ABSTRACT
(1) Background: coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been linked to hematological dysfunctions, but there are little experimental data that explain this. Spike (S) and Nucleoprotein (N) proteins have been putatively associated with these dysfunctions. In this work, we analyzed the recruitment of hemoglobin (Hb) and other metabolites (hemin and protoporphyrin IX-PpIX) by SARS-Cov2 proteins using different approaches. (2) Methods: shotgun proteomics (LC-MS/MS) after affinity column adsorption identified hemin-binding SARS-CoV-2 proteins. The parallel synthesis of the peptides technique was used to study the interaction of the receptor bind domain (RBD) and N-terminal domain (NTD) of the S protein with Hb and in silico analysis to identify the binding motifs of the N protein. The plaque assay was used to investigate the inhibitory effect of Hb and the metabolites hemin and PpIX on virus adsorption and replication in Vero cells. (3) Results: the proteomic analysis by LC-MS/MS identified the S, N, M, Nsp3, and Nsp7 as putative hemin-binding proteins. Six short sequences in the RBD and 11 in the NTD of the spike were identified by microarray of peptides to interact with Hb and tree motifs in the N protein by in silico analysis to bind with heme. An inhibitory effect in vitro of Hb, hemin, and PpIX at different levels was observed. Strikingly, free Hb at 1mM suppressed viral replication (99%), and its interaction with SARS-CoV-2 was localized into the RBD region of the spike protein. (4) Conclusions: in this study, we identified that (at least) five proteins (S, N, M, Nsp3, and Nsp7) of SARS-CoV-2 recruit Hb/metabolites. The motifs of the RDB of SARS-CoV-2 spike, which binds Hb, and the sites of the heme bind-N protein were disclosed. In addition, these compounds and PpIX block the virus's adsorption and replication. Furthermore, we also identified heme-binding motifs and interaction with hemin in N protein and other structural (S and M) and non-structural (Nsp3 and Nsp7) proteins.
Subject(s)
COVID-19/etiology , Hemoglobins/metabolism , SARS-CoV-2/metabolism , Viral Nonstructural Proteins/metabolism , Viral Structural Proteins/metabolism , COVID-19/blood , Hemin/metabolism , Hemoglobins/ultrastructure , Humans , Molecular Docking Simulation , Protein Binding , Protein Domains , Proteomics , Protoporphyrins/metabolism , SARS-CoV-2/pathogenicity , Viral Nonstructural Proteins/ultrastructure , Viral Structural Proteins/ultrastructure , Virus Attachment , Virus ReplicationABSTRACT
The outbreak of coronavirus disease 2019 (COVID-19) requires urgent need for effective treatment. Severe COVID-19 is characterized by a cytokine storm syndrome with subsequent multiple organ failure (MOF) and acute respiratory distress syndrome (ARDS), which may lead to intensive care unit and increased risk of death. While awaiting a vaccine, targeting COVID-19-induced cytokine storm syndrome appears currently as the efficient strategy to reduce the mortality of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The stress-responsive enzyme, heme oxygenase-1 (HO-1) is largely known to protect against inflammatory response in animal models. HO-1 is induced by hemin, a well-tolerated molecule, used for decades in the treatment of acute intermittent porphyria. Experimental studies showed that hemin-induced HO-1 mitigates cytokine storm and lung injury in mouse models of sepsis and renal ischemia-reperfusion injury. Furthermore, HO-1 may also control numerous viral infections by inhibiting virus replication. In this context, we suggest the hypothesis that HO-1 cytoprotective pathway might be a promising target to control SARS-CoV-2 infection and mitigate COVID-19-induced cytokine storm and subsequent ARDS.