ACBD3-mediated recruitment of PI4KB to picornavirus RNA replication sites

2B, 2BC, 2C, 3A, and 3AB interact with the C-terminal region of ACBD3

To determine the region in ACBD3 required for binding to the viral proteins, the ability of various deletion mutants of ACBD3 to interact with 2B, 2BC, 2C, 3A, or 3AB was investigated in the mammalian two-hybrid system (Figure 2A and B). For 2B, 2C, 3A, or 3AB, mut3 and mut5, both of which contain a C-terminal region (aa 327–528), maintained the ability to induce a greater than ∼15-fold increase in luciferase activity compared with negative controls. Also for 2BC, 7- and 4-fold increases in luciferase activity were detected with mut3 and mut5, respectively. On the other hand, mutants without the C-terminal region lacked the ability to enhance luciferase expression. These results suggest that the C-terminal region of ACBD3 is essential for binding to 2B, 2BC, 2C, 3A, and 3AB. Further detailed experiments to identify the binding domain to these viral proteins were carried out using a series of deletion mutants of mut3 (Figure 2C and D). For the five viral proteins, mut3Δ1 with the deletion of aa 328–373 enhanced luciferase expression to a similar level to mut3, but the other mutants did not. This suggests that aa 374–528 of ACBD3 are important for binding to the viral proteins.

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Figure 2

The C-terminal region of ACBD3 interacts with 2B, 2BC, 2C, 3A, and 3AB. (A, C) Schematic representation of (A) full-length ACBD3 (WT) and its mutants (mut1–mut5) and (C) mut3 (amino acids 328–528) and its mutants (mut3Δ1–Δ5). ACBD3 contains characteristic domains as follows: PR, proline-rich domain; ACB, ACB region; CAR, charged amino acid-rich domain; QR, glutamine-rich domain; and GOLD, Golgi dynamic domain. Numbers indicate amino-acid positions. The region between amino acids 373 and 528 is required for binding giantin. (B, D) Mammalian two-hybrid analyses were performed to examine interactions (B) between the ACBD3 mutants (mut1–5) and 2B, 2BC, 2C, 3A, or 3AB, and (D) between the mut3 mutants (mut3Δ1–Δ5) and 2B, 2BC, 2C, 3A, or 3AB, and the results are represented as described in Figure 1A. All experiments were repeated at least three times. Standard deviation bars are shown.

ACBD3 colocalizes with 2B, 2C, 3A, and dsRNA in AiV RNA-transfected cells

To investigate whether ACBD3 colocalizes with viral proteins or replicating viral RNA, an AiV replicon RNA (AV-FL-Luc-5′rzm) containing a firefly luciferase gene was transfected into Vero cells by electroporation, and then an immunofluorescence assay was carried out. The replicating viral RNA was detected by staining double-stranded replicative intermediates and replicative forms using an antibody recognizing dsRNA in which the helix length is >40 bp, as carried out for other positive-stranded RNA viruses including picornavirus (Miller et al, 2006; Harwood et al, 2008; Knoops et al, 2008; Berger et al, 2009; DeWitte-Orr et al, 2009, Hyde et al, 2009). 2B, 2C, and 3A were detected as patchy clusters in the cytoplasm at 4 h after transfection (Figure 3A), and accumulated in the perinuclear region at 6 h (only the data for 3A are shown in Figure 3A). At 4 h, when viral RNA replicates actively (see Figure 5A or C), 2B, 2C, and 3A colocalized with dsRNA (Figure 3B). ACBD3, which also formed patchy clusters in the cytoplasm at 4 h after transfection, colocalized with 2B, 2C, 3A (Figure 3A), and dsRNA (Figure 3B). These results indicate that ACBD3, 2B, 2C, and 3A (and possibly also the precursor proteins 2BC and 3AB) are present in the viral RNA replication sites.

