The 3 A protein from multiple picornaviruses utilizes the Golgi Adaptor Protein ACBD 3 1 to Recruit PI 4

11 12 The activity of phosphatidylinositol 4-kinase class III beta (PI4KIIIβ) has been shown to 13 be required for the replication of multiple picornaviruses, however it is unclear whether a 14 physical association between PI4KIIIβ and the viral replication machinery exists and if it 15 does, whether association is necessary. We examined the ability of the 3A protein from 16 18 different picornaviruses to form a complex with PI4KIIIβ by affinity purification of 17 Strep-tagged transiently transfected constructs followed by mass spectrometry and 18 western blotting for putative interacting targets. We found that the 3A proteins of Aichi 19 virus, bovine kobuvirus, poliovirus, Coxsackievirus B3, and human rhinovirus 14 all 20 copurify with PI4KIIIβ. Furthermore we found that multiple picornavirus 3A proteins 21 copurify with with the Golgi adaptor protein acyl-CoA binding domain protein 3 22 (ACBD3/GPC60), including those from Aichi virus, bovine kobuvirus, human rhinovirus 23 14, poliovirus, coxsackievirus B2, B3, and B5. Affinity purification of ACBD3 24 confirmed interaction with multiple picornaviral 3As and revealed the ability to bind 25 PI4KIIIβ in the absence of 3A. Mass spectrometric analysis of transiently expressed 26 Aichi virus, bovine kobuvirus, and human klassevirus 3A proteins demonstrated that the 27 N-terminal glycines of these 3A proteins are myristoylated. Alanine scanning 28 mutagenesis along the entire length of Aichi 3A followed by transient expression and 29 affinity purification revealed that copurification of PI4KIIIβ could be eliminated by 30 mutation of specific residues, with little or no effect on recruitment of ACBD3. One 31 mutation at the N-terminus, I5A, significantly reduced copurification of both ACBD3 and 32 PI4KIIIβ. The dependence of Aichi virus replication on the activity of PI4KIIIβ was 33 confirmed by both chemical and genetic inhibition. Knockdown of ACBD3 by siRNA 34 also prevented replication of both Aichi virus and poliovirus. Point mutations in 3A that 35 abrogate PI4KIIIβ association sensitized Aichi virus to PIK93, suggesting that disruption 36 of the 3A/ACBD3/PI4KIIIβ complex may represent a novel therapeutic intervention 37 target that would be complementary to the inhibition of the kinase activity itself.