Characterization of the c10orf76‐PI4KB complex and its necessity for Golgi PI4P levels and enterovirus replication

The lipid kinase PI4KB, which generates phosphatidylinositol 4‐phosphate (PI4P), is a key enzyme in regulating membrane transport and is also hijacked by multiple picornaviruses to mediate viral replication. PI4KB can interact with multiple protein binding partners, which are differentially manipulated by picornaviruses to facilitate replication. The protein c10orf76 is a PI4KB‐associated protein that increases PI4P levels at the Golgi and is essential for the viral replication of specific enteroviruses. We used hydrogen–deuterium exchange mass spectrometry to characterize the c10orf76‐PI4KB complex and reveal that binding is mediated by the kinase linker of PI4KB, with formation of the heterodimeric complex modulated by PKA‐dependent phosphorylation. Complex‐disrupting mutations demonstrate that PI4KB is required for membrane recruitment of c10orf76 to the Golgi, and that an intact c10orf76‐PI4KB complex is required for the replication of c10orf76‐dependent enteroviruses. Intriguingly, c10orf76 also contributed to proper Arf1 activation at the Golgi, providing a putative mechanism for the c10orf76‐dependent increase in PI4P levels at the Golgi. Synopsis Enteroviruses hijack host PI4KB and GBF1 to build replication organelles. This study reveals that the direct interaction between c10orf76 and PI4KB is required for c10orf76 recruitment to the Golgi and the replication of c10orf76‐dependent enteroviruses. c10orf76 is localized to the Golgi through a direct interaction with the kinase linker of PI4KB. c10orf76 regulates GBF1 and active Arf1 dynamics that control Golgi PI4P levels. Enteroviruses that rely on c10orf76 for replication depend on formation of the c10orf76‐PI4KB complex. Graphical Abstract Enteroviruses hijack host PI4KB and GBF1 to build replication organelles. This study reveals that the direct interaction between c10orf76 and PI4KB is required for c10orf76 recruitment to the Golgi and the replication of c10orf76‐dependent enteroviruses.