Structural and mechanistic basis of key intracellular steps of enterovirus infection

ongoing

Enteroviruses (EVs) are positive-sense, single stranded RNA virus from the Picornaviridae family. With 106 types, they can cause mild to severe diseases in human, especially children, such as common cold (Rhinovirus), myocarditis (Coxsackievirus B3, CVB3) or poliomyelitis (Poliovirus, PV). EVs are known to remodel the cytoplasm of host cells to create replication organelles allowing efficient viral genome replication. They also hijack the secretory autophagy pathway for replication and viral release. Recent cryo-electron tomography (cryo-ET) work done in the Carlson lab has highlight that PV virions directly assemble on replication membrane through a tethering complex where the tether is thought to be viral ATPase 2C. Moreover, they were able to show one class of autophagosomes is carrying bundles of filament proteins, suspected as filamentous actin (F-actin). My project will address two questions in EV replication: (i) how does the viral protein 2C assist membrane-localized assembly? (ii) what is the identity and role of the filaments in virus-induced autophagy? I will use an integrative approach of cell biology, biochemistry and structural biology and PV and CVB3 will be used as model viruses for this project. I first aim to characterize the viral assembly complex by in vitro reconstitution of the interaction between model membranes, purified 2C, capsid proteins and RNA through cross-linking mass spectrometry (XL-MS) and cryo-ET. By combining focus-ion-beam (FIB) milling and cryo-ET to perform subtomogram averaging, I will study the structure of the tethering complex and the one of the filament proteins found in autophagosomes of infected cells. In parallel, to aid the identification of the filament, I will perform proteomic studies through MS study on isolated autophagosomes infected cells. Finally, combined with knock-out cells, I will be able to identify potential protein candidates and test their effect on EV infection through cryo-ET and live cell imaging.