The 3A protein from multiple picornaviruses utilizes the golgi adaptor protein ACBD3 to recruit PI4KIIIβ

Abstract

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

Fig 1

The N termini of Aichi virus 3A, klassevirus, and bovine kobuvirus 3A are myristoylated. Strep-Tagged kobuvirus 3A proteins were transiently transfected into 293T cells, affinity purified using the Strep-Tag system, and then subjected to mass-spectrometric peptide sequencing. Mass spectra from LC-MS/MS analysis show myristoylation of Aichi virus 3A (A), klassevirus 3A (B), bovine kobuvirus 3A (C), and I5A mutant Aichi virus 3A (D); however, only the acetylated form was observed for the Aichi virus 3A R3A mutant (E).

Fig 2

Picornaviral 3A proteins interact differentially with PI4KIIIβ and ACBD3. (A) Strep-Tagged 3A proteins across the picornavirus family were affinity captured under fully equilibrated binding conditions and then blotted with anti-ACBD3, anti-PI4KIIIβ, and anti-Strep-Tag antibodies, revealing stable interactions between kobuvirus and enterovirus 3A proteins and ACBD3. (B) When the affinity capture conditions were changed to rapid capture and wash conditions (described in Materials and Methods), transient interactions between the enterovirus 3A proteins and PI4KIIIβ were also observed.

Fig 3

Affinity purification of Strep-Tagged ACBD3. Strep-Tagged ACBD3 and FLAG-tagged enterovirus and kobuvirus 3A proteins were transiently cotransfected into 293T cells, and ACBD3 complexes were affinity captured under rapid kinetic conditions. Samples were then serially Western blotted for PI4KIIIβ, Strep-Tag, and Flag tag. ACBD3 copurifies with PI4KIIIβ in the absence or presence of picornavirus 3A. In the presence of 3A, ACBD3 copurifies with 3A proteins from multiple entero- and kobuviruses, with the exception of EV71. In the presence of Aichi virus 3A, an SDS-resistant complex consistent with ACBD3 bound to 3A is visible (black triangle). A separate Western blot of the input samples was serially blotted for GAPDH and Flag-tag (bottom) to control for expression and loading levels.

Fig 4

Site-directed mutagenesis of Aichi virus 3A identifies residues required for interaction with PI4KIIIβ and ACBD3. (A) Alanine scanning of individual and multiple residues in Aichi virus 3A was performed. Mutated Aichi virus 3A was transiently transfected into 293T cells and affinity purified using the Strep-Tag system. The eluate was blotted with anti-PI4KIIIβ, anti-ACBD3, and anti-Strep-Tag to normalize for bait expression. (B) Map of residues contributing to Aichi virus 3A and ACBD3 and PI4KIIIβ interaction. Residues labeled in red abolished myristoylation when converted to alanine. The predicted transmembrane region is highlighted in purple, and predicted alpha-helices are highlighted in teal. Black boxes indicate residues that reduce binding to ACBD3 or PI4KIIIβ by >90% when converted to alanine. Gray boxes indicate residues that do not change binding by >90%, and white boxes indicate residues for which no information is available.

Fig 5

Chemical and genetic inhibition of PI4KIIIβ decreases Aichi virus replication. PIK93, an inhibitor of PI4KIIIβ, demonstrates a dose-dependent block in poliovirus (A) and Aichi virus replication (B). Genetic inhibition of PI4KIIIβ by stable shRNA blocks poliovirus (C) and Aichi virus replication (D) only when PI4KIIIβ levels are strongly reduced. RLU, relative light units. qRT-PCR (E) and Western blotting (F) of shRNA constructs demonstrate 10%, 90%, and 99% knockdown of PI4KIIIβ transcript levels and 0%, 60%, and 98% knockdown of PI4KIIIβ protein levels.

Fig 6

Genetic inhibition of physically interacting genes reduces viral replication. siRNA knockdown of GBF1, PI4KIIIβ, and ACBD3 reduces poliovirus (A) and Aichi virus (B) replicon growth in HeLa cells relative to control siRNA. RLU, relative light units. (C) Western blot of GBF1, PI4KIIIβ, ACBD3, and GAPDH in siRNA knockdown and control knockdown HeLa cells.

Fig 7

Reduced recruitment of PIKIIIβ correlates with delayed replication kinetics of Aichi virus replicons. 3A mutants were cloned into Aichi virus replicon and examined for replication efficiency. (A) The Aichi virus 3A E11A mutant is unable to replicate. (B) The Aichi virus 3A N2A and R3A mutants replicate, suggesting that myristoylation of Aichi virus 3A is not required for replication. (C to F) Aichi virus 3A mutants with mutations that reduce PI4KIIIβ binding by >90% are still are capable of replicating, albeit with slower kinetics. (G) The Aichi virus 3A E22A mutant, which retains PI4KIIIβ and ACBD3 binding, demonstrates replication kinetics similar to those of wild-type, while the P59A mutant demonstrates significantly delayed and reduced replication kinetics. RLU, relative light units.

Fig 8

Aichi virus replicons with 3A mutants with reduced ability to recruit PI4KIIIβ are sensitized to PIK93 inhibition. (A) The EC₅₀ for PIK93 against wild-type Aichi virus replicon was 0.60 μM at 330 min posttransfection. PIK93 inhibition of Aichi virus replication can be overcome at lower concentrations given a longer replication window. While 1 μM and 0.5 μM PIK93 significantly reduce and delay Aichi virus replication, lower concentrations of PIK93 merely delay replication of wild-type Aichi virus, though the virus eventually reaches the same level of replication. RLU, relative light units. (B) 3A mutants with significantly reduced binding of PI4KIIIβ are sensitized to PIK93 inhibition; the EC₅₀ is reduced to 0.24 μM in the I5A mutant and to 0.03 μM in the NRVI2AAAA mutant.