SARS-CoV-2 USES ENDOSOMAL MEMBRANES TO BUILD REPLICATION ORGANELLES

Background: The health emergency caused by the COVID-19 pandemic has evidenced that the frequency of spillover episodes of viruses infecting bats to other species, including humans, has significantly increased compared to previous decades. Besides SARS-CoV-2, six other human coronaviruses (NL63, 229E, OC43, HKU1, SARS-CoV and MERS-CoV) emerged in the 20th and 21st century, most likely because of cross-species transmission events from bats. While many of these coronaviruses cause mild respiratory infections, MERS-CoV, SARS-CoV and SARS-CoV-2 can cause severe respiratory distress, particularly in immunocompromised individuals. However, unlike SARS-CoV and MERS-CoV, SARS-CoV-2 is highly contagious, very stable, with many person-to-person transmissions, which can occur even before individuals exhibit any symptoms. While vaccines are readily available, the emergence of new SARS-CoV-2 variants along with the increasing incidence of individuals developing long COVID urge to develop antivirals specific to treat COVID-19. To reach this goal, we need to have a working knowledge of the host-SARS-CoV-2 interactions to identify targets for therapeutic intervention. Method(s): Following that rationale, we focused on understanding how SARSCoV- 2 generates replication organelles (ROs). All coronaviruses need to remodel cellular membranes to create these structures to allow the active replication and transcription of their genome. Due to their relevance for virus replication, disabling RO formation represents a promising strategy to fight SARS-CoV-2. However, the biogenesis mechanism, the origin, and type of these replication organelles are still a major focus of debate. To identify the cellular membranes that SARS-CoV-2 uses to generate ROs we used multiple cell lines and primary cells that were evaluated by fluorescence microscopy, genetic engineering, compounds that specifically inhibit cellular processes, and immunoprecipitation assays to validate protein-protein interactions. We also used RT-qPCR to assess viral genome replication. Result(s): SARS-CoV-2 uses the viral protein NSP6 to remodel endosomal membranes juxtaposed to the ER to generate replication organelles. Specifically, the virus depends on Clathrin, COPB1, and Rab5 for efficient SARSCoV- 2 RNA synthesis. Conclusion(s): Uncovering the origins and mechanism(s) by which SARS-CoV-2 assembles ROs opens new avenues to develop strategies to interfere with RO biogenesis and halt virus replication.

Morphological, Cellular, and Molecular Basis of Brain Infection in COVID-19 Patients and Its Association to Psychiatric Symptoms

Background: Although increasing evidence confirms neuropsychiatric manifestations associated mainly with severe COVID-19 infection, long-term neuropsychiatric dysfunction (recently characterized as part of "long COVID-19" syndrome) has been frequently observed after mild infection. Method(s): We performed a broad translational investigation, employing brain imaging and cognitive tests in 81 living COVID-19 patients (mildly infected individuals) as well as flow cytometry, respirometry, microscopy, proteomics, and metabolomics in postmortem brain samples, and in preclinical in vitro and ex vivo models. Result(s): We observed orbitofrontal cortical atrophy, neurocognitive impairment, excessive fatigue and anxiety symptoms in living individuals. Postmortem brain tissue from 26 individuals who died of COVID-19 revealed histopathological signs of brain damage. Five individuals out of the 26 exhibited foci of SARS- CoV-2 infection and replication, particularly in astrocytes. Supporting the hypothesis of astrocyte infection, neural stem cell-derived human astrocytes in vitro are susceptible to SARS-CoV-2 infection through a non-canonical mechanism that involves spike-NRP1 interaction. SARS-CoV-2-infected astrocytes manifested changes in energy metabolism and in key proteins and metabolites used to fuel neurons, as well as in the biogenesis of neurotransmitters. Moreover, human astrocyte infection elicits a secretory phenotype that significantly reduces neuronal viability. Conclusion(s): Our data support the model in which COVID-19 alter cortical thickness, promoting psychiatric symptoms. In addition, SARS-CoV-2 is able to reach the brain, infects astrocytes, and consequently, leads to neuronal death or dysfunction. These deregulated processes could contribute to the structural and functional alterations seen in the brains of COVID-19 patients. Funding Source: Sao Paulo Research Foundation (FAPESP) Keywords: COVID-19, Anxiety, Astrocytes, Multi-omics, Brain Magnetic Resonance Imaging (MRI)Copyright © 2023

Peroxisomes and Viruses: Overview on Current Knowledge and Experimental Approaches.