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Figure 3

(A) Colocalization of ACBD3 with 2B, 2C, and 3A. Vero cells were electroporated with replicon RNA, AV-FL-Luc-5′rzm. At 4 or 6 h after electroporation, the cells were fixed and double stained with rabbit anti-ACBD3 and guinea pig anti-3A, anti-2B, or anti-2C antibodies. (B) Colocalization of dsRNA with ACBD3, 2B, 2C, or 3A. Vero cells were electroporated with replicon RNA. At 4 h, the cells were fixed and double stained with anti-dsRNA (mouse) and anti-2B, anti-2C, anti-3A, or anti-ACBD3 antibodies (rabbit). Bars represent 30 μm. (C) Effect of knockdown of ACBD3 on AiV replication. Vero cells were transfected with 80 nM of either control siRNA or siRNA against ACBD3. At 72 h after transfection, lysates were prepared and subjected to immunoblotting to assess the levels of ACBD3 and α-tubulin (left panel). At 72 h post transfection with siRNAs, the cells were transfected with replicon RNA and then luciferase activity in cell lysates harvested at the indicated times was measured (right panel). The maximum value obtained for cells treated with control siRNA was taken as 100%. The experiment was repeated at least three times. Standard deviation bars are shown.

Knockdown of ACBD3 inhibits AiV RNA replication

To investigate whether ACBD3 is involved in AiV RNA replication, replication of AV-FL-Luc-5′rzm RNA was examined in Vero cells treated with small interfering RNA (siRNA) targeting ACBD3 or control siRNA. At 72 h after treatment with siRNA targeting ACBD3, the amount of ACBD3, but not that of α-tubulin, was apparently decreased (Figure 3C). An effect of knockdown of ACBD3 on cell viability was not observed (data not shown). At 72 h after treatment with siRNA targeting ACBD3 or control siRNA, cells were transfected with AV-FL-Luc-5′rzm RNA by lipofection, and then cell lysates were prepared at various times after transfection and subjected to the luciferase assay. Knockdown of ACBD3 resulted in a decrease in luciferase activity. At 10 h after transfection, luciferase activity was decreased by ∼70% (Figure 3C). Here, Vero cells were used to exclude the effect of siRNA-induced interferon response, but a similar result was obtained using HeLa cells (data not shown). These results indicate that ACBD3 plays an important role in AiV RNA replication.

Localization of other Golgi proteins in AiV RNA-transfected cells

ACBD3 localizes to the Golgi through interaction with giantin, a Golgi protein, and the giantin-binding domain of ACBD3 has been mapped to the C-terminal region (aa 373–528) (Sohda et al, 2001). This region overlaps with the region important for binding to the AiV proteins (aa 374–528) (Figure 2). We then examined localization of giantin in viral RNA-replicating cells (Figure 4A). To distinguish RNA-transfected cells, a viral non-structural protein, the L protein, was immunostained. In mock-transfected cells, both ACBD3 and giantin were located in the Golgi. At 2 h after electroporation with viral RNA, the dispersion of ACBD3 and giantin was observed in some cells; however, the L protein could not be detected, because of its insufficient accumulation. At this time point, ACBD3 colocalized with giantin. As infection progressed, the redistribution of ACBD3 and giantin from the Golgi to the cytoplasm became more apparent. Importantly, at 4 h, giantin was dispersed throughout the cytoplasm, whereas ACBD3 formed clusters. As shown in Figure 3, ACBD3 colocalized with dsRNA or the viral proteins in such structures. At 4 h after electroporation, the intensity of immunofluorescence for giantin seems to be decreased compared with that in mock-transfected cells; however, no degradation of giantin was observed in an immunoblot analysis using anti-giantin antibody (data not shown).

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Figure 4

Dynamics of different Golgi proteins during AiV RNA replication. Vero cells were mock electroporated or electroporated with the replicon RNA, AV-FL-Luc-5′rzm. At 2 or 4 h after electroporation, the cells were fixed and triple stained with rabbit anti-ACBD3, guinea pig anti-L, and (A) mouse anti-giantin, (B) mouse anti-GM130, or (C) sheep anti-TGN46 antibodies. Bars represent 30 μm.

