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

Abstract

The enterovirus genus of the picornavirus family includes a large number of important human pathogens such as poliovirus, coxsackievirus, enterovirus A71, and rhinoviruses. Like all other positive-strand RNA viruses, genome replication of enteroviruses occurs on rearranged membranous structures called replication organelles (ROs). Phosphatidylinositol 4-kinase IIIβ (PI4KB) is required by all enteroviruses for RO formation. The enteroviral 3A protein recruits PI4KB to ROs, but the exact mechanism remains elusive. Here, we investigated the role of acyl-coenzyme A binding domain containing 3 (ACBD3) in PI4KB recruitment upon enterovirus replication using ACBD3 knockout (ACBD3^KO) cells. ACBD3 knockout impaired replication of representative viruses from four enterovirus species and two rhinovirus species. PI4KB recruitment was not observed in the absence of ACBD3. The lack of ACBD3 also affected the localization of individually expressed 3A, causing 3A to localize to the endoplasmic reticulum instead of the Golgi. Reconstitution of wild-type (wt) ACBD3 restored PI4KB recruitment and 3A localization, while an ACBD3 mutant that cannot bind to PI4KB restored 3A localization, but not virus replication. Consistently, reconstitution of a PI4KB mutant that cannot bind ACBD3 failed to restore virus replication in PI4KB^KO cells. Finally, by reconstituting ACBD3 mutants lacking specific domains in ACBD3^KO cells, we show that acyl-coenzyme A binding (ACB) and charged-amino-acid region (CAR) domains are dispensable for 3A-mediated PI4KB recruitment and efficient enterovirus replication. Altogether, our data provide new insight into the central role of ACBD3 in recruiting PI4KB by enterovirus 3A and reveal the minimal domains of ACBD3 involved in recruiting PI4KB and supporting enterovirus replication.IMPORTANCE Similar to all other positive-strand RNA viruses, enteroviruses reorganize host cellular membranes for efficient genome replication. A host lipid kinase, PI4KB, plays an important role in this membrane rearrangement. The exact mechanism of how enteroviruses recruit PI4KB was unclear. Here, we revealed a role of a Golgi-residing protein, ACBD3, as a mediator of PI4KB recruitment upon enterovirus replication. ACBD3 is responsible for proper localization of enteroviral 3A proteins in host cells, which is important for 3A to recruit PI4KB. By testing ACBD3 and PI4KB mutants that abrogate the ACBD3-PI4KB interaction, we showed that this interaction is crucial for enterovirus replication. The importance of specific domains of ACBD3 was evaluated for the first time, and the domains that are essential for enterovirus replication were identified. Our findings open up a possibility for targeting ACBD3 or its interaction with enteroviruses as a novel strategy for the development of broad-spectrum antienteroviral drugs.

Keywords: acyl-coenzyme A binding domain containing 3 (ACBD3); enterovirus; phosphatidylinositol 4-kinase IIIβ (PI4KB); replication organelles; viral replication; virus-host interactions.

FIG 1

ACBD3 is crucial for enterovirus replication. (A) Growth curves of enteroviruses in HeLa^wt and ACBD3^KO 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 ACBD3^KO cells. HeLa^wt and ACBD3^KO 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 ACBD3^KO cells. HeLa^wt and ACBD3^KO 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) HeLa^wt (A and B) and ACBD3^KO (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) HeLa^wt (A) and ACBD3^KO (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 HeLa^WT cells and ACBD3^KO cells. (A) Golgi and ER integrity in ACBD3^KO cells. HeLa^wt and ACBD3^KO cells were fixed and stained with antibodies against the Golgi markers GM130 and TGN46 or the ER marker calreticulin. (B to D) HeLa^wt and ACBD3^KO 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 ACBD3^KO 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) HeLa^wt and ACBD3^KO 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 PI4KB^KO cells. (A) RNA replication of CVB3 mutant in PI4KB^KO cells. HeLa^wt cells and two PI4KB^KO 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 PI4KB^KO 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) HeLa^wt and PI4KB^KO cells were transfected with plasmids encoding EGFP-tagged GalT, FLAG-tagged PI4KB wt, PI4KB-I⁴³A or -D⁴⁴A mutant. At 24 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-GalT control using a one-way ANOVA. ***, P < 0.001; N.S., not significant.

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) HeLa^wt and ACBD3^KO 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 ACBD3^KO 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). Nuclei were stained with DAPI (blue). Bars represent 10 µm.