The general interest in the study of the interplay between peroxisomes and viruses has increased in recent years, with different reports demonstrating that distinct viruses modulate peroxisome-related mechanisms to either counteract the cellular antiviral response or support viral propagation. Nevertheless, mechanistical details are still scarce, and information is often incomplete. In this chapter, we present an overview of the current knowledge concerning the interplay between peroxisomes and different viruses. We furthermore present, compare, and discuss the most relevant experimental approaches and tools used in the different studies. Finally, we stress the importance of further, more detailed, and spatial-temporal analyses that encompass all the different phases of the viruses' infection cycles. These studies may lead to the discovery of novel peroxisome-related cellular mechanisms that can further be explored as targets for the development of novel antiviral therapies.

Clinical, Biochemical, and ATR-FTIR Spectroscopic Parameters Associated with Death or Survival in Patients with Severe COVID-19

The wide range of symptoms of the coronavirus disease 2019 (COVID-19) makes it challenging to predict the disease evolution using a single parameter. Therefore, to describe the pathophysiological response to SARS-CoV-2 infection in hospitalized patients with severe COVID-19, we compared according to survival or death, the sociodemographic and clinical characteristics, the biochemical and immunological attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectra from saliva samples and their correlation with chemometric findings. Herein, we demonstrate that ATR-FTIR spectroscopy allows the description of the events related to cell damage, such as lipids biogenesis and the secondary structure of proteins associated with lactate dehydrogenase and albumin levels. Moreover, humoral (IgM) and cellular (IFN-gamma, TNF-alpha, IL-10, and IL-6) responses were also increased in patients who died from COVID-19. Copyright © 2023 Adriana Martinez-Cuazitl et al.

Plant-derived nanovesicles: further exploration of biomedical function and application potential

Extracellular vesicles (EVs) are phospholipid bilayer vesicles actively secreted by cells, that contain a variety of functional nucleic acids, proteins, and lipids, and are important mediums of intercellular communication. Based on their natural properties, EVs can not only retain the pharmacological effects of their source cells but also serve as natural delivery carriers. Among them, plant-derived nanovesicles (PNVs) are characterized as natural disease therapeutics with many advantages such as simplicity, safety, eco-friendliness, low cost, and low toxicity due to their abundant resources, large yield, and low risk of immunogenicity in vivo. This review systematically introduces the biogenesis, isolation methods, physical characterization, and components of PNVs, and describes their administration and cellular uptake as therapeutic agents. We highlight the therapeutic potential of PNVs as therapeutic agents and drug delivery carriers, including anti-inflammatory, anticancer, wound healing, regeneration, and antiaging properties as well as their potential use in the treatment of liver disease and COVID-19. Finally, the toxicity and immunogenicity, the current clinical application, and the possible challenges in the future development of PNVs were analyzed. We expect the functions of PNVs to be further explored to promote clinical translation, thereby facilitating the development of a new framework for the treatment of human diseases.Copyright © 2023 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences

Characterization of extracellular vesicle and virus-like particles by single vesicle tetraspanin analysis

Extracellular vesicles (EVs) are nano-sized membranous particles secreted by cells. EVs have been classified into subpopulations according to their presumed biogenesis pathway, but their detailed biogenesis mechanisms still need to be fully elucidated. Enveloped viruses are another type of cell-derived nano-vesicles, and their biogenesis processes are much better known than that of EVs. Recently, studies on the similarity between enveloped viruses and EVs have been increasingly reported. The biogenesis of EVs could be better understood if these similarities are adequately investigated. In this study, we utilized a single vesicle imaging technique to visualize the protein expressions of individual nano-sized vesicles and analyzed expression patterns within single vesicles. Using this technique, we identified unique tetraspanin expression patterns in single EVs and that these patterns were closely related to their subcellular origins. The expression of CD9 or CD81 in EVs implied that they originated from the plasma membrane, and the expression of CD63 in EVs implied that they originated from endosomal organelles. We further analyzed the tetraspanin expressions of two different types of virus-like particles (VLPs) and demonstrated that the HIV-Gag-induced VLPs were more similar to EVs than SARS-CoV-2-NP/M/E-induced VLPs. In addition, HIV-Gag-GFP-expressing VLPs were highly colocalized with CD9, CD63, and CD81 signals, whereas SARS-CoV-NP-GFP-expressing VLPs were not. Based on these observations, we could assume that tetraspanin-expressing EVs might be produced through a similar process by which HIV is produced. © 2023