We also examined the localization of other Golgi proteins, GM130, a cis-Golgi marker, and TGN46, a trans-Golgi marker. At 2 h after transfection with replicon RNA, dispersion of GM130 and TGN46 appears to begin like giantin, and they colocalized with ACBD3 (Figure 4B and C). GM130 was redistributed to form clusters in the cytoplasm at 4 h after transfection, but did not colocalize with ACBD3 (Figure 4B). TGN46 also became dispersed in the cytoplasm with the progression of infection, and did not colocalize with ACBD3 (Figure 4C). Thus, in viral RNA-replicating cells, other Golgi proteins examined did not colocalize with ACBD3.

AiV RNA replication requires PI4KB activity

It was recently reported that PI4KB is an essential host factor for enterovirus RNA replication (Hsu et al, 2010; Arita et al, 2011). We investigated whether PI4KB is also important for AiV replication. First, we examined the effect of a PI4KB-specific inhibitor, T-00127-HEV1 (Arita et al, 2011), on viral RNA replication. Vero cells were electroporated with AV-FL-Luc-5′rzm RNA, and then cultured in medium containing 0, 1, or 5 μM T-00127-HEV1. T-00127-HEV1 inhibited AiV RNA replication in a dose-dependent manner, and the replication was almost completely inhibited at 5 μM (Figure 5A). Cell viability was not affected by treatment with 5 μM (data not shown). It is noted that at 1 h after electroporation, the level of luciferase activity observed for T-00127-HEV1-treated cells was similar to that for cells treated with DMSO, and was significantly higher than that for mock-transfected cells. At this time point, viral RNA replication does not occur, and the obtained luciferase activity mainly results from translation of input RNA (Sasaki and Taniguchi, 2008). Thus, this result suggests that PI4KB activity is important for AiV RNA replication, but not for translation.

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Figure 5

(A) T-00127-HEV1 inhibits AiV RNA replication. Vero cells were mock electroporated or electroporated with the replicon RNA, AV-FL-Luc-5′rzm, and then treated with 0 (DMSO), 1 or 5 μM T-00127-HEV1. After incubation for the indicated times, the cells were assayed for luciferase activity. (B) Depletion of endogenous PI4KB severely reduces AiV RNA replication. Vero cells were transfected with 40 nM control siRNA or siRNA against PI4KB. At 72 h after transfection, the levels of PI4KB and α-tubulin were determined by immunoblotting (right panel), and the subsequent analysis was performed as in Figure 3C (left panel). (C) AiV RNA replication is insensitive to BFA. Vero cells were electroporated with poliovirus (PV) (upper panel) or AiV (lower panel) replicon RNA and then incubated with or without 10 μg/ml of BFA. After incubation for the indicated times, the cells were assayed for luciferase activity. All experiments were repeated at least three times. Standard deviation bars are shown.

Furthermore, we investigated viral RNA replication in PI4KB knockdown cells. Vero cells were treated with siRNA targeting PI4KB or control siRNA. At 72 h after treatment with siRNA targeting PI4KB, a decrease in the amount of PI4KB was observed (Figure 5B). Cell viability was not affected by treatment with siRNA targeting PI4KB (data not shown). At 72 h after siRNA treatment, AV-FL-Luc-5′rzm RNA was transfected by lipofection, and RNA replication was examined by a luciferase assay. Knockdown of PI4KB almost completely inhibited AiV RNA replication, and at 10 h after transfection, luciferase activity was decreased by 99% (Figure 5B). A similar result was obtained using HeLa cells (data not shown). These results indicate that PI4KB activity is essential for AiV RNA replication.

