalian cells. In this study, we used transmission electron microscopy and electron tomography to investigate the main structural alterations in the cytoplasm of Vero cells infected with a SARS-CoV-2 isolate from São Paulo state (Brazil). RESULTS: Different membranous structures derived from the zippered endoplasmic reticulum were observed along with virus assembly through membrane budding. Also, we demonstrated the occurrence of annulate lamellae in the cytoplasm of infected cells and the presence of virus particles in the perinuclear space. CONCLUSIONS AND SIGNIFICANCE: This study contributes to a better understanding of the cell biology of SARS-CoV-2 and the mechanisms of the interaction of the virus with the host cell that promote morphological changes, recruitment of organelles and cell components, in a context of a virus-induced membrane remodelling.

The SARS-CoV-2 envelope and membrane proteins modulate maturation and retention of the spike protein, allowing assembly of virus-like particles.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a ß-coronavirus, is the causative agent of the COVID-19 pandemic. Like for other coronaviruses, its particles are composed of four structural proteins: spike (S), envelope (E), membrane (M), and nucleoprotein (N) proteins. The involvement of each of these proteins and their interactions are critical for assembly and production of ß-coronavirus particles. Here, we sought to characterize the interplay of SARS-CoV-2 structural proteins during the viral assembly process. By combining biochemical and imaging assays in infected versus transfected cells, we show that E and M regulate intracellular trafficking of S as well as its intracellular processing. Indeed, the imaging data reveal that S is relocalized at endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) or Golgi compartments upon coexpression of E or M, as observed in SARS-CoV-2-infected cells, which prevents syncytia formation. We show that a C-terminal retrieval motif in the cytoplasmic tail of S is required for its M-mediated retention in the ERGIC, whereas E induces S retention by modulating the cell secretory pathway. We also highlight that E and M induce a specific maturation of N-glycosylation of S, independently of the regulation of its localization, with a profile that is observed both in infected cells and in purified viral particles. Finally, we show that E, M, and N are required for optimal production of virus-like-particles. Altogether, these results highlight how E and M proteins may influence the properties of S proteins and promote the assembly of SARS-CoV-2 viral particles.

Coronavirus infection induces progressive restructuring of the endoplasmic reticulum involving the formation and degradation of double membrane vesicles.

Coronaviruses rearrange endoplasmic reticulum (ER) membranes to form a reticulovesicular network (RVN) comprised predominantly of double membrane vesicles (DMVs) involved in viral replication. While portions of the RVN have been analyzed by electron tomography (ET), the full extent of the RVN is not known, nor how RVN formation affects ER morphology. Additionally the precise mechanism of DMV formation has not been observed. In this work, we examined large volumes of coronavirus-infected cells at multiple timepoints during infection using serial-section ET. We provide a comprehensive 3D analysis of the ER and RVN which gives insight into the formation mechanism of DMVs as well as the first evidence for their lysosomal degradation. We also show that the RVN breaks down late in infection, concurrent with the ER becoming the main budding compartment for new virions. This work provides a broad view of the multifaceted involvement of ER membranes in coronavirus infection.

SARS-CoV-2 structure and replication characterized by in situ cryo-electron tomography.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the COVID19 pandemic, is a highly pathogenic ß-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.

Eating the unknown: Xenophagy and ER-phagy are cytoprotective defenses against pathogens.

Autophagy is an evolutionary conserved catabolic process devoted to the removal of unnecessary and harmful cellular components. In its general form, autophagy governs cellular lifecycle through the formation of double membrane vesicles, termed autophagosomes, that enwrap and deliver unwanted intracellular components to lysosomes. In addition to this omniscient role, forms of selective autophagy, relying on specialized receptors for cargo recognition, exert fine-tuned control over cellular homeostasis. In this regard, xenophagy plays a pivotal role in restricting the replication of intracellular pathogens, thus acting as an ancient innate defense system against infections. Recently, selective autophagy of the endoplasmic reticulum (ER), more simply ER-phagy, has been uncovered as a critical mechanism governing ER network shape and function. Six ER-resident proteins have been characterized as ER-phagy receptors and their orchestrated function enables ER homeostasis and turnover overtime. Unfortunately, ER is also the preferred site for viral replication and several viruses hijack ER machinery for their needs. Thus, it is not surprising that some ER-phagy receptors can act to counteract viral replication and minimize the spread of infection throughout the organism. On the other hand, evolutionary pressure has armed pathogens with strategies to evade and subvert xenophagy and ER-phagy. Although ER-phagy biology is still in its infancy, the present review aims to summarize recent ER-phagy literature, with a special focus on its role in counteracting viral infections. Moreover, we aim to offer some hints for future targeted approaches to counteract host-pathogen interactions by modulating xenophagy and ER-phagy pathways.