Viral and Host Small RNA Response to SARS-CoV-2 Infection

After two years into the pandemic of the coronavirus disease 2019 (COVID-19), it remains unclear how the host RNA interference (RNAi) pathway and host miRNAs regulate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and impact the development of COVID-19. In this study, we profiled small RNAs in SARS-CoV-2-infected human ACE2-expressing HEK293T cells and observed dysregulated host small RNA groups, including specific host miRNAs that are altered in response to SARS-CoV-2 infection. By comparing dysregulated miRNAs in different SARS-CoV-2-infected samples, we identified miRNA-210-3p, miRNA-30-5p, and miR-146a/b as key host miRNAs that may be involved in SARS-CoV-2 infection. Furthermore, by comparing virally derived small RNAs (vsmRNAs) in different SARS-CoV-2-infected samples, we observed multiple hot spots in the viral genome that are prone to generating vsmRNAs, and their biogenesis can be dependent on the antiviral isoform of Dicer. Moreover, we investigated the biogenesis of a recently identified SARS-CoV-2 viral miRNA encoded by ORF7a and found that it is differentially expressed in different infected cell lines or in the same cell line with different viral doses. Our results demonstrate the involvement of both host small RNAs and vsmRNAs in SARS-CoV-2 infection and identify these small RNAs as potential targets for anti-COVID-19 therapeutic development. © 2022 by the authors.

Landscape Analysis of Quercetin: A Potential Candidate Against SARS-CoV-2

Fruit, vegetables, and green tea contain quercetin (a flavonoid). Some of the diet's most signifi-cant sources of quercetin are apples, onions, tomatoes, broccoli, and green tea. Antioxidant, anticancer, anti-inflammatory, antimicrobial, antibacterial, and anti-viral effects have been studied of quercetin. The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus, ribonucleic acid (RNA) polymer-ase, and other essential viral life-cycle enzymes are all prevented from entering the body by quercetin. Despite extensive in vitro and in vivo investigations on the immune-modulating effects of quercetin and vitamin C treatment. 3-methyl-quercetin has been shown to bind to essential proteins necessary to convert minus-strand RNA into positive-strand RNAs, preventing the replication of viral RNA in the cytoplasm. Quercetin has been identified as a potential SARS-CoV-2 3C-like protease (3CLpro) suppressor in recent molecular docking studies and in silico assessment of herbal medicines. It has been demonstrated that quercetin increases the expression of heme oxygenase-1 through the nuclear factor erythroid-related factor 2 (Nrf2) signal network. Inhibition of heme oxygenase-1 may increase bilirubin synthesis, an endoge-nous antioxidant that defends cells. When human gingival fibroblast (HGF) cells were exposed to lipo-polysaccharide (LPS), inflammatory cytokine production was inhibited. The magnesium (Mg+2) cation complexation improves quercetin free radical scavenging capacity, preventing oxidant loss and cell death. The main objective of this paper is to provide an overview of the pharmacological effects of quercetin, its protective role against SARS-CoV-2 infection, and any potential molecular processes.Copyright © 2022 Bentham Science Publishers.

P06-21 MEHP treatment induces a decrease in SIRT1 expression, mitochondrial dysfunction, and transcriptomic alterations in the human trophoblastic cell line HTR-8/svneo

