High-Order Epistasis and Functional Coupling of Infection Steps Drive Virus Evolution toward Independence from a Host Pathway

The phosphatidylinositol-4 kinase III beta (PI4KB)/oxysterol-binding protein (OSBP) family I pathway serves as an essential host pathway for the formation of viral replication complex for viral plus-strand RNA synthesis; however, poliovirus (PV) could evolve toward substantial independence from this host pathway with four mutations. Recessive epistasis of the two mutations (3A-R54W and 2B-F17L) is essential for viral RNA replication. Quantitative analysis of effects of the other two mutations (2B-Q20H and 2C-M187V) on each step of infection reveals functional couplings between viral replication, growth, and spread conferred by the 2B-Q20H mutation, while no enhancing effect was conferred by the 2C-M187V mutation. The effects of the 2B-Q20H mutation occur only via another recessive epistasis between the 3A-R54W/2B-F17L mutations. These mutations confer enhanced replication in PI4KB/OSBP-independent infection concomitantly with an increased ratio of viral plus-strand RNA to the minus-strand RNA. This work reveals the essential roles of the functional coupling and high-order, multitiered recessive epistasis in viral evolution toward independence from an obligatory host pathway. IMPORTANCE Each virus has a different strategy for its replication, which requires different host factors. Enterovirus, a model RNA virus, requires host factors PI4KB and OSBP, which form an obligatory functional axis to support viral replication. In an experimental evolution system in vitro, virus mutants that do not depend on these host factors could arise only with four mutations. The two mutations (3A-R54W and 2B-F17L) are required for the replication but are not sufficient to support efficient infection. Another mutation (2B-Q20H) is essential for efficient spread of the virus. The order of introduction of the mutations in the viral genome is essential (known as "epistasis"), and functional couplings of infection steps (i.e., viral replication, growth, and spread) have substantial roles to show the effects of the 2B-Q20H mutation. These observations would provide novel insights into an evolutionary pathway of the virus to require host factors for infection.