In this study, we have identified TBC1D22A/B as a new interacting partner of ACBD3, a protein of central importance in Golgi organization and picornaviral replication. TBC1D22A/B is a Golgi membrane-localized putative Rab33 RabGAP (9
). Altered ER-Golgi morphology has been associated with overexpression of TBC1D22B and is dependent on the presence of TBC1D22B RabGAP activity (10
). We found that the interaction between TBC1D22A/B and ACBD3 was dependent on a narrow region that maps to a predicted helix in the N terminus of TBC1D22A/B. This interaction may determine localization of these RabGAPs to the Golgi membrane and their ability to affect Golgi morphology. TBC1D22A/B bound to the same region on ACBD3 as did PI4KB, and we found that the same residues that were critical for TBC1D22A/B binding were also critical for PI4KB binding. Together with our finding that TBC1D22A/B directly competed with PI4KB for ACBD3 binding, this suggests that, in addition to putatively regulating Rab33 GTP binding at the Golgi membrane, these RabGAPs may in part determine the Golgi membrane localization of PI4KB, thereby influencing cellular phosphatidylinositol 4-phosphate (PI4P) levels and Golgi morphology. It is also of note that other RNA viruses and intracellular pathogens use TBC domain-containing proteins and their binding partners to manipulate Rab dynamics at the ER-Golgi interface for their replication, including hepatitis C virus, Legionella
species, and pathogenic Escherichia coli
Given that both TBC1D22A/B and PI4KB share very similar binding sequences and that no ACBD3 mutant could be found that discriminates between these two partners, it remains an open question whether direct regulation of either of these proteins produces modified binding to ACBD3. Mutation of the main phosphorylation site on ACBD3 (SS344) to alanine or glutamic acid had no differential effect on TBC1D22A/B versus PI4KB binding. Conversion of the identified phosphorylation sites on TBC1D22B and PI4KB to glutamic acid also did not disrupt interaction with ACBD3. TBC1D22A/B and PI4KB are organized in similar fashions, with an N-terminal ACBD3 binding domain, a 14-3-3 binding phosphorylation site in the middle, and a C-terminal enzymatic domain. Because 14-3-3 does not bind to phosphomimetic mutations, we were unable to test the effect of 14-3-3 binding on interaction with ACBD3 without phosphorylation (15
). At present, in the absence of three-dimensional structure information for each of these proteins, it is not clear what regulates the binding of TBC1D22A/B versus PI4KB to the glutamine-rich region of ACBD3.
The picornaviral 3A proteins may inform the cellular biology that regulates whether TBC1D22A/B or PI4KB is bound to ACBD3. An open question in picornavirus biology is why enteroviruses retain a specific GBF1 recruitment domain and are sensitive to brefeldin A while kobuviruses do not bind GBF1 and are insensitive to brefeldin A. Given that multiple picornaviruses rely on ACBD3 and PI4KB, direct competition by TBC1D22A/B suggests that viruses have evolved a mechanism to subvert cellular regulation of these two proteins. Our data show that the kobuviral 3A proteins appear to abrogate the binding of TBC1D22A/B to ACBD3, thus favoring the formation of a stable 3A-ACBD3-PI4KB complex that remains even after 25 to 30 min of washing. This stable complex may obviate the need for GBF1 recruitment and activity and allow kobuviruses to directly influence PI4KB localization and activity with respect to viral replication in a manner similar to, yet distinct from, that of enterovirus 3A. It is also remarkable that a protein that binds entirely to the GOLD domain can manipulate the binding of proteins in other domains of ACBD3, suggesting cross talk between ACBD3’s various domains. Although we have mapped the critical binding region of TBC1D22A/B to the Q-rich region of ACBD3, we cannot discount the role of the GOLD domain, as it appears to have an influence on the binding of these proteins as shown in .
It is not clear if or how enteroviruses manipulate the TBC1D22A/B-ACBD3 interaction, given that enterovirus 3A and TBC1D22A/B appear to occupy the same ACBD3 in our affinity purifications. The ACBD3-PI4KB-TBC1D22A/B system may provide a clue as to why enterovirus 3A proteins directly bind to and require GBF1 for replication. Mapping of the ACBD3-interacting region on the poliovirus 3A protein demonstrated an ACBD3 binding domain C-terminal to the region responsible for binding GBF1, suggesting that poliovirus 3A’s recruitment of GBF1 to Golgi membranes might be for recruitment of GBF1 to ACBD3. GBF1-Arf1 dynamics at the membrane may be responsible for recruiting another regulator that determines whether TBC1D22A/B or PI4KB is bound to ACBD3. Our affinity purification conditions may not promote the formation of a 3A-GBF1-ACBD3-PI4KB complex, thus allowing TBC1D22A/B to compete off PI4KB from ACBD3.
The reliance on overexpressed proteins in these protein-protein interaction studies is an important caveat. For example, nonphysiological levels of ACBD3 and/or TBC1D22A/B could produce an interaction that would be unlikely to occur at native levels. While our attempts to address this have been hampered by the lack of immunoprecipitation-competent antibodies to the untagged version of TBC1D22A/B, we have previously published experiments which indicate that ACBD3 and TBC1D22A/B interact at native expression levels. Specifically, we note that native TBC1D22A/B copurified with native ACBD3 in the presence of affinity-purified enterovirus 3A proteins, suggesting that the interaction is not entirely an artifact of overexpressing ACBD3 or TBC1D22A/B (see Table S7 in reference 5
Future experiments with the PI4KB mutants isolated in this study will help illuminate whether different PI4KB mutants can rescue enterovirus replication after depletion of native PI4KB. However, it is of note that enteroviruses selected for resistance to enviroxime family PI4KB inhibitors contain mutations in the ACBD3 binding region of 3A (V45A and H57Y) (25
). How these mutants affect ACBD3 binding by 3A and whether PI4KB can compete TBC1D22A/B off ACBD3 are important outstanding questions.
In conclusion, multiple picornaviruses coopt the same central ACBD3-PI4KB axis for replication but utilize different cellular mechanisms to manipulate the system. The kobuviruses and enteroviruses may reflect convergent evolutionary strategies to manipulate this key lipid regulator. The mutants obtained in this study will be useful tools to test whether PI4KB localization to ACBD3 is required for picornavirus replication; to test the effect of ACBD3, TBC1D22A/B, and GBF1 on PI4P levels in the cell; and to discover regulatory mechanisms that govern the ACBD3 interactome.