Project 4

SUMMARYTo prevent deadly CoV infections we propose to study the molecular basis of coronavirus-induced lung edema and its resolution, through the identification of (i) Signaling pathwaysresulting in severe lung disease to inform inhibitors as antiviral candidates; and (ii) Virusvirulence genes. Deletion of these genes will lead to attenuated vaccine candidates. Three aimsare proposed: Aim 1. To determine the factors involved in edema induction and resolutionduring CoV infection. We have shown that both E and 3a proteins of SARS-CoV, and proteins Eand 5 of MERS-CoV include two sequence domains involved in virulence, one containing a PDZbinding motif (PBM), and another one encoding ion channel (IC) activity. The binding of theSARS-CoV E protein PBM to proteins containing the PDZ motif causes Acute RespiratoryDisease Syndrome in infected animals. The importance of the PBM is likely associated with itsability to bind to more than 400 cellular proteins and, therefore, to regulate many cell signalingpathways. We will study the whole-proteome interactions between PBMs in MERS-CoV, andcellular PDZs. This interactome will be the basis for the identification of peptides interfering withPBM-PDZ binding, using peptide libraries. The mechanism of inflammasome activation byMERS-CoV proteins with IC activity will be studied. Edema resolution is possible by twoenzymatic activities: epithelial sodium channel activity (ENa+C) and Na+/K+ ATPase that moveNa+ ions from the alveolar fluid into the interstitium promoting water elimination. We showedthat SARS-CoV E protein binds Na+/K+ ATPase and have postulated that this binding reducesNa+/K+ ATPase activity, leading to lung edema; this will be investigated in this project. Aim 2.We propose to identify viral and host non-coding RNAs involved in MERS-CoV pathogenesisand lung inflammation, as potential targets in antiviral and anti-edema strategies. Aim 3. Todevelop safe live-attenuated vaccines for MERS-CoV. The construction of MERS-CoVsdefective in propagation, and the generation of attenuated, dissemination competent rMERS-CoVs are proposed. Maximizing biosafety and genetic stability of the vaccine candidatesare main goals of the project.