PI4KB interacts with ACBD3

BFA is an inhibitor of enterovirus RNA replication (Irurzun et al, 1992; Maynell et al, 1992). BFA inactivates GBF1 (Peyroche et al, 1999) and inhibits recruitment of GBF1/Arf1 to 3A, resulting in inhibition of recruitment of PI4KB to the replication sites of enteroviruses (Belov et al, 2008; Hsu et al, 2010). We investigated whether BFA inhibits AiV replication. Treatment with 10 μg/ml of BFA inhibited poliovirus RNA replication, as known, but did not affect AiV RNA replication at all (Figure 5C). This result suggests that PI4KB is recruited independently of GBF1/Arf1 to the AiV replication sites. As described above, ACBD3 was localized to the Aichi viral RNA replication sites through binding to some viral non-structural proteins. We examined whether ACBD3 interacts with PI4KB. In the mammalian two-hybrid assay, interaction between ACBD3 and PI4KB resulted in a 138-fold increase in luciferase activity, indicating a strong interaction between the two proteins (Figure 6A). No apparent activation of luciferase expression was detected for viral non-structural proteins (3.5-fold increase for 3A and less for other proteins). To further confirm the interaction between ACBD3 and PI4KB, HA-tagged PI4KB was co-expressed with FLAG-tagged ACBD3 or FLAG-tagged 3A in 293T cells, and then co-immunoprecipitation analysis was performed. As shown in Figure 6B, PI4KB was co-immunoprecipitated with ACBD3, but not with 3A, confirming the interaction between ACBD3 and PI4KB.

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Figure 6

PI4KB interacts with ACBD3. (A) The mammalian two-hybrid assay was performed to examine interactions between PI4KB and ACBD3 or the virus proteins and the results are represented as in Figure 1A. (B) Co-immunoprecipitation of PI4KB with ACBD3. HA-tagged PI4KB was co-expressed with FLAG-tagged ACBD3 or FLAG-tagged 3A in 293T cells, and the subsequent analysis was performed as in Figure 1B. (C) Mapping of the PI4KB-binding region of ACBD3. Schematic representation of full-length ACBD3 (WT) and its mutants (mut6 and mut7) (upper panel). Mammalian two-hybrid analysis was performed to examine interactions between PI4KB and ACBD3 WT or its mutants (mut1–7), and the results are represented as described in Figure 1A (lower panel). Figure source data can be found in Supplementary data.

PI4KB interacts with the central part of ACBD3

To determine the PI4KB-binding domain of ACBD3, interactions of deletion mutants of ACBD3 with PI4KB were investigated in the mammalian two-hybrid system (Figure 6C). Of the five mutants shown in Figure 2A, mut2 and mut5 activated luciferase expression strongly (∼15- and 45-fold, respectively), implying the importance of a wider region including aa 171–327 of ACBD3 for binding to PI4KB. Additional two mutants, mut6 and mut7, which contain aa 116–327 and aa 116–428, respectively, exhibited the ability to strongly activate luciferase expression (24- and 32-fold, respectively). These results suggest that the central part of ACBD3 (aa 116–327) is important for binding to PI4KB. In addition, the PI4KB-binding domain was suggested not to overlap with the binding domain for 2B, 2BC, 2C, 3A, and 3AB (aa 374–528).

PI4KB colocalizes with ACBD3, 2B, 2C, 3A, and dsRNA in AiV RNA-transfected cells

We examined whether PI4KB localizes to the viral RNA replication sites. In mock-transfected cells, PI4KB mainly localized to the Golgi, and colocalized with ACBD3 (Figure 7A). When ACBD3 expression was knockdown by using siRNA, PI4KB was dispersed in the cytoplasm and did not localize to the Golgi (Figure 7B), strongly suggesting a critical role of ACBD3 in Golgi recruitment of PI4KB. In viral RNA-replicating cells, PI4KB colocalized with ACBD3, 2B, 2C, 3A, and dsRNA (Figure 7A and C). Treatment of mock-transfected cells with BFA resulted in dispersion of ACBD3 and PI4KB to the cytoplasm (Figure 7D). In contrast, in viral RNA-transfected cells with BFA treatment, ACBD3 and PI4KB formed clusters in the cytoplasm, and colocalized with dsRNA (Figure 7E). These results indicate that PI4KB is present in the viral RNA replication sites through interaction with ACBD3, which is recruited through interaction with 2B, 2BC, 2C, 3A, and 3AB. In addition, it was shown that recruitment of PI4KB and ACBD3 to the AiV RNA replication sites was not affected by BFA.