Background: Mono(ethylhexyl) phthalate (MEHP) is the main metabolite of Di(2-ethylhexyl) phthalate (DEHP), a chemical worldwide used as a plastic softener to increase the malleability, flexibility, and durability of several types of plastic, including those employed in bottled water, medical devices, and food wrapping, among others. Importantly, the consumption of these products has dramatically increased during the COVID-19 pandemic. MEHP has been classified as an endocrine disruptor chemical (EDC) and its involuntary intake has been associated with pregnancy complications such as preeclampsia and miscarriages. The placenta is a transitory organ that provides sustainability to the fetus, as well as the transportation of nutrients, hormones, and oxygen. Recent studies have proposed that MEHP may impair placental development and functionality. Nevertheless, little is known about its molecular mechanisms and effects on the placenta. Recent data has suggested that Sirtuin 1 (SIRT1) might be a molecular target. The aim of this study was to analyze the toxic and transcriptomic effects of MEHP in the human trophoblastic cell line HTR-8/svneo, focusing on the SIRT1-related pathways. Methods and Results: The HTR-8/svneo cell line was used as an extravillous trophoblast model to investigate MEHP effects. MEHP concentrations employed in this study were 0.5, 5, 50, 100, and 200 microM. Cell viability was evaluated by two methods: fixable viability staining, using eFluor 780, and MTT assay. Only the MTT assay suggested a significant decrease in cell viability at 48 hours with MEHP treatments of 5, 50, 100, and 200 microM. Mitochondrial biogenesis was analyzed by qPCR amplifying a region of the MT-ND1 mitochondrial gene. GAPDH promoter was used as a reference control. The results suggested a decrease in mitochondrial DNA at 48 hours. The transcriptomic analysis was performed in an Illumina Next-seq 500 with a coverage of 10 million reads. Doses of 5 and 200 microM of MEHP at 48 hours were analyzed. The results show that 41 and 341 genes were differentially expressed, respectively. These genes are involved in trophoblast function and pathophysiology and, according to previous reports, a portion of them are regulated by SIRT1. Finally, the effect of MEHP on SIRT1 was explored at both protein and mRNA levels by western blot and RT-qPCR, respectively. The results for mRNA levels exhibited a significant decline at 24 hours for all treatments, while protein levels were significantly reduced by 200 microM MEHP treatment at 48 hours. Conclusion(s): The present study demonstrates that MEHP treatments promote mitochondrial dysfunction in HTR-8/svneo cells. Moreover, the transcriptome analysis showed that MEHP modifies important signals for placental function and pathophysiology. The decline in SIRT1 levels correlates with the mitochondrial alterations as well as a portion of the transcriptomic changes, suggesting that SIRT1 may have an important role in MEHP effects in trophoblastic cells. Copyright © 2022 Elsevier B.V.

Translating extracellular vesicle packaging into therapeutic applications.

Extracellular vesicles (EVs) are membrane-bound particles released by cells in various (patho)physiological conditions. EVs can transfer effector molecules and elicit potent responses in recipient cells, making them attractive therapeutic agents and drug delivery platforms. In contrast to their tremendous potential, only a few EV-based therapies and drug delivery have been approved for clinical use, which is largely attributed to limited therapeutic loading technologies and efficiency. As EV cargo has major influence on their functionality, understanding and translating the biology underlying the packaging and transferring of biomolecule cargos (e.g. miRNAs, pathogen antigens, small molecule drugs) into EVs is key in harnessing their therapeutic potential. In this review, through recent insights into EVs' content packaging, we discuss different mechanisms utilized by EVs during cargo packaging, and how one might therapeutically exploit this process. Apart from the well-characterized EVs like exosomes and microvesicles, we also cover the less-studied and other EV subtypes like apoptotic bodies, large oncosomes, bacterial outer membrane vesicles, and migrasomes to highlight therapeutically-diverse opportunities of EV armoury.

The Role of Microparticles in Polycystic Ovarian Syndrome. An Updated Review

Microparticles (MPs) are vesicles of less than 1 mu m in diameter (submicron vesicles) shed from plasma membranes to cell activation, injury, and apoptosis response. They consisted of membrane proteins and cytosolic material from the cell they originated. These vesicles are vital mediators of pathological and physiological cellular processes. Polycystic ovary syndrome (PCOS) is a regular endocrine, menstrual and metabolic condition that affects 10-15% of females in their reproductive period. Numerous researches have described the association between low-grade chronic inflammation and PCOS;however, the relation is not well understood. Chronic lowgrade inflammation is reflected as a risk factor for cardiovascular disease, atherosclerosis, and endothelial dysfunction, and it is linked to abdominal obesity and insulin resistance (IR). MPs may be useful biomarkers for the early detection of cardiovascular disease and thrombosis in PCOS patients. In March 2020, the novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) became pandemic, wreaking havoc on healthcare systems worldwide and the global economy. Obesity, diabetes, and cardiovascular disease have all been linked to COVID-19 increased risk of infection. PCOS patients have recently been identified as an underserved and potentially high-risk demographic for COVID-19 problems. This article tried to review and present recent studies that explored the role of microparticles in polycystic ovarian syndrome.

