Mechanisms Of Golgi Protein ACBD3 In Enterovirus Replication

Enterovirus 71(EV71)is the major causative pathogen of hand,foot,and mouth disease(HFMD).Infection with EV71 is often associated with neurological complications,ranging from aseptic meningitis,brainstem and cerebellar encephalitis,to acute flaccid paralysis.In recent years,the frequency and the severity of EV71 infection are increasing in China and pose a threat to human health and social stability.EV71 belongs to Picornavirus which can utilize intracellular membranes,such as endoplasmic reticulum(ER),the ER-Golgi intermediate compartment,the Golgi,endosomes,or lysosomes,for genome replication.Virus infections results in remodelling of intracellular membranes to form "replication organelles" and increasing the local concentrations of components required for replication.GBF1 and ARF1 has been reported to participate in viral replication,initially.Recent research has revealed that Kobuvirus can recruits PI4KB to viral RNA replication sites through interaction between 3 A and ACBD3.Although ACBD3 can interact with different enteroviral 3 A protein,the regulatory mechanism remains elusive.By screening human cDNA library in the yeast two-hybrid system,we uncover that ACBD3 serves as a target of the 3 A protein of EV71.As such,our project will provide the theoretical basis for the elucidation of pathogenesis and the discovery of antiviral drug targets of EV71.The interaction between 3A and ACBD3 occurs in cells expressing 3A or infected with EV71.Although aggregated in the Golgi in mock infected cells,ACBD3 spread out in the cytoplasm upon EV71 infection.Genetic inhibition or deletion of ACBD3 drastically impairs viral RNA replicaion,protein expression and plaque formation.Such defects are corrected upon restoration of ACBD3.The C-terminal domain of ACBD3 and C-terminal domain of 3A play important role in the interaction.I44A or H54Y substitution in 3 A interrupts the binding to ACBD3.Our results demonstrate that EV71-3A selectively utilizes ACBD3 to facilitate viral replication.PI4KB is a membrane-modifying host factor essential for enterovirus replication.However,it is unclear whether a physical association between PI4KB and EV71 replication machinery exists.We examined the ablity of PI4KB to participate in EV71 replication,and found PI4KB inhibitor can reduce EV71 replication.Genetic inhibition or deletion of PI4KB drastically impairs viral RNA replicaion,protein expression and plaque formation.Furthermore,we found that EV71-3A can enhance the interaction between PI4KB and ACBD3.In infected cells,EV71 3A redirects ACBD3,along with PI4KB,to the replication site,which facilitaties PI4P production.Consequently,the viral proteins and host proteins can form a large complex that is necessary for RNA synthesis at replication sites.I44A or H54Y substitution in 3A also interrupts the interaction between ACBD3 and PI4KB.Further analysis suggests that ACBD3 is also indispensable for the replication of enterovirus 68 but disadvantageous to human rhinovirus 16.Overall,viral and host proteins can form 3A-ACBD3-PI4KB complex for replication of EV71 and EV68.These results provide a new insight into the molecular network of enterovirus replication.Collectively,our data indicate that a 3A/ACBD3/PI4KB complex is formed to synthesize PI4P at EV71 RNA replication sites and plays an essential role in viral RNA replication.ACBD3 also differentially mediates the replication of enterovirus 68 and rhinovirus 16.Our project reveal a mechanism of enterovirus replication that involves a selective utilization of host ACBD3 for viral benefit,and provide a new target for antiviral drugs.