Systematic functional analysis of SARS-CoV-2 proteins uncovers viral innate immune antagonists and remaining vulnerabilities

Hayn, Manuel; Hirschenberger, Maximilian; Koepke, Lennart; Nchioua, Rayhane; Straub, Jan Hendrik; Klute, Susanne; Hunszinger, Victoria; Zech, Fabian; Bozzo, Caterina Prelli; Aftab, Wasim; Christensen, Maria H; Conzelmann, Carina; Müller, Janis A.; Badarinarayan, Smitha Srinivasachar; Stürzel, Christina M.; Forné, Ignasi; Stenger, Steffen; Conzelmann, Karl‐Klaus; Münch, Jan; Schmidt, Florian; Sauter, Daniel; Imhof, Axel; Kirchhoff, Frank; Sparrer, Konstantin Maria Johannes

doi:10.1016/j.celrep.2021.109126

Cited by 206 publications

(299 citation statements)

References 84 publications

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“…Other explanations for limited autophagy in SARS-CoV-2infected cells include recently described ORF3a, ORF7a, NSP15 involvement 65 , and particularly NSP1-dependent host protein translational shutoff 26,27 that might prevent the synthesis of autophagy-regulating proteins, maintain a high ATP/AMP ratio, and increase amino acid availability. The reduction of multiple Fig.…”

Section: Discussionmentioning

confidence: 99%

SARS-CoV-2-mediated dysregulation of metabolism and autophagy uncovers host-targeting antivirals

et al. 2021

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Viruses manipulate cellular metabolism and macromolecule recycling processes like autophagy. Dysregulated metabolism might lead to excessive inflammatory and autoimmune responses as observed in severe and long COVID-19 patients. Here we show that SARS-CoV-2 modulates cellular metabolism and reduces autophagy. Accordingly, compound-driven induction of autophagy limits SARS-CoV-2 propagation. In detail, SARS-CoV-2-infected cells show accumulation of key metabolites, activation of autophagy inhibitors (AKT1, SKP2) and reduction of proteins responsible for autophagy initiation (AMPK, TSC2, ULK1), membrane nucleation, and phagophore formation (BECN1, VPS34, ATG14), as well as autophagosome-lysosome fusion (BECN1, ATG14 oligomers). Consequently, phagophore-incorporated autophagy markers LC3B-II and P62 accumulate, which we confirm in a hamster model and lung samples of COVID-19 patients. Single-nucleus and single-cell sequencing of patient-derived lung and mucosal samples show differential transcriptional regulation of autophagy and immune genes depending on cell type, disease duration, and SARS-CoV-2 replication levels. Targeting of autophagic pathways by exogenous administration of the polyamines spermidine and spermine, the selective AKT1 inhibitor MK-2206, and the BECN1-stabilizing anthelmintic drug niclosamide inhibit SARS-CoV-2 propagation in vitro with IC50 values of 136.7, 7.67, 0.11, and 0.13 μM, respectively. Autophagy-inducing compounds reduce SARS-CoV-2 propagation in primary human lung cells and intestinal organoids emphasizing their potential as treatment options against COVID-19.

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Section: Discussionmentioning

confidence: 99%

SARS-CoV-2-mediated dysregulation of metabolism and autophagy uncovers host-targeting antivirals

et al. 2021

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“…In a recent paper [1], we showed that infection of human cells with SARS-CoV-2 reduces autophagic flux. This is characterized by two hallmarks, an accumulation of SQSTM1 and the increased presence of the processed form of LC3B, LC3B-II.…”