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Figure 7

PI4KB is recruited to the AiV RNA replication sites through interaction with ACBD3 associated with the viral proteins. (A) Colocalization of PI4KB with ACBD3. Vero cells were mock electroporated or electroporated with the replicon RNA, AV-FL-Luc-5′rzm. At 4 h after electroporation, the cells were fixed and triple stained with mouse anti-PI4KB, rabbit anti-ACBD3, and guinea pig anti-L antibodies. (B) Effect of ACBD3 knockdown on the localization of PI4KB. Vero cells were transfected with siRNA against ACBD3. At 72 h after transfection, cells were fixed and double stained with anti-ACBD3 and anti-PI4KB. Asterisks indicate cells where knockdown of the expression of ACBD3 was observed. (C) PI4KB colocalizes with 2B, 2C, 3A, or dsRNA. At 4 h after electroporation with the replicon RNA, the Vero cells were fixed and double stained with mouse anti-PI4KB and rabbit anti-2B, -2C, or -3A antibodies, or rabbit anti-PI4KB and mouse anti-dsRNA antibodies. (D, E) Recruitment of PI4KB and ACBD3 to the AiV RNA replication sites is not affected by BFA. Vero cells were (D) mock electroporated or (E) electroporated with the replicon RNA, and then incubated in medium with or without 10 μg/ml of BFA. At 4 h after electroporation, the cells were fixed and immunostained with the indicated antibodies. Bars represent 30 μm.

Replicon RNAs of AiV and poliovirus

The AiV and poliovirus replicon RNAs containing a firefly luciferase gene (AV-FL-Luc-5′rzm RNA and PV-Fluc mc RNA, respectively) were generated by in-vitro transcription using a T7 RiboMAX express large scale RNA production system (Promega) from HindIII-linearized pAV-FL-Luc-5′rzm (Sasaki and Taniguchi, 2003; Nagashima et al, 2005) and DraI-linearized pPV-Fluc mc (Arita et al, 2006), respectively. Lysates of cells transfected with the replicon RNAs were prepared using a Passive lysis buffer (Promega) and subjected to a luciferase assay using a Luciferase assay system (Promega) and a luminometer, Lumat LB9507 (Berthold).

Plasmids

The construction of pBIND-L, -2A, -2B, -2BC, -2C, -3A, -3AB, -3Cm, -3CD, and -3D, and pACT-L, -2A, -2B, -2BC, -2C, -3A, -3AB, -3Cm, -3CD, and -3D was described previously (Ishikawa et al, 2010). The coding region of ACBD3 was amplified by PCR from a cDNA clone (Toyobo, FCC137B01), while the PI4KB-coding sequence was amplified by RT–PCR using total RNA prepared from HeLa cells. Each PCR product was cloned into pBIND and pACT (Promega) as described previously (Ishikawa et al, 2010), yielding pBIND-ACBD3 and -PI4KB, and pACT-ACBD3 and -PI4KB. pBIND and pACT constructs encoding ACBD3 deletion mutants (pBIND- or pACT-ACBD3-mut1, -mut2, -mut3, -mut4, -mut5, -mut6, -mut7, -mut3Δ1, -mut3Δ2, -mut3Δ3, -mut3Δ4, and -mut3Δ5) were generated by cloning each DNA fragment amplified by PCR from pACT-ACBD3 into pBIND and pACT. The DNA fragments encoding L, 2B, 2BC, 2C, 3A, and 3AB were obtained by PCR from pAV-FL-Luc-5′rzm. The PCR-amplified DNA fragment encoding L, 2B, 2BC, 2C, 3A, 3AB, PI4KB, or ACBD3 was cloned into pCI mammalian expression vector (Promega) containing an HA or FLAG tag-coding sequence to produce an N-terminally HA- or FLAG-tagged protein (pCI-HA–L, pCI-HA–2B, pCI-HA–2BC, pCI-HA–2C, pCI-HA–3A, pCI-HA–3AB, and pCI-HA–PI4KB, and pCI-FLAG–3A and pCI-FLAG–ACBD3). To construct bait plasmids for the yeast two-hybrid assays, the 2B-, 2C- and 3A-coding regions were amplified by PCR and then cloned into pGBKT7 (Clontech), resulting in pGBKT7-2B, -2C, and -3A. The nucleotide sequences of all the PCR products were verified. For expressing MBP-fused 2B, 2C, or 3A in E. coli, these genes were amplified by PCR and cloned into pMAL-c2X (New England Biolabs). pMAL-3AB was described previously (Nagashima et al, 2008). For expressing GST-fused ACBD3, PCR-amplified ACBD3-coding sequence was cloned into pGEX-6P3 (Amersham Pharmacia Biotech).

