In vitroreconstitution reveals membrane clustering and double-stranded RNA recruitment by the enteroviral AAA+ ATPase 2C

Abstract

AbstractEnteroviruses are a vast genus of positive-sense RNA viruses that cause diseases ranging from common cold to poliomyelitis and viral myocarditis. They encode a membrane-bound AAA+ ATPase, 2C, that has been suggested to serve several roles in virus replication, e.g. as an RNA helicase and capsid assembly factor. As an oligomeric peripheral membrane protein, the low solubility of 2C has hitherto impaired biochemical studies of the full-length protein. Here, we report a biochemical reconstitution poliovirus 2C association with membranes. We show that a conserved glycine divides the N-terminal membrane-binding domain of 2C into two amphipathic helix regions, AH1 and AH2. AH2 is the main mediator of 2C oligomerization, and is sufficient for its membrane binding. AH1 is the main mediator of a novel function of 2C: clustering of membranes. Cryo-electron tomography reveal that several 2C copies mediate this function by localizing to vesicle-vesicle interfaces. 2C- mediated clustering is partially outcompeted by nucleic acids, suggesting a way by which 2C can switch from an early role in coalescing replication organelles, and recruiting lipid droplets to them, to a later role where 2C assists RNA replication and particle assembly. 2C is sufficient to recruit RNA to membranes, with a preference for double-stranded RNA, thus being a candidate for the factor that localizes viral RNA replication to membranes. Finally, thein vitroreconstitution revealed that full- length 2C localized to a membrane has weak RNA chaperoning activity, dependent on having a free N terminus, but does not possess ATP-dependent helicase activity. Together, this study suggests two novel roles for 2C in membrane clustering and double-stranded RNA recruitment to membranes, and calls into question a role of 2C as an RNA helicase. The reconstitution of functional, 2C-decorated vesicles provides a platform for further experimental studies into this protein and its roles in enterovirus replication.