RAPALOGS DOWNMODULATE INTRINSIC IMMUNITY and PROMOTE CELL ENTRY of SARS-CoV-2

Background: SARS-CoV-2 infection in immunocompromised individuals has been associated with prolonged virus shedding and the development of novel viral variants. Rapamycin and rapamycin analogs (rapalogs, including everolimus, temsirolimus, and ridaforolimus) are FDA-approved for use as mTOR inhibitors in multiple clinical settings, including cancer and autoimmunity, but a common side effect of these drugs is immunosuppression and increased susceptibility to infection. Immune impairment caused by rapalog use is traditionally attributed to their impacts on T cell signaling and cytokine production. Methods: We used replication-competent SARS-CoV-2 and HIV pseudotyped with betacoronavirus Spike proteins to assess how rapalog pretreatment of cells ex vivo and rodent animals in vivo impacts susceptibility to Spike-mediated infection. Results: We show that exposure to rapalogs increases cellular susceptibility to SARS-CoV-2 infection by antagonizing components of the constitutive and interferon-induced cell-intrinsic immune response. Pre-treatment of cells (including human lung epithelial cells and primary human small airway epithelial cells) with rapalogs promoted the early stages of SARS-CoV-2 infection by facilitating Spike-mediated virus entry. Rapalogs also boosted infection mediated by Spike from SARS-CoV and MERS-CoV in addition to hemagglutinin of influenza A virus and glycoprotein from vesicular stomatitis virus, suggesting that rapalogs downmodulate antiviral defenses that pose a common barrier to these viral fusion proteins. By identifying one rapalog (ridaforolimus) that lacks this function, we demonstrate that the extent to which rapalogs promote virus entry is linked to their capacity to trigger the lysosomal degradation of IFITM2 and IFITM3, intrinsic inhibitors of virus-cell membrane fusion. Mechanistically, rapalogs that promote virus entry inhibit the mTOR-mediated phosphorylation of TFEB, a transcription factor controlling lysosome biogenesis and lysosomal degradation pathways such as autophagy. In contrast, TFEB phosphorylation by mTOR was not inhibited by ridaforolimus. In the hamster model of SARS-CoV-2 infection, injection of rapamycin four hours prior to virus exposure resulted in elevated virus titers in lungs, accelerated weight loss, and decreased survival. Conclusion: Our findings indicate that preexisting use of certain rapalogs may elevate host susceptibility to SARS-CoV-2 infection and disease by activating a lysosome-mediated suppression of intrinsic immunity.

Oleanolic acid and ursolic acid

According to the World Health Organization (WHO), more than 70% of worldwide mortality rates can be attributed to noncommunicable diseases (NCDs) and these figures are going to be more alarming after the Covid-19 outbreak. The careful examination of in-vitro tests, in-vivo studies, and clinical models undertaken establish that the intake of fruits/vegetables rich in bioactives can reduce the risk of NCD. Oleanolic acid (OA) and its isomer ursolic acid (UA) fall under the ubiquitous class of pentacyclic triterpenoid bioactives mainly present in medicinal plants, herbs, fruit, and vegetables. In addition to antioxidant, antiinflammatory, and anticancer activities, the diet rich in OA and UA may regulate the human immune system. The protective role and clinically useful activities of OA and UA motivated us to highlight various features, points, and critical concerns regarding biogenesis, extraction, isolation, or purification of these valuable bioactives. Moreover, this chapter consolidates and expands stories of clinical trials undertaken during the last couple of decades to evaluate the biological activities and toxicities of these triterpenoids. © 2021 Elsevier Inc.

Investigating the Anti-Viral and Anti-Bacterial activities of Jordanian Medicinal plants: A narrative review

The diversity in Jordan’s flora due to its geographical areas make is well noted in the scientific literature. The challenge of disease and death caused by infectious diseases like viruses and bacteria, and as infectious diseases evolve and pathogens develop resistance to existing pharmaceuticals, the search for new novel leads, possibly with different modes of action, against bacterial and viral diseases has intensified in recent years. The intent of this review is to provide prevalent information on the antibacterial and antiviral potential in medicinal plants in Jordan, mode of action, type of viruses and bacteria, and phytochemical contents. It has been demonstrated by several studies presented in this review that medicinal plants in Jordan are rich in phytochemicals and possess antiviral and antibacterial properties.