Antibodies

Rabbit or guinea pig polyclonal antibody to 3A was obtained by immunization with the His-tagged 3A protein expressed in Escherichia coli. Rabbit or guinea pig polyclonal antibodies to 2B and 2C were obtained by immunization with a mixture of two peptides (2B: GLLTLSADTETNQTLNKC and SQFDLSGPANSVSLAASC; and 2C: CPFDPTNALNPIPGTQSK and IRGKAKTDPQAKLADVHC). The preparation of guinea pig polyclonal antibody to L was described previously (Sasaki et al, 2003). Mouse monoclonal antibodies to HA tag, FLAG tag, ACBD3 (clone G2G), and α-tubulin, and rabbit polyclonal antibodies to FLAG tag and ACBD3 were purchased from Sigma; mouse monoclonal antibody to GST from Wako; rabbit polyclonal antibody to HA tag from Bethyl; rabbit polyclonal antibody to PI4KB from Millipore; mouse monoclonal antibodies to GM130 and PI4KB from BD Biosciences; mouse monoclonal antibody to giantin from Abcam; sheep polyclonal antibody to TGN46 was from AbD serotec; anti-PI4P antibody (mouse monoclonal IgM) from Echelon Biosciences; mouse monoclonal antibody to dsRNA (clone J2) from English and Scientific Consulting; Alexa Fluor 488-conjugated anti-mouse IgG and anti-rabbit IgG antibodies, Alexa Fluor 594-conjugated anti-mouse IgG, anti-mouse IgM, anti-rabbit IgG, anti-sheep IgG, and anti-guinea pig IgG antibodies from Molecular Probes; AMCA-conjugated anti-guinea pig IgG from Millipore; and horseradish peroxidase (HRP)-conjugated antibodies from Zymed.

Yeast two-hybrid assays

Yeast two-hybrid assays were performed with a Matchmaker two-hybrid system 3 (Clontech) according to the manufacturer's instructions. The bait plasmid encoding 2B, 2C or 3A, pGBKT-2B, -2C or -3A, was transformed into Saccharomyces cerevisiae strain AH109, and the individual transformants were mated with S. cerevisiae strain Y187 pretransformed with a human HeLa cDNA library (Clontech) constructed in pGADT7. The resulting diploids were screened on agar plates of dropout medium lacking adenine, histidine, leucine, and tryptophan, supplemented with X-alpha-Gal (5-bromo-4-chloro-3-indolyl-alpha-O-galactopyranoside). The insert DNAs of yeast clones grown on plates were amplified by PCR and sequenced directly.

Mammalian two-hybrid assays

The mammalian two-hybrid assays were performed as described previously using a Checkmate mammalian two-hybrid system (Promega) (Ishikawa et al, 2010). The ability of viral protein gene-containing constructs to express fusion proteins was examined previously (Ishikawa et al, 2010). Expression of the ACBD3 constructs and deletion mutants were also examined by immunoblotting as described previously (Ishikawa et al, 2010) and confirmed expression of fusion proteins for mutants that exhibited no increase in luciferase activity (data not shown).

