EXTENDED EXPERIMENTAL PROCEDURES
Cell and Virus Propagation
HeLa cells (ATCC, MD) were maintained in Dulbecco's modified Eagle's medium (DMEM) supplement with 10% fetal bovine serum (FBS), 25mM HEPES buffer, 2mM glutamine, 100units/ml of penicillin and 100 μg/ml of streptomycin. NNeo/3-5B(RG) cells were provided by Dr. Stanley Lemon (Univ. Texas-Galveston). This Huh7–derived cell line contains autonomously replicating, subgenomic, dicistronic, selectable HCV RNAs from an infectious molecular clone HCV-N of genotype 1b virus and expresses the HCV non-structural proteins NS3-NS5B.The replicon-bearing cell line was cultured in DMEM, supplemented with 2 mM L-glutamine, 5% heat-inactivated FBS, 100 U/ml penicillin, 100 μg/ml streptomycin and 300 μg/mL G418. Huh7 was maintained in the same media described above in the absence of G418. To generate CVB3 viral stock, confluent HeLa cells were infected by CVB3 for 24 hr. The infected cells underwent freeze-and-thaw cycle 3 times, supernatant was collected and stored at −80° C. Virus titer was determined by plaque assays.
Plasmids, Antibodies, and Chemicals
Arf1-GFP, Arf1-RFP, εCOP-GFP, GalT-YFP, Sar1T39N plasmids and anti-GBF1 antibodies were gifts of Jennifer Lippincott-Schwartz, NIH; Sac1 plasmid was gift of Peter Mayinger, OHSU;ERGIC53-GFP was gift of Theresa Ward, London School of Tropical Medicine; anti- CVB3_3A was gift of J.Lindsay Whitton, Scripps Research Institute; anti-GalT was gift of EG Berger, University of Zurich); anti-Arf1 was gift of Julie Donaldson, NIH; anti-HCV NS5A and HCV replicon J6/JFH (p7-Rluc2A)/delta E1 E2 were gifts of Charles Rice, The Rockefeller University; and PIK93 was gift of Kevin Shokat, UC Berkeley. Following reagents were commercially obtained: Anti-PI4KIIIβ and anti-myc (Cell Signaling, MA); anti-βCOP (Sigma, MO); anti-TGN46 (Abd Serotec, NC); anti-ERGIC53 (Sigma); anti-GFP (Millipore Corp., MA), anti- sec31 (BD Biosciences) and all secondary antibodies (Invitrogen Corp. CA; Jackson ImmunoResearch Labs Inc, PA). Brefeldin A, saponin (Sigma, MO), Live cell Renilla substrate Endu-Ren (Promega Corp. WI), Bovine serum albumin (BSA) (Fisher Scientific, PA), DMEM (Mediatech, VA), FBS (Atlanta biologicals, GA).
All imaging was performed on a Zeiss LSM510META confocal laser scanning confocal microscope (Carl Zeiss, USA) equipped with lasers emitting 458nm, 488nm, 514nm, 565nm and 633nm laser lines. For all live-cell imaging cells were grown in coverslip bottomed Lab-Tek chambers (ThermoFisher, NY), and selected plasmids were transfected using FuGENE6 (Roche Applied science, IN). After 16 hr, incubation medium were replaced with imaging medium (phenol red free complete media buffered with 25mM HEPES pH7.3). Cells were maintained on the microscope stage in an environmental chamber where temperature (37° C) and CO2 (5%) and humidity were controlled for the duration of experiment. For high-resolution light-level imaging only 40X and 63X oil immersion objectives with 1.4 numerical aperture were utilized with pinhole set at 1.2 Airy units. Time lapsed images were acquired every 5 min. For fluorescence quantification pinhole was opened up to collect fluorescence from the entire depth of fluorescent structure of interest. Fluorescence on selected regions of interest within images was quantified using either Zeiss LSM image analysis (Carl Zeiss, USA) or Image J (NIH, MD) software.
Immunofluorescence and Analysis
Cells were plated on glass coverslips, fixed with 4% formaldehyde PBS solution (10 mM sodium phosphate, 150 mM sodium chloride, pH 7.4) at room temperature. Coverslips were permeabilized with either 0.2% Triton or 0.2% saponin and sequentially incubated with primary and fluorophore-tagged secondary antibodies. To quantify the relative fluorescence of Arf1, GBF1, PI4KIIIβ, PI4KIIIα or βCOP in 3A-myc labeled structures, images were analyzed using LSM-FCS software (Carl Zeiss, USA). The Golgi and cytosol regions of interest were selected for both 3A and neighboring non-3A expressing cells, and mean fluorescence intensity of Arf1, GBF1, PI4KIIIβ, PI4KIIIα and βCOP in those regions were obtained. The relative fluorescence was determined by following formula: [(IGolgi-ICytosol) 3A cell/(IGolgi-ICytosol)non-3A expressing cell] *100, where IGolgi is the mean fluorescence intensity per pixel at the Golgi and ICytosol is the mean fluorescence intensity in the cytosol. PI4P staining on 3-5B cells was done as described previously (Jovic et al., 2009). Cells were fixed with 3.7% formaldehyde PBS solution for 10 min and incubated with anti-PI4P (Echelon Biosciences, UT) and HCV NS5A antibodies in 0.2% Saponin, 0.5%BSA PBS solution for 1 hr followed by fluorophore –tagged secondary antibodies for detection.
Fluorescence In Situ Hybridization
Codetection of protein (Arf1, GBF1) and RNA (CVB3 plus-strand RNA) in cells was adapted from http://www.singerlab.org/protocols
. Alexa555 labeled CVB3 plus strand RNA specific ~500nt length probes were made using FISH Tag RNA kit (Invitrogen, CA). HeLa cells were infected with CVB3 for 4 hr, fixed with 4% formaldehyde followed by overnight permeabilization with 70% ethanol at 4°C. Cells were re-hydrated in 2XSSC buffer (300 mM sodium chloride, 30 mM sodium citrate, pH 7.0), 50% formamide and hybridized with Alexa555 labeled CVB3 plus strand RNA specific probe at 37°C overnight in hybridization buffer (10%dextran sulfate, 2 mM vanadyl-ribonucleoside complex, 0.02% RNase-free BSA, 40 μg E. coli
tRNA, 2×SSC, 50%formamide, 30ng of probe). The excess probes were washed, and Arf1 or GBF1 was subsequently immunostained with primary and Alexa488-secondary antibodies in PBS/BSA in the absence of detergents.
Arf1 siRNA was part of the human on-target plus membrane trafficking and remodeling siRNA library (Dharmacon, CO); GBF1, PI4KIIIβ and nontargeting siRNA were purchased separately (QIAGEN,CA). HeLa cells were seeded at 10000/well in a 96-well plate (for the replicon assay) or in 200000/well in a 12-well plate for Western blot analysis to verify knockdown efficiency of proteins one day before siRNA transfection. 50 nM of siRNA were transfected via Dharmafect1 transfection reagent (Dharmacon, CO) and incubated for 48 hr.
Cell Viability Quantification
Optimal plasmid expression times, siRNA concentrations, siRNA incubation times and PIK93 concentration and incubation times that maximize cell viability were assessed by both quantifying cell number and by CellTiter-Glo cell viability assays (Promega Corp, WI). Plasmid concentration range tested was 0.1 μg/μl-1 μg/μl; and siRNA concentration range tested was 25 nM-100 nM.
The CVB3 pRib-Rluc, PV pXpA-RenR, and J6/JFH (p7-Rluc2A) plasmids with Renilla luciferase gene as reporters in place of structural genes were utilized to measure viral RNA replication. Plasmids were linearized and in vitro-transcribed and RNA purified with MEGAscript system and MEGAclear Kit respectively (Applied BioSystems/Ambion, TX). RNA was quantified by spectrophotometry and quality of runoff transcripts were estimated by agarose gels. Replicon RNAs were transfected into HeLa cells grown in 96-well white plates with flat bottom (Corning Corp., NY) at 15 ng/well with Trans-it mRNA transfection reagent (Mirus Bio LLC, WI). Incubation media contained 80 μM live-cell Renilla substrate Endu-Ren (Promega Corp., WI). Renilla light signal was recorded with Biotek HT multi-well plate reader (Biotek, VT) at 15 min intervals up to 16 hr at 37°C.
PI4P Lipid Extraction and Quantification
Cells were harvested, and PI4P lipids were extracted in cold 0.5M of Trichloroacetic acid (TCA) for 5 min. Cell pellets were washed twice with 3 ml of 5% TCA/1 mM EDTA solution and pellets were suspended in 3 ml of methanol:chloroform (2:1) solution, incubated at room temperature for ten minutes and subjected to centrifugation. To obtain phosphatidylinositol lipids, the 2.25 ml of methanol: chloroform: 12N hydrochloric acid solution (80:40:1) was added to pellets followed by fifteen minutes incubation at room temperature. Supernatant was mixed with 0.75 ml of chloroform before adding 1.35 ml of 0.1N hydrochloric acid to generate organic and aqueous phases. The organic phase containing the phosphatidylinositide was dried in a vacuum dryer, and subjected to PI4P quantification by protein lipid overlay assay (Echelon Biosciences, UT). PI4P samples from different post-infection time points were spotted on nitrocellulose membrane strips and blocked with 3% BSA 0.1% tween-20 PBS solution for 1 hr to remove non-specific binding. PI4P lipids were detected by PI4P detectors derived from FAPP1 proteins (Dowler et al., 2002
) followed by secondary and tertiary antