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Inhibiting coronavirus replication by chemical ER stress, including SARS-CoV-2 and MERS-CoV

ABSTRACT: Coronaviruses (CoVs) are important human pathogens for which no specific treatment is available. Here, we provide evidence that pharmacological reprogramming of ER stress pathways can be exploited to suppress CoV replication. We found that the ER stress inducer thapsigargin efficiently inhibits coronavirus (HCoV-229E, MERS-CoV, SARS-CoV-2) replication in different cell types, (partially) restores the virus-induced translational shut-down, and counteracts the CoV-mediated downregulation of IRE1α and the ER chaperone BiP. Proteome-wide data sets revealed specific pathways, protein networks and components that likely mediate the thapsigargin-induced antiviral state, including HERPUD1, an essential factor of ER quality control, and ER-associated protein degradation complexes. The data show that thapsigargin hits a central mechanism required for CoV replication, suggesting that thapsigargin (or derivatives thereof) may be developed into broad-spectrum anti-CoV drugs.

INSTRUMENT(S): timsTOF Pro

ORGANISM(S): Homo Sapiens (human) Middle East Respiratory Syndrome-related Coronavirus Severe Acute Respiratory Syndrome Coronavirus 2 Chlorocebus Sabaeus

TISSUE(S): Permanent Cell Line Cell, Cell Culture

DISEASE(S): Severe Acute Respiratory Syndrome Coronavirus 2

SUBMITTER: Uwe Linne

LAB HEAD: Uwe Linne

PROVIDER: PXD021222 | Pride | 2021-06-24

REPOSITORIES: Pride

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Project description:Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) infection causes an immune-mediated disease. We have recently shown that SARS-CoV-induced epithelial Calu-3 cytokines could exacerbate and dampen host inflammatory and T cell responses, respectively, through modulating the functions of macrophages and dendritic cells, thereby suggesting that not only are lung epithelial cells the primary cells of SARS-CoV infection, but they also involve in initiating and orchestrating the host innate and adaptive immunity. Comprehensive evaluation of the complex epithelial signaling to SARS-CoV is, thus, crucial for paving the way to better understand SARS pathogenesis and develop the innovative therapeutics against SARS. Here, based on the microarray-based functional genomics, we reported that 2B4 cells, a clonal derivative of Calu-3 cells, elicited a temporal and spatial activation of nuclear factor (NF)kappaB, activator protein (AP)-1 (ATF2/c-Jun), and interferon regulatory factor (IRF)-3/-7 at 12-, 24-, and 48-hrs post infection (p.i.), respectively, resulting in the activation of many antiviral genes, including interferon (IFN)-Î², -Î»s, SARS-related inflammatory mediators, and various IFN-stimulated genes (ISGs). While elevated responses of IFN-Î² and IFN-Î»s were not detected until 48-hrs p.i., as a consequence of a delayed IRF-3/-7 activation, we showed, for the first time, that both types of IFNs exerted previously under-described non-redundant, complementary, and/or synergistic effects on the epithelial defense against SARS-CoV. Collectively, our results highlight the molecular mechanisms of the sequential activation of virus- and IFN-dependent signaling of lung epithelial cells against SARS-CoV and identify novel cellular targets for future studies, aiming at advancing strategies against SARS. DNA microarrays were performed in the Molecular Genomics (MG) Core Facilities at UTMB. The detailed information about the procedures of microarray analysis has been posted on the MG Core Facilities website (genomics@scms.utmb.edu). To characterize the dynamic, spatial, and temporal changes of the gene expression induced by SARS-CoV, confluent 2B4 cells grown in T-75 flasks were infected with SARS-CoV (MOI=0.1) or remained uninfected (as control) for 12, 24, and 48hrs. Because 2B4 cells were also permissive to the productive infection of Dhori virus (DHOV), a member of the Orthomyxoviridae family within the Thogotovirus genus, resulting in robust responses of IFNs and other pro-inflammatory mediators, we also established parallel cultures of DHOV-infected 2B4 cells (MOI=0.1) for the comparative analysis of global gene expression elicited by SARS-CoV- versus DHOV-infected 2B4 cells. To meet the minimal number required for application of statistical algorithms, we performed the study in triplicate at each time point for mock-, SARS-CoV-, and DHOV-infected cultures, yielding a total of 27 arrays. Briefly, supernatants were harvested from differentially treated cultures at 12-, 24-, and 48-hrs p.i. for subsequent analyses of viral yields and cytokine profiling, whereas the cells were subjected to total RNA extraction by using an RNAqueous-4PCR kit and following the protocol recommended by the manufacturer (Ambion, Austin, TX). Purified RNA samples were sent to our core facility for conversion to cDNA, biotin-labeled, and hybridized to 27 Affymetrix Human Genome U133 Plus 2.0 âGene Chipsâ each of which contained 54,675 probe set identifiers representing more than ~47,400 transcripts that identify ~38,500 well-characterized genes, and various internal controls (Affymetrix, Santa Clara, CA). Mock-infected cells were compared to cells infected with SARS-CoV or DHOV at each time point.

