Role of host factors regulating membrane homeostasis in enterovirus replication

Abstract

Enteroviruses are causative agents of diverse diseases. Besides poliovirus, there are more than 280 serotypes of non-polio enteroviruses that can cause various mild and more severe diseases, especially in young children and immunocompromised individuals. There are no approved antivirals that can treat enterovirus infections. Given the large number of (sero-)types, development of vaccines against all enteroviruses is unlikely attainable. Antivirals with broad-acting efficacy are most desired to combat numerous enterovirus infections. The formation of replication organelles (ROs) is a universal feature that can be found in infected cells by any positive-strand RNA viruses. Both viral proteins and host factors are required for the membrane reorganization process. For enteroviruses, viral protein 3A and the cellular lipid kinase PI4KB have been suggested to be the key factors that drive this process. This thesis aims to provide mechanistic insights on the role of PI4KB and its interactors in enterovirus replication and RO formation. We employed a mutant coxsackievirus (3A-H57Y) that is insensitive to PI4KB inhibition and to OSBP inhibition to understand the functions of PI4KB and its product, PI4P, in RO biogenesis and the potential escape mechanism. OSBP is a lipid transfer protein that controls cholesterol/PI4P exchange and is essential for enterovirus replication. Under PI4KB inhibition, the mutant virus could replicate its genome in the absence of ROs, using instead the intact Golgi, yet enhanced innate immune responses were not observed. These findings suggest that ROs are not absolutely required for genome replication and question their role as a physical barrier against innate immune sensors. Growing evidence suggests that alterations in lipid homeostasis affect the proteolytic processing of the enterovirus polyprotein. We show that both PI4KB and OSBP inhibitors specifically affected the cleavage at the 3A-3B junction, and that mutation 3A-H57Y could recover impaired proteolytic processing at this junction as one escape mechanism. 3A protein recruits PI4KB to ROs, but the exact mechanism remained elusive. We studied the role of the Golgi-resident protein ACBD3 as a mediator of the 3A-PI4KB interaction. Our data provide new insight into the central role of ACBD3 in recruiting PI4KB by enterovirus 3A and reveal the minimal domains of ACBD3 involved in recruiting PI4KB and supporting enterovirus replication. By utilizing mutant versions of ACBD3 and PI4KB that cannot bind to each other, we revealed the importance of the ACBD3-PI4KB interaction for replication of all enteroviruses. Furthermore, we determined the crystal structure of the ACBD3 GOLD domain together with 3A proteins from different enteroviruses to gain a better understanding of structural determinants of ACBD3 recruitment to the viral replication sites. Protein c10orf76 was poorly characterized in terms of its localization and its function, except for being an interactor of PI4KB. We studied the canonical role of c10orf76 in cells as well as its role in enterovirus replication. We generated mutants that prevent the interaction between c10orf76 and PI4KB to define the role of the c10orf76-PI4KB complex in PI4KB activity and enterovirus replication. The findings that have been made in this thesis will contribute to developing host targeting antivirals.