DNA transfection, immunoprecipitation, and immunoblotting

Transfection of plasmid DNA was carried out using a FuGENE HD transfection reagent (Roche) according to the manufacturer's protocol. For the immunoprecipitation assays, 293T cells were grown in 6-well plates for 24 h and then transfected with HA- and FLAG-tagged constructs (2 μg/well). At 24 h (for co-expression of HA–2B, HA–2C, HA–3A, or HA–PI4KB) or 48 h (for co-expression of HA–2BC or HA–3AB) after transfection, cells were washed three times with phosphate-buffered saline (PBS) and then lysed in 350 μl lysis buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1% or 0.2% Nonidet P-40 (NP-40), 1 mM phenylmethylsulphonyl fluoride) supplemented with a protease inhibitor cocktail and a phosphatase inhibitor cocktail (Sigma). Cell lysates were centrifuged at 12 000 g for 10 min, and the supernatants were immunoprecipitated with 50 μl of Dynabeads Protein G (Dynal) coated with 1 μg of antibody at 4°C for 1 h. After washing three times with lysis buffer and then one time with wash buffer (50 mM Tris–HCl pH 8.0), the beads were boiled in SDS–PAGE sample buffer. The immunoprecipitates were separated by 12.5% or 7.5% SDS–PAGE and then transferred onto polyvinylidene difluoride membranes (Bio-Rad). The membranes were probed with appropriate primary antibodies, followed by incubation with HRP-conjugated secondary antibodies. The chemiluminescence signals were visualized using ECL Advance reagents (Amersham Biosciences) and an LAS-4000UVmini (Fujifilm).

MBP pull-down assay

MBP, MBP–2B, MBP–2C, MBP–3A, and MBP–3AB, and GST–ACBD3 were expressed in E. coli BL21 cells according to the manufacturer's protocols. For purifying GST–ACBD3, harvested cells were suspended in Buffer A (140 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH 7.4) and lysed by sonication. The lysate was mixed with 50% glutathione-Sepharose slurry (GE Healthcare) and incubated for 4 h at 4°C. After the beads were washed three times with Buffer A, GST–ACBD3 were eluted with elution buffer (50 mM Tris–HCl, 10 mM reduced glutathione, pH 8.0). Cells expressing MBP or MBP-fused viral proteins were suspended in Column buffer (20 mM Tris–HCl, 200 mM NaCl, 1 mM EDTA, pH 7.5) and lysed by sonication, and the lysates were incubated with 7.5 μl of 50% amylose resin slurry (New England Biolabs) for 90 min at 4°C. Then, the resin was washed three times with Column buffer, resuspended in NP-40 buffer (50 mM Tris–HCl, 150 mM NaCl, 1% NP-40, pH 8.0), and then mixed with purified GST–ACBD3. After incubation for 90 min at 4°C, the resin was washed five times with NP-40 buffer. Proteins binding to the resin were eluted with Column buffer containing 10 mM maltose, and analysed by SDS–PAGE, followed by immunoblotting with anti-GST antibody and Coomassie brilliant blue (CBB) staining.

Electroporation and immunofluorescence microscopy

Electroporation was performed as described previously (Sasaki et al, 2001). Vero cells mock electroporated or electroporated with replicon RNA were cultured on slide glasses. For BFA treatment, the electroporated cells were cultured in medium containing 10 μg/ml of BFA for 4 h. At appropriate times after electroporation, the cells were fixed with PBS containing 4% paraformaldehyde for 45 min and then permeabilized with PBS containing 0.5% Triton X-100 for 15 min or PBS containing 20 μM digitonin for 5 min only for staining PI4P. The cells were incubated with appropriate primary antibodies and then with appropriate secondary antibodies diluted in PBS containing 3% bovine serum albumin or can get signal immunostain solution A (Toyobo). The slide glasses were mounted with Fluoromount Plus (Diagnostic BioSystems) and then viewed under a fluorescence microscope, BZ-8000 (Keyence).

This study was supported in part by Grant-in-Aid for Scientific Research (C), and Grant-in-Aid for Scientific Research on Priority Areas: Matrix of Infection Phenomena from the Ministry of Education, Culture, Sports, Science and Technology, and Grant-in-Aid from the Ministry of Health, Labor and Welfare, Japan.

Author contributions: JS designed the experiments, performed experiments shown in Figure 1C, 5A and C, and wrote the manuscript. KI performed most of the experiments, analysed the results, and wrote the manuscript. MA contributed with important reagents and discussed the data. KT coordinated the study and oversaw the research.