Project description:Human coronaviruses (hCoV) have become a threat to global health and society, as evident from the SARS outbreak in 2002 caused by SARS-CoV-1 and the most recent COVID-19 pandemic caused by SARS-CoV-2. Despite high sequence similarity between SARS-CoV-1 and -2, each strain has distinctive virulence. A better understanding of the basic molecular mechanisms mediating changes in virulence is needed. Here, we profile the virus-host protein-protein interactions of two hCoV non-structural proteins (nsps) that are critical for virus replication. We use tandem mass tag-multiplexed quantitative proteomics to sensitively compare and contrast the interactomes of nsp2 and nsp4 from three betacoronavirus strains: SARS-CoV-1, SARS-CoV-2, and hCoV-OC43 – an endemic strain associated with the common cold. This approach enables the identification of both unique and shared host cell protein binding partners and the ability to further compare the enrichment of common interactions across homologs from related strains. We identify common nsp2 interactors involved in endoplasmic reticulum (ER) Ca2+ signaling and mitochondria biogenesis. We also identify nsp4 interactors unique to each strain, such as E3 ubiquitin ligase complexes for SARS-CoV-1 and ER homeostasis factors for SARS-CoV-2. Common nsp4 interactors include N-linked glycosylation machinery, unfolded protein response (UPR) associated proteins, and anti-viral innate immune signaling factors. Both nsp2 and nsp4 interactors are strongly enriched in proteins localized at mitochondrial-associated ER membranes suggesting a new functional role for modulating host processes, such as calcium homeostasis, at these organelle contact sites. Our results shed light on the role these hCoV proteins play in the infection cycle, as well as host factors that may mediate the divergent pathogenesis of OC43 from SARS strains. Our mass spectrometry workflow enables rapid and robust comparisons of multiple bait proteins, which can be applied to additional viral proteins. Furthermore, the identified common interactions may present new targets for exploration by host-directed anti-viral therapeutics.

Project description:Human coronaviruses have become an increasing threat to global health; three highly pathogenic strains have emerged since the early 2000s, including most recently SARS-CoV-2, the cause of COVID-19. A better understanding of the molecular mechanisms of coronavirus pathogenesis is needed, including how these highly virulent strains differ from those that cause milder, common-cold like disease. While significant progress has been made in understanding how SARS-CoV-2 proteins interact with the host cell, non-structural protein 3 (nsp3) has largely been omitted from the analyses. Nsp3 is a viral protease with important roles in viral protein biogenesis, replication complex formation, and modulation of host ubiquitinylation and ISGylation. Herein, we use affinity purification-mass spectrometry to study the host-viral protein-protein interactome of nsp3 from five coronavirus strains: pathogenic strains SARS-CoV-2, SARS-CoV, and MERS-CoV, and endemic common-cold strains hCoV-229E and hCoV-OC43. We divide each nsp3 into three fragments and use tandem mass tag technology to directly compare the interactors across the five strains for each fragment. We find that that few interactors are common across all variants for a particular fragment, but we identify shared patterns between select variants, such as ribosomal proteins enriched in the N-terminal fragment (nsp3.1) analysis for SARS-CoV-2 and SARS-CoV. We also identify unique biological processes enriched for individual homologs, for instance nuclear protein important for the middle fragment of hCoV-229E, as well as ribosome biogenesis of the MERS nsp3.1 homolog. Lastly, we further investigate the interaction of the SARS-CoV-2 nsp3.1 N-terminal fragment with ATF6, a regulator of the unfolded protein response. We show that SARS-CoV-2 nsp3.1 directly binds to ATF6 and can suppress the ATF6 stress response. Characterizing the host interactions of nsp3 widens our understanding of how coronaviruses co-opt cellular pathways and presents new avenues for host-targeted antiviral therapeutics.

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Inhibiting coronavirus replication by chemical ER stress, including SARS-CoV-2 and MERS-CoV

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