A unifying structural and functional model of the coronavirus replication organelle: Tracking down RNA synthesis.

Zoonotic coronavirus (CoV) infections, such as those responsible for the current severe acute respiratory syndrome-CoV 2 (SARS-CoV-2) pandemic, cause grave international public health concern. In infected cells, the CoV RNA-synthesizing machinery associates with modified endoplasmic reticulum membranes that are transformed into the viral replication organelle (RO). Although double-membrane vesicles (DMVs) appear to be a pan-CoV RO element, studies to date describe an assortment of additional CoV-induced membrane structures. Despite much speculation, it remains unclear which RO element(s) accommodate viral RNA synthesis. Here we provide detailed 2D and 3D analyses of CoV ROs and show that diverse CoVs essentially induce the same membrane modifications, including the small open double-membrane spherules (DMSs) previously thought to be restricted to gamma- and delta-CoV infections and proposed as sites of replication. Metabolic labeling of newly synthesized viral RNA followed by quantitative electron microscopy (EM) autoradiography revealed abundant viral RNA synthesis associated with DMVs in cells infected with the beta-CoVs Middle East respiratory syndrome-CoV (MERS-CoV) and SARS-CoV and the gamma-CoV infectious bronchitis virus. RNA synthesis could not be linked to DMSs or any other cellular or virus-induced structure. Our results provide a unifying model of the CoV RO and clearly establish DMVs as the central hub for viral RNA synthesis and a potential drug target in CoV infection.

Morphology and morphogenesis of SARS-CoV-2 in Vero-E6 cells.

BACKGROUND: The coronaviruses (CoVs) called the attention of the world for causing outbreaks of severe acute respiratory syndrome (SARS-CoV), in Asia in 2002-03, and respiratory disease in the Middle East (MERS-CoV), in 2012. In December 2019, yet again a new coronavirus (SARS-CoV-2) first identified in Wuhan, China, was associated with a severe respiratory infection, known today as COVID-19. This new virus quickly spread throughout China and 30 additional countries. As result, the World Health Organization (WHO) elevated the status of the COVID-19 outbreak from emergency of international concern to pandemic on March 11, 2020. The impact of COVID-19 on public health and economy fueled a worldwide race to approve therapeutic and prophylactic agents, but so far, there are no specific antiviral drugs or vaccines available. In current scenario, the development of in vitro systems for viral mass production and for testing antiviral and vaccine candidates proves to be an urgent matter. OBJECTIVE: The objective of this paper is study the biology of SARS-CoV-2 in Vero-E6 cells at the ultrastructural level. METHODS: In this study, we documented, by transmission electron microscopy and real-time reverse transcription polymerase chain reaction (RT-PCR), the infection of Vero-E6 cells with SARS-CoV-2 samples isolated from Brazilian patients. FINDINGS: The infected cells presented cytopathic effects and SARS-CoV-2 particles were observed attached to the cell surface and inside cytoplasmic vesicles. The entry of the virus into cells occurred through the endocytic pathway or by fusion of the viral envelope with the cell membrane. Assembled nucleocapsids were verified inside rough endoplasmic reticulum cisterns (RER). Viral maturation seemed to occur by budding of viral particles from the RER into smooth membrane vesicles. MAIN CONCLUSIONS: Therefore, the susceptibility of Vero-E6 cells to SARS-CoV-2 infection and the viral pathway inside the cells were demonstrated by ultrastructural analysis.