Fig 1.
PI4KA is essential for cardiovirus replication.
(A-D) Effects of PI4KA knockdown on EMCV infection. HeLa R19 cells were reverse transfected with siRNA against PI4K2A, PI4K2B, PI4KA, PI4KB or scrambled siRNA as a control (A) or siRNA against PI4KA, PI4KB or scrambled siRNA (B-D). At 48 h post transfection (p.t.), cells were infected with EMCV (A-C) or RLuc-EMCV (D) at an MOI of 0.1 or transfected with full-length infectious EMCV in vitro-transcribed RNA (C). After 8 h, cells were freeze-thawed to release intracellular virus particles and the total virus titers were determined by endpoint titration (A-C). Alternatively, cells were lysed and Renilla luciferase activity was determined as a measure of viral RNA replication (D). In parallel, a cell viability assay was performed to evaluate the cytotoxicity of the siRNA treatment (E). (F) Western blot analysis showing efficient knockdown of PI4KA and PI4KB. Actin was used as loading control. (G-I) AL-9 treatment inhibits cardiovirus replication. HeLa R19 cells were infected with virus at an MOI of 0.1 (G and H) or 1 (I), followed by AL-9 treatment for 8 h, after which cells were lysed and virus replication was measured by endpoint titration (G and I) or by determining the Renilla luciferase activity (H). Cytotoxicity of AL-9 was determined in a cell viability assay run in parallel (J). Bars represent mean values of triplicates ± standard error of the means (SEM). Means were statistically compared using unpaired t tests. *P < 0.05, **P<0.01; ***P<0.001.
Fig 2.
PI4KA is recruited to EMCV replication sites.
HeLa R19 cells were transfected with plasmids encoding GFP-PI4KA (A and B) or GFP-PI4KB (C). The next day, cells were mock-infected or infected with EMCV at an MOI of 250. At 6 h post infection (p.i.), cells were fixed and stained with antibodies against dsRNA as a marker of infection (A) or 3AB as a RO marker (B and C). Nuclei were stained with DAPI (blue). The crop panels at the bottom depict enlargements of boxed areas. Scale bars represent 10 μm.
Fig 3.
Localization of early secretory membranes in EMCV-infected cells.
HeLa R19 cells were mock-infected or infected with EMCV-2C-HA (A-C) or EMCV (D-F) at MOI 10. 6 h later, cells were fixed and co-stained with antibodies against viral 3AB or HA as RO markers and antibodies against endogenous GM130 (cis-Golgi marker; A), TGN46 (trans-Golgi marker, B), ERGIC53 (ERGIC marker, C), Sec13 (ER exit site marker, D) or calreticulin (ER marker, E and F). Nuclei were stained with DAPI (blue). The crop panels at the bottom depict enlargements of boxed areas. Scale bars represent 10 μm.
Fig 4.
(A) Only EMCV 3A and 3AB can interact with PI4KA. Huh7-Lunet/T7 cells were transfected with empty vector or plasmids encoding myc-tagged EMCV nonstructural proteins and co-transfected with pTM-HA-PI4KA where indicated. 7 h later, cells were radiolabeled for 16 h with [35S] methionine/cysteine-containing medium, lysed and subjected to immunoprecipitation using anti-myc antibodies. Samples were analyzed by SDS-PAGE and visualized by autoradiography. An enlargement of the boxed area at the top shows co-precipitation of HA-PI4KA with 3A- and 3AB-myc, but not with 2C-myc. (B) Huh7-Lunet/T7 cells were transfected with plasmids encoding HA-PI4KA and either myc-tagged EMCV 3A or CVB3 3A proteins. One day later, cells were lysed and subjected to immunoprecipitation using two different anti-myc antibodies. Captured complexes were separated by SDS-PAGE and subjected to western blot analysis using specific antibodies against the myc- or HA-tags. (C) Specific recruitment of PI4KA to 3A-positive membranes. Huh7-Lunet/T7 cells were cotransfected with the HA-PI4KA expression construct and either empty vector or plasmids encoding myc-tagged EMCV 2B, 3A or 3AB. The next day, cells were fixed and co-stained with antibodies against myc and HA to detect the overexpressed proteins. The crop panels at the bottom depict an enlargement of the boxed areas. Scale bars represent 10 μm.
Fig 5.
PI4P homeostasis is affected upon EMCV infection.
