Recruitment Of PI4KB And Formation Of 3A-3D^pol-PI4KB Replication Complex In The Cells Infected With Foot-and-mouth Disease Virus

In the cells infected with picornavirus, transport of intracellular material such as the bidirectionaltransport between the endoplasmic reticulum and Golgi is destroyed, and intracellular membranes arereorganized into the membrane mostly containing phosphatidylinositol-4-phosphate(PI4P). Upon thereorganized membrane, viral replication complex is formed through vial nonstructural proteinsspecifically recruiting cellular proteins, thereby initiating efficient viral RNA synthesis. PI4 P issynthesized by phosphatidylinositol-4-kinase IIIβ(PI4KB), which is important for viral replicationcomplex formation. The mechanism by which PI4 KB is recruited to picornavirus replication sites hasbeen focused. Picornaviral RNA-dependent RNA polymerase(Rd Rp, 3Dpol) is respobsible forreplicating viral genomic RNA and thus the core of viral replication complex. Studies on the formationof replication complex contribute to understanding the viral replication mechanism in infected cells.Foot-and-mouth disease virus(FMDV) is the causative agent of foot-and-mouth disease(FMD), ahighly contagious and economically important disease of cloven-hoofed animals such as pigs, bovines,sheep and goats. FMDV, a member of picornaviridae family, is characterized by fast replication andwide host tropism, which is likely to be associated with uniquely viral replication complex. The presentstudy is aimed to explore mechanisms of PI4 KB being recruited to replication sites and FMDVreplication complex formation.In the IBRS-2 cells, the knockdown of PI4 KB impaired FMDV nonstructural protein 3A expression,viral titer, and viral RNA synthesis; the overexpression of PI4 KB increased the 3A expression, viral titer,and viral RNA synthesis, demonstrating that PI4 KB is required for FMDV replication. Co-IP and GSTpulldown revealed that FMDV 3A interacted indirectly with PI4 KB, and the 3A colocalized withendogenous PI4 KB at replication sites in the FMDV-infected cells, showing that the 3A recruits PI4 KBto the replication sites and suggesting that another protein is involved in the recruitment. It was furthershowed that the C-terminal GOLD domain of cellular Acyl-Co-A Binding Domain containing 3(ACBD3) mediated the interaction between FMDV 3A and PI4 KB, and that the binding affinity of the3A-ACBD3 association was higher than that of the ACBD3-PI4 KB association in GST pulldowncompetitive assays, demonstrating that FMDV 3A binds competitively to PI4KB-associated ACBD3 and suggesting that ACBD3 mediates the PI4 KB recruitment. However, the knockdown of ACBD3 didnot impair the colocalization of FMDV 3A with PI4 KB and FMDV replication in immunofluorescenceand small interference RNA assay, showing that the recruitment of PI4 KB to replication organelles bythe 3A was ACBD3-independent. These results suggest that there is a unknown protein involved in thePI4 KB recruitment. During the screening of FMDV 3Dpol-interacting cellular proteins, PI4 KB wasidentified as a candidate protein associating with the 3Dpol. Co-IP and GST pulldown showed 3Dpol wasa novel direct interaction partner of PI4 KB, the 3Dpol colocaized with endogenous PI4 KB at replicationsites upon FMDV infection, and the 3Dpol was able to associate with the 3A, demonstrating theformation of FMDV replication complex at least comprising the 3A, 3Dpol, and PI4 KB, whichcontributes to viral efficient replication.In conclusion, FMDV 3A is responsible for recruiting PI4 KB to replication sites, the latter is essentialfor viral replication. The PI4 KB recruitment by FMDV 3A is ACBD3-independent, despite the 3A bindscompetitively to PI4KB-associated ACBD3. The replication complex of the 3A-3Dpol-PI4 KB is formedin the cells infected with FMDV, which is prerequisite of FMDV replication after entering cells.