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confidence: 94%

Manipulation of autophagy by SARS-CoV-2 proteins

et al. 2021

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As part of innate immune defenses, macroautophagy/autophagy targets viruses and viral components for lysosomal degradation and exposes pathogen-associated molecular patterns to facilitate recognition. However, viruses evolved sophisticated strategies to antagonize autophagy and even exploit it to promote their replication. In our recent study, we systematically analyzed the impact of individual SARS-CoV-2 proteins on autophagy. We showed that E, M, ORF3a, and ORF7a cause an accumulation of autophagosomes, whereas Nsp15 prevents the efficient formation of autophagosomes. Consequently, autophagic degradation of SQSTM1/p62 is decreased in the presence of E, ORF3a, ORF7a, and Nsp15. Notably, M does not alter SQSTM1 protein levels and colocalizes with accumulations of LC3B-positive membranes not resembling vesicles. Infection with SARS-CoV-2 prevents SQSTM1 degradation and increases lipidation of LC3B, indicating overall that the infection causes a reduction of autophagic flux. Our mechanistic analyses showed that the accessory proteins ORF3a and ORF7a both block autophagic degradation but use different strategies. While ORF3a prevents the fusion between autophagosomes and lysosomes, ORF7a reduces the acidity of lysosomes. In summary, we found that Nsp15, E, M, ORF3a, and ORF7a of SARS-CoV-2 manipulate cellular autophagy, and we determined the molecular mechanisms of ORF3a and ORF7a.

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“…One can envisage that escape of IFN-mediated restriction by betacoronaviruses is species-specific. For instance, SARS-CoV-2 Nsp14 targets human IFNAR1 for lysosomal degradation [62], but may be unable to degrade bat IFNAR1. This inability to evade IFN response in Eptesicus serotinus kidney cells and in Myotis myotis nasal epithelial cells may contribute to the cellular control of infection in FLG-ID and MmNep cells, as in experimentally infected Eptesicus fuscus [65].…”

Section: Discussionmentioning

confidence: 99%

“…SARS-CoV-2 has evolved numerous synergetic mechanisms to evade the IFN response in human cells [62], resulting in an absence of IFN expression in some cells, including A549 cells [51,63]. The virus is unable to counteract ISG induction in Eptesicus serotinus kidney cells and in Myotis myotis nasal epithelial cells.…”

Section: Discussionmentioning

confidence: 99%

Species-specific molecular barriers to SARS-CoV-2 replication in bat cells

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Streicher

Chazal

et al. 2021

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Bats are natural reservoirs of numerous coronaviruses, including the potential ancestor of SARS-CoV-2. Knowledge concerning the interaction between coronaviruses and bat cells is sparse. We investigated the susceptibility of primary cells from Rhinolophus ferrumequinum and Myotis species, as well as of established and novel cell lines from Myotis myotis, Eptesicus serotinus, Tadarida brasiliensis and Nyctalus noctula, to SARS-CoV-2 infection. None of these cells were sensitive to infection, not even the ones expressing detectable levels of angiotensin-converting enzyme 2 (ACE2), which serves as the viral receptor in many mammalian species. The resistance to infection was overcome by expression of human ACE2 (hACE2) in three cell lines, suggesting that restriction to viral replication was due to a low expression of bat ACE2 (bACE2) or absence of bACE2 binding in these cells. Infectious virions were produced but not released from hACE2-transduced M. myotis brain cells. E. serotinus brain cells and M. myotis nasal epithelial cells expressing hACE2 efficiently controlled viral replication. This ability to control viral replication correlated with a potent interferon response. Our data highlight the existence of species-specific molecular barriers to viral replication in bat cells. These novel chiropteran cellular models are valuable tools to investigate the evolutionary relationships between bats and coronaviruses.

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Systematic functional analysis of SARS-CoV-2 proteins uncovers viral innate immune antagonists and remaining vulnerabilities

Abstract: Highlights d Numerous SARS-CoV-2 proteins synergize to suppress immune sensing and signaling d Nsp14 targets IFNAR1 for lysosomal degradation d ORF3a and ORF7a block autophagy by different mechanisms d Synergistic treatment with IFN-g and -l1 is highly effective against SARS-CoV-2

Cited by 206 publications

References 84 publications

SARS-CoV-2-mediated dysregulation of metabolism and autophagy uncovers host-targeting antivirals

SARS-CoV-2-mediated dysregulation of metabolism and autophagy uncovers host-targeting antivirals

Manipulation of autophagy by SARS-CoV-2 proteins

Species-specific molecular barriers to SARS-CoV-2 replication in bat cells

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