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 aspects1–3. 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.
Reporter Assay and IFN Bioassay The following reporter constructs were used in this study: pISRE-luc was purchased from Stratagene, EF1-α-ren from Engin Gürlevik, pCAGGS-Flag-RIG-I from Chris Basler, pIRF1-GAS-ff-luc, pWPI-SMN1-flag and pWPI-NS5 (ZIKV)-HA was described previously.
For the reporter assay, HEK293RI cells were plated in 24-well plates 24 hours prior to transfection. Firefly reporter and Renilla transfection control were transfected together with plasmids expressing viral proteins using polyethylenimine (PEI, Polysciences) for untreated and treated conditions. In 18 hours cells were stimulated for 8 hours with a corresponding inducer and harvested in the passive lysis buffer (Promega). Luminescence of Firefly and Renilla luciferases was measured using dual-luciferase-reporter assay (Promega) according to the manufacturer’s instructions in a microplate reader (Tecan). Total amounts of IFN-α/β in cell supernatants were measured by using 293T cells stably expressing the firefly luciferase gene under the control of the mouse Mx1 promoter (Mx1-luc reporter cells). Briefly, HEK293RI cells were seeded, transfected with pCAGGS-flag-RIG-I plus viral protein constructs and stimulated as described above. Cell supernatants were harvested in 8 hour. Mx1-luc reporter cells were seeded into 96-well plates in triplicates and were treated 24 hours later with supernatants. At 16 hours post incubation, cells were lysed in the passive lysis buffer (Promega), and luminescence was measured with a microplate reader (Tecan). The assay sensitivity was determined by a standard curve.
Viral inhibitors assay A549-ACE2 cells were seeded into 96-well plates in DMEM medium (10% FCS, 100 ug/ml Streptomycin, 100 IU/ml Penicillin) one day before infection. Six hours before infection, or at the time of infection, the medium was replaced with 100ul of DMEM medium containing either the compounds of interest or DMSO as a control. Infection was performed by adding 10ul of SARS-CoV-2-GFP (MOI 3) per well and plates were placed in the IncuCyte S3 Live-Cell Analysis System where whole well real-time images of mock (Phase channel) and infected (GFP and Phase channel) cells were captured every 4h for 48h. Cell viability (mock) and virus growth (mock and infected) were assessed as the cell confluence per well (Phase area) and GFP area normalized on cell confluence per well (GFP area/Phase area) respectively using IncuCyte S3 Software (Essen Bioscience; version 2019B Rev2).
Reference & Source information: https://www.biorxiv.org/
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