ACBD3 Is an Essential Pan-enterovirus Host Factor That Mediates the Interaction between Viral 3A Protein and Cellular Protein PI4KB

FIG 1. ACBD3 is crucial for enterovirus replication. (A) Growth curves of enteroviruses in HeLawt and ACBD3KO cells. After infection for 30 min at an MOI of 5, cells were incubated for the indicated times. Then, cells were freeze-thawed three times to harvest infectious virus particles. Total virus titers were determined by endpoint dilutions. (B) RNA replication of CVB3 and EV-A71 virus in HeLa ACBD3KO cells. HeLawt and ACBD3KO cells were transfected with in vitro-transcribed RNA of CVB3 or EV-A71 subgenomic replicons encoding firefly luciferase in place of the capsid region. After 7 h, cells were lysed to determine the intracellular luciferase activity. (C) Replication of the CVB3 3A-H57Y mutant in ACBD3KO cells. HeLawt and ACBD3KO cells were infected with wild-type (wt) or 3A-H57Y mutant CVB3 reporter viruses encoding Renilla luciferase (RlucCVB3) at an MOI of 0.1. After 8 h, cells were lysed to determine luciferase activity. Values are the means plus standard errors of the means (SEM) (error bars) for triplicate values. Values were statistically evaluated using a two-tailed paired t test. **, P < 0.01; ***, P < 0.001.

FIG 2. PI4KB recruitment to virus replication sites depends on ACBD3. (A to D) HeLawt (A and B) and ACBD3KO (C and D) cells were infected with CVB3 wt at an MOI of 5. At the indicated time points, cells were fixed and stained with antibodies against CVB3 3A and ACBD3 (A and C) or CVB3 3A and PI4KB (B and D). Nuclei were stained with DAPI (blue). White asterisks indicate infected cells. Bars represent 10 µm.

FIG 3. Effects of ACBD3 knockout on the localization of enterovirus 3A proteins and the recruitment of PI4KB. (A and B) HeLawt (A) and ACBD3KO (B) cells were transfected with plasmids encoding myc-tagged EV-A71 3A, CVB3 3A, or PV-1 3A or EGFP-tagged EV-D68 3A, RV-2 3A, RV-14 3A. The next day, cells were fixed and stained with antibodies against the myc tag to detect 3A (in the case of myc-3A), ACBD3, or PI4KB. White asterisks indicate 3A-expressing cells. Nuclei were stained with DAPI (blue). Bars represent 10 µm.

FIG 4. The localization of 3A differs between HeLaWT cells and ACBD3KO cells. (A) Golgi and ER integrity in ACBD3KO cells. HeLawt and ACBD3KO cells were fixed and stained with antibodies against the Golgi markers GM130 and TGN46 or the ER marker calreticulin. (B to D) HeLawt and ACBD3KO cells were transfected with plasmid encoding myc-tagged CVB3 3A. The next day, cells were fixed and stained with an antibody against the myc tag to detect 3A and with antibodies against GM130 (B), TGN46 (C), or calreticulin (D). Nuclei were stained with DAPI (blue). Bars represent 10 µm.

FIG 5. Reconstitution of wt ACBD3 but not ACBD3-FQ mutant rescues PI4KB recruitment and CVB3 replication. (A and B) HeLa ACBD3KO cells were cotransfected with plasmids encoding myc-tagged CVB3 3A and EGFP-tagged GalT, ACBD3 wt, or ACBD3-FQ mutant. The next day, cells were fixed and stained with antibodies against the myc tag to detect 3A (A) or PI4KB (B). Nuclei were stained with DAPI (blue). Bars represent 10 µm. (C) HeLawt and ACBD3KO cells were transfected with plasmids encoding EGFP-tagged GalT, ACBD3 wt, or ACBD3-FQ mutant. At 24 h posttransfection (p.t.), cells were infected with RlucCVB3 at an MOI of 0.1. After 8 h, cells were lysed to determine luciferase activity. Values are the means plus SEM of triplicate values. Values were statistically evaluated compared to the EGFP-GalT control using a one-way ANOVA. ***, P < 0.001.

FIG 6. A PI4KB mutant, which does not interact with ACBD3, cannot be recruited by 3A and cannot restore virus replication in PI4KBKO cells. (A) RNA replication of CVB3 mutant in PI4KBKO cells. HeLawt cells and two PI4KBKO cell clones were infected with wt or 3A-H57Y mutant CVB3 reporter viruses carrying a Renilla luciferase (RlucCVB3) at an MOI of 0.1. Guanidine hydrochloride (GuHCl), a replication inhibitor, was included as a control. After 8 h, cells were lysed to determine luciferase activity (in relative light units [RLUs]). Values are means plus SEM of triplicate values. Values were statistically evaluated using a two-tailed paired t test. **, P < 0.01; ***, P < 0.001. (B and C) HeLa PI4KBKO cells were cotransfected with plasmids encoding myc-tagged CVB3 3A and FLAG-tagged PI4KB wt and PI4KB-I43A or -D44A mutants. The next day, cells were fixed and stained with antibodies against the FLAG tag to detect PI4KB and against the myc tag to detect 3A (B) or ACBD3 (C). Nuclei were stained with DAPI (blue). Bars represent 10 µm. (D) HeLawt and PI4KBKO cells were transfected with plasmids encoding EGFP-tagged GalT, FLAG-tagged PI4KB wt, PI4KB-I43A or -D44A mutant. At 24 p.t., cells were…

FIG 7. The Q and GOLD domains of ACBD3 are sufficient to support proper 3A localization, PI4KB recruitment, and enterovirus replication. (A) Schematic representation of full-length ACBD3 and its N-terminal deletion mutants (mut1 to mut3). ACBD3 contains the acyl-CoA binding (ACB) domain, the charged-amino-acid region (CAR), the glutamine-rich (Q) domain, and the Golgi dynamics domain (GOLD). The numbers indicate the amino acid positions. (B) HeLawt and ACBD3KO cells were transfected with plasmids encoding EGFP-tagged GalT, ACBD3 wt, ACBD3-FQ mutant, or ACBD3 N-terminal deletion mutants (mut1 to mut3). At 24 h p.t., cells were infected with RlucCVB3 at an MOI of 0.1. After 8 h, cells were lysed to determine luciferase activity. Values are means plus SEM of triplicate values. Values were statistically evaluated compared to the EGFP control using a one-way ANOVA. ****, P < 0.0001; N.S., not significant. (C) HeLa ACBD3KO cells were cotransfected with plasmids encoding myc-tagged CVB3 3A and EGFP-tagged ACBD3 wt, or ACBD3 N-terminal deletion mutants (mut1 to -3). The next day, the cells were fixed and stained with antibodies against PI4KB (red) and the myc tag to detect 3A (light blue).…