TARGETING LIPID METABOLISM in COVID-19

Viruses are obligate intracellular parasites that make use of the host metabolic machineries to meet their biosynthetic needs. Thus, identifying host pathways essential for the virus replication may lead to potential targets for therapeutic intervention. We demonstrate major effects of SARS-CoV- 2 to modulate cellular lipid metabolism in human cells favoring increased de novo lipid synthesis and lipid remodeling, leading to increased lipid droplet (LD) accumulation in human cells. We provided evidence that LDs participate at two levels of host pathogen interaction in SARS-CoV-2 infection: first, they are important players for virus replication;and second, they are central cell organelles in the amplification of inflammatory mediator production. We demonstrated that SARS-CoV-2 modulates pathways of lipid uptake and lipogenesis leading to increased LD accumulation in human host cells. We further showed that LDs are in close proximity with SARS-CoV-2 suggestive that LDs are recruited as part of replication compartment. Moreover, we demonstrated that inhibition of DGAT-1 blocked LD biogenesis, and reduced virus replication, cell-death and pro-inflammatory mediator production. Collectively, our findings support major roles for LDs in SARS-CoV-2 replication cycle and immune response. Moreover, the finding that the host lipid metabolism and LDs are required for SARS-CoV-2 replication suggests a potential strategy to interfere with SARS-CoV-2 replication and pathogenesis by targeting lipid metabolic pathway enzymes.

Exosomes: Isolation, characterization, and biomedical applications.

Exosomes are nano-sized bioactive vesicles of 30-150 nm in diameter. They are secreted by exocytosis of nearly all type of cells in to the extracellular fluid. Thereby, they can be found in many biological fluids. Exosomes regulate intracellular communication between cells via delivery of their cargo which include lipids, proteins, and nucleic acid. Many desirable features of exosomes made them promising candidates in several therapeutic applications. In this review, we discuss the use of exosomes as diagnostic tools and their possible biomedical applications. Additionally, current techniques used for isolation, purification, and characterization of exosomes from both biological fluids and in vitro cell cultures were discussed.

The Role and Mechanism of Histone Deacetylases in Acute Kidney Injury.

Acute kidney injury (AKI) is a common clinical complication with an incidence of up to 8-18% in hospitalized patients. AKI is also a complication of COVID-19 patients and is associated with an increased risk of death. In recent years, numerous studies have suggested that epigenetic regulation is critically involved in the pathophysiological process and prognosis of AKI. Histone acetylation, one of the epigenetic regulations, is negatively regulated by histone deacetylases (HDACs). Increasing evidence indicates that HDACs play an important role in the pathophysiological development of AKI by regulation of apoptosis, inflammation, oxidative stress, fibrosis, cell survival, autophagy, ATP production, and mitochondrial biogenesis (MB). In this review, we summarize and discuss the role and mechanism of HDACs in the pathogenesis of AKI.

The SARS-CoV-2 subgenome landscape and its novel regulatory features.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is currently a global pandemic. CoVs are known to generate negative subgenomes (subgenomic RNAs [sgRNAs]) through transcription-regulating sequence (TRS)-dependent template switching, but the global dynamic landscapes of coronaviral subgenomes and regulatory rules remain unclear. Here, using next-generation sequencing (NGS) short-read and Nanopore long-read poly(A) RNA sequencing in two cell types at multiple time points after infection with SARS-CoV-2, we identified hundreds of template switches and constructed the dynamic landscapes of SARS-CoV-2 subgenomes. Interestingly, template switching could occur in a bidirectional manner, with diverse SARS-CoV-2 subgenomes generated from successive template-switching events. The majority of template switches result from RNA-RNA interactions, including seed and compensatory modes, with terminal pairing status as a key determinant. Two TRS-independent template switch modes are also responsible for subgenome biogenesis. Our findings reveal the subgenome landscape of SARS-CoV-2 and its regulatory features, providing a molecular basis for understanding subgenome biogenesis and developing novel anti-viral strategies.

Ipomoeassin-F inhibits the in vitro biogenesis of the SARS-CoV-2 spike protein and its host cell membrane receptor.

In order to produce proteins essential for their propagation, many pathogenic human viruses, including SARS-CoV-2, the causative agent of COVID-19 respiratory disease, commandeer host biosynthetic machineries and mechanisms. Three major structural proteins, the spike, envelope and membrane proteins, are amongst several SARS-CoV-2 components synthesised at the endoplasmic reticulum (ER) of infected human cells prior to the assembly of new viral particles. Hence, the inhibition of membrane protein synthesis at the ER is an attractive strategy for reducing the pathogenicity of SARS-CoV-2 and other obligate viral pathogens. Using an in vitro system, we demonstrate that the small molecule inhibitor ipomoeassin F (Ipom-F) potently blocks the Sec61-mediated ER membrane translocation and/or insertion of three therapeutic protein targets for SARS-CoV-2 infection; the viral spike and ORF8 proteins together with angiotensin-converting enzyme 2, the host cell plasma membrane receptor. Our findings highlight the potential for using ER protein translocation inhibitors such as Ipom-F as host-targeting, broad-spectrum antiviral agents.This article has an associated First Person interview with the first author of the paper.