Strating, J. R. P. M. | Search bioRxiv

bioRxiv2019

Characterisation 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 was also required for proper Arf1 activation at the Golgi, providing a putative mechanism for the c10orf76-dependent increase in PI4P levels at the Golgi.\n\nHighlightsO_LIc10orf76 forms a direct complex with PI4KB, with the interface formed by a disorder-to-order transition in the kinase linker of PI4KB\nC_LIO_LIThe c10orf76 binding site of PI4KB can be phosphorylated by PKA, with phosphorylation leading to decreased affinity for c10orf76\nC_LIO_LIComplex-disrupting mutants of PI4KB and c10orf76 reveal that PI4KB recruits c10orf76 to the Golgi/TGN\nC_LIO_LIDepletion of c10orf76 leads to decreases in both active Arf1 and Golgi PI4P levels\nC_LIO_LIEnteroviruses that rely on c10orf76 for replication depend on formation of the c10orf76-PI4KB complex\nC_LI

biochemistry↗

bioRxiv2018

ACBD3 is an essential pan-enterovirus host factor that mediates the interaction between viral 3A protein and cellular protein PI4KB

The enterovirus genus of the picornavirus family includes a large number of important human pathogens such as poliovirus, coxsackievirus, enterovirus-A71, and rhinoviruses. Like all other positive-strand RNA viruses, genome replication of enteroviruses occurs on rearranged membranous structures called replication organelles (ROs). Phosphatidylinositol 4-kinase III{beta} (PI4KB) is required by all enteroviruses for RO formation. The enteroviral 3A protein recruits PI4KB to ROs, but the exact mechanism remains elusive. Here, we investigated the role of Acyl-coenzyme A binding domain containing 3 (ACBD3) in PI4KB recruitment upon enterovirus replication using ACBD3-knockout (ACBD3KO) cells. ACBD3 knockout impaired replication of representative viruses from four enterovirus and two rhinovirus species. PI4KB recruitment was not observed in the absence of ACBD3. The lack of ACBD3 also affected the localization of individually expressed 3A, causing 3A to localize to the endoplasmic reticulum instead of the Golgi. Reconstitution of wt ACBD3 restored PI4KB recruitment and 3A localization, while an ACBD3 mutant that cannot bind to PI4KB restored 3A localization, but not virus replication. Consistently, reconstitution of a PI4KB mutant that cannot bind ACBD3 failed to restore virus replication in PI4KBKO cells. Finally, by reconstituting ACBD3 mutants lacking specific domains in ACBD3KO cells, we show that Acyl-coenzyme A binding (ACB) and charged amino acids region (CAR) domains are dispensable for 3A-mediated PI4KB recruitment and efficient enterovirus replication. Altogether, 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.\n\nImportanceAs all other RNA viruses, enteroviruses reorganize host cellular membranes for efficient genome replication. A host lipid kinase, PI4KB, plays an important role on this membrane rearrangement. The exact mechanism of how enteroviruses recruit PI4KB was unclear. Here, we revealed a role of a Golgi-residing protein, ACBD3, as a mediator of PI4KB recruitment upon enterovirus replication. ACBD3 is responsible for proper localization of enteroviral 3A proteins in host cells which is important for 3A to recruit PI4KB. By testing ACBD3 and PI4KB mutants that abrogate the ACBD3-PI4KB interaction, we showed that this interaction is crucial for enterovirus replication. The importance of specific domains of ACBD3 was evaluated for the first time, and the essential domains for enterovirus replication were identified. Our findings open up a possibility for targeting ACBD3 or its interaction with virus as a novel strategy for a broad-spectrum antiviral drug.

microbiology↗