Role of the interaction of SARS-CoV E protein with Na+/K+ ATPase in edema resolution

Abstract

SARS-CoV caused a worldwide epidemic infecting 8000 people with a mortality of around 10%. SARS-CoV is a pathogenic respiratory virus, whose infection led in its most severe form to acute respiratory distress syndrome (ARDS), which is characterized by interstitial and alveolar edema leading to death. We demonstrated that a recombinant mouse adapted SARS-CoV lacking the envelope protein (E) was attenuated in vivo. The SARS-CoV E protein is a virulence factor that includes two relevant motifs: its ion cannel (IC) activity encoded within the transmembrane domain and a PDZ binding motif (PBM) located at its carboxy-terminus. The main objective of this project is to study the virulence mechanism of SARS-CoV E protein by analizing the role of this protein in lung edema resolution. A recombinant SARS-CoV virus that encodes an E protein with two marker sequences (FLAG and HA) at its carboxyterminus was engineered (rSARS-CoV-Etag). Cellular proteins binding to SARS-CoV E protein were identified by tandem affinity purification (TAP) and mass spectometry (MS). To asses whether the tag sequences had an effect on virus replication, the levels of subgenomic mRNA (sgmRNA), growth kinetics and virus titers were analyzed in comparison with the mouse adapted SARS-CoV (SARS-CoV-MA15). It was shown that the rSARS-CoV-Etag was as functional as the SARS-CoV-MA15. Interestingly we identified the binding of E protein to a main mediator of edema resolution, the Na+/K+ ATPase. Moreover, it was demonstrated that this was a direct interaction by using the FAR-Western blotting assay. In addition, it was shown that in the lungs of BALB/c mice infected with SARS-CoV-MA15 the architecture of the epithelial layer was not maintained leading to the mislocalization of the Na+/K+ ATPase from its basolateral position within the plasma membrane of epithelial cells. We are investigating whether the interaction of SARS-CoV E protein with the Na+/K+ ATPase is responsible for the lack of edema resolution after SARS-CoV infection, either by inhibiting its activity or by relocating the enzyme to another subcellular compartment. Studying the consequences of the interaction of SARS-CoV E protein with Na+/K+ ATPase in edema resolution may lead to the identification of inhibitors of the lung edema induced by SARS-CoV infection.