(A) EMCV alters the distribution of intracellular PI4P lipids. Huh7-Lunet/T7 cells were mock-infected or infected with GFP-EMCV at an MOI of 10. At 6 h p.i., cells were fixed and stained with antibodies against PI4P using specific protocols for detection of plasma membrane and intracellular PI4P pools, as described previously [50]. Nuclei were stained with DAPI (blue). (B) Quantification of PI4P levels by immunofluorescence analysis. Huh7-Lunet/T7 cells were mock-infected or infected with EMCV at an MOI of 10. At 6 h p.i., cells were fixed and stained for intracellular PI4P and viral dsRNA. The intensities of fluorescent PI4P signals from whole-cell z-stacks were quantified using ImageJ. Shown are mean values ± SEM of 30 cells per condition. Means of mock and infected cells were statistically analyzed using the Mann–Whitney test. ***P<0.001. (C) EMCV ROs contain PI4P. Huh7-Lunet/T7 cells were mock-infected or infected with EMCV-2C-HA at an MOI of 10. At 5.5 h p.i., cells were treated with DMSO or 1 μM BF738735 (PI4KB inhibitor) for 30 min, then fixed and stained for intracellular PI4P and HA using specific antibodies. Nuclei were stained with DAPI (blue). The crop panels at the bottom depict an enlargement of the boxed areas. Scale bars represent 10 μm.
Fig 6.
OSBP is a PI4KA effector important for EMCV replication.
(A) OSBP knockdown reduces EMCV replication. HeLa R19 cells were reverse transfected with siRNA against OSBP or scrambled siRNA as a control. At 48 h p.t., cells were infected with EMCV at MOI 0.1. After 8 h, cells were freeze-thawed to release intracellular virus particles and total virus titers were determined by endpoint titration. OSBP knockdown efficiency was verified by western blot analysis. Actin was used as loading control. (B) Antiviral effects of the OSBP inhibitor OSW-1 against EMCV. HeLa R19 cells were infected with RLuc-EMCV at MOI 0.1, followed by treatment with OSW-1 at the indicated concentrations. Renilla luciferase levels at 8 h p.i were determined as a measure of virus genome replication. Cell viability was measured in parallel. (C) HeLa R19 cells were infected with RLuc-EMCV at an MOI of 1 and treated at the indicated time points after infection with DMSO, OSW-1 or 25-HC at the specified concentrations. At 7 h p.i., cells were lysed and Renilla luciferase activity was measured. (D) OSBP localization at EMCV ROs is PI4P-dependent. HeLa R19 cells were infected with EMCV at MOI 10. At 5.5 h p.i., cells were treated for 30 min with DMSO or 10 μM AL-9 to inhibit PI4KA activity, then fixed and subjected to immunofluorescence analysis using antibodies against OSBP and viral 3AB. Colocalization of OSBP with 3AB was determined by calculating the Pearson’s correlation coefficients for at least 15 cells for each condition. (E) Specific recruitment of OSBP to 3A-positive membranes. Huh7-Lunet/T7 cells were transfected with empty vector or plasmids encoding myc-tagged EMCV 2B, 3A or 3AB. One day later, cells were fixed and co-stained with antibodies against myc and endogenous OSBP. The crop panels at the bottom depict enlargements of boxed areas. Scale bars represent 10 μm. Shown are mean values ± SEM. Means were statistically compared using either unpaired t tests (A and B) or the Mann–Whitney test (D). *P < 0.05, ***P<0.001.
Fig 7.
Cholesterol delivery to EMCV ROs depends on PI4KA and OSBP activities.
(A) HeLa R19 cells were infected with EMCV at MOI 10. At 4 h p.i., cells were treated with DMSO, 10 μM AL-9 or 3nM OSW-1. After 2 h, cells were fixed, followed by staining with antibodies against 3AB or filipin for cholesterol detection. The merged panels also depict the outline of the cell (white line). Scale bars represent 10 μm. Pearson’s correlation coefficients of colocalization between filipin and 3AB were calculated for at least 15 cells for each condition. Shown are mean values ± SEM. Means were statistically compared using the Mann–Whitney test. ***P<0.001.
Fig 8.
Evolution of the recruitment of lipid-modulating pathways by picorna- and flaviviruses.
Our current understanding of the major elements of lipid-modulating pathways targeted by proteins of picorna- and flaviviruses in the context of the virus phylogeny is presented. Only viruses characterized so far with respect to this function are depicted. The tree branches are of arbitrary scale, while the branching of picornaviruses reflects the topology of maximum-likelihood RdRp-based tree according to Gorbalenya & Lauber, 2010 [80], and the entire tree is rooted according to Gibrat et al., 2013 [81]. *In addition to NS5A, NS5B has also been suggested to be involved in the interaction with PI4KA [24,46]. Abbreviation of virus names are as follows: DENV, dengue virus; HCV, hepatitis C virus; EMCV, encephalomyocarditis virus; ERAV, equine rhinitis A virus; CV, coxsackievirus; PV, poliovirus; RV, rhinovirus; AiV, Aichivirus; HAV, hepatitis A virus.