The Foot-and-Mouth Disease Virus Replication Complex: Dissecting the Role of the Viral Polymerase(3Dpol) and Investigating Interactions with Phosphatidylinositol-4-kinase (PI4K)

Replication of many positive-strand RNA viruses have been shown to occur within intracellular membrane-associated compartments termed replication complexes. Replication of viral RNA occurs within these intracellular compartments as a way for the virus to concentrate the structural and non-structural components into a small area to facilitate replication as well as protecting the virus components from host cell pathogen recognition and innate immune responses. Using immunofluorescent confocal and electron microscopy, foot-and-mouth disease virus (FMDV) has been shown to dysregulate Golgi and ER-derived membranes, but to date, no distinct membrane-bound replication complex comprised of viral RNA, structural and nonstructural proteins, and host-cell proteins have yet to be identified for FMDV. The FMDV RNA-dependent RNA polymerase, 3Dpol, is the primary protein involved in virus genome replication and has been previously shown to form higher-order fibril like structures in vitro in the presence of RNA. These 3Dpol fibril structures could act to ‘scaffold’ replication complex formation. Here, several mutations were made in 3Dpol to assess their role in higher-order complex formation. The ability for the different 3Dpol mutations to function was assessed biochemically, structurally and in cell culture. The results point towards the necessity for a fully functional (catalytically active) polymerase in the formation of the higher-order structures. Furthermore, complementation studies indicate that 3Dpol has two distinct functions necessary for replication within cells. Additionally, it was pertinent to investigate the role of membrane-associated kinases,such as PI4K, as a number of related viruses utilise this cellular pathway to form an optimal environment within which viral replication can occur by upregulating the formation of lipids used in the building of intracellular membranes. Investigation of translation and replication of FMDV RNA within cells show that FMDV does not appear to utilise the PI4K pathway. These results highlight differences between FMDV and other related picornaviruses and provide a basis to investigate alternative methods for replication complex formation.