Multilevel proteomics reveals host perturbations by SARS-CoV-2 and SARS-CoV

Abstract

The sudden global emergence of SARS-CoV-2 urgently requires an in-depth understanding of molecular functions of viral proteins and their interactions with the host proteome. Several omics studies have extended our knowledge of COVID-19 pathophysiology, including some focused on proteomic aspects 1-3 . To understand how SARS-CoV-2 and related coronaviruses manipulate the host we here characterized interactome, proteome and signaling processes in a systems-wide manner. This identified connections between the corresponding cellular events, revealed functional effects of the individual viral proteins and put these findings into the context of host signaling pathways. We investigated the closely related SARS-CoV-2 and SARS-CoV viruses as well as the influence of SARS-CoV-2 on transcriptome, proteome, ubiquitinome and phosphoproteome of a lung-derived human cell line. Projecting these data onto the global network of cellular interactions revealed relationships between the perturbations taking place upon SARS-CoV-2 infection at different layers and identified unique and common molecular mechanisms of SARS coronaviruses. The results highlight the functionality of individual proteins as well as vulnerability hotspots of SARS-CoV-2, which we targeted with clinically approved drugs. We exemplify this by identification of kinase inhibitors as well as MMPase inhibitors with significant antiviral effects against SARS-CoV-2. Main text:To identify interactions of SARS-CoV-2 and SARS-CoV with cellular proteins, we transduced A549 lung carcinoma cells with lentiviruses expressing individual HA-tagged viral proteins (Figure 1a;Extended data Fig. 1a; Supplementary Table 1). Affinity purification followed by mass spectrometry analysis (AP-MS) and statistical modelling of the MS1-level quantitative data allowed identification of 1484 interactions between 1086 cellular proteins and 24 SARS-CoV-2 and 27 SARS-CoV bait proteins (Figure 1b; Extended data Fig. 1b; Supplementary Table 2). The resulting virus-host interaction network revealed a wide range of cellular activities intercepted by SARS-CoV-2 and SARS-CoV (Figure 1b; Extended data Table 1; Supplementary Table 2). In particular, we discovered Extended data Figure 1 | Expression of viral proteins in transduced A549 cells induces changes to the host proteome. (a) Expression of HA-tagged viral proteins, in stably transduced A549 cells, used in AP-MS and proteome expression measurements. (b) The extended version of the virus-host protein-protein interaction network with 24 SARS-CoV-2 and 27 SARS-CoV proteins, as well as ORF3 of HCoV-NL63 and ORF4 and 4a of HCoV-229E, used as baits. Host targets regulated upon viral protein overexpression or SARS-CoV-2 infection (based on the analysis of all data of this study) are highlighted (see the in-plot legend). (c-f) Co-precipitation experiments in HEK 293T cells showing a specific enrichment of (c) endogenous MAVS co-precipitated with c-term HA-tagged ORF7b of SARS-CoV-2 and SARS-CoV (negative controls: SARS-CoV-2 ORF6-HA, ORF7a-HA), (d) ORF7b-H...

Consensus transcriptional regulatory networks of coronavirus-infected human cells

“…Consistent with a robust signature for the glucocorticoid receptor across all CoVs (GR; q-values: SARS1, 3e-35; SARS2, 1e-35; MERS, 7e-32), while this paper was under review, studies were published showing the GR agonist dexamethasone was a successful therapeutic for SARS2 infection 31 . Finally, and also while this paper was under review, in vitro analyses confirmed our predictions of the modulation by SARS2 infection of ErbB/EGFR 20,32 and TGFBR 16,32 signaling systems (Fig. 2).…”

The coding capacity of SARS-CoV-2

“…ORF9b appears in UniProt annotations and was detected by Bojkova et al 14 infected cells 14,15 did not detect peptides that originate from the out-of-frame ORFs we annotated, likely due to challenges in detecting trypsin-digested products from short coding regions 24 . Indeed, two canonical SARS-CoV-2 proteins, ORF7b (43aa) and ORF-E (75aa) were also not detected by mass-spectrometry 14,15,26,27 , and our ribosome profiling data is the first to show these SARS-CoV-2 proteins are indeed expressed.…”

Inhibiting coronavirus replication in cultured cells by chemical ER stress

“…They also indicate that it will not be trivial to identify the essential targets that mediate thapsigargin's antiviral effects. Our data provide a rich resource for further drug target analysis, also in conjunction with the few deep protein sequencing studies available for SARS-CoV-2 (but not MERS-CoV) (Bojkova et al, 2020;Bouhaddou et al, 2020;Grenga et al, 2020;Stukalov et al, 2020). Our study fills an important knowledge gap by providing a direct side-by-side comparison of pharmacologically targeted cells infected with two highly pathogenic human coronaviruses.…”

“…Green colors highlight HERPUD1 and BiP (HSPA5) values. Orange colors highlight SQSMT1 which was also identified as SARS-CoV-2regulated protein in an independent study (Stukalov et al, 2020). (D) Overrepresentation analysis showing the top 10 pathways mapping to gene IDs with increased (180 proteins, red) or decreased (61 proteins, blue) expression levels in thapsigargin-treated and infected cells compared to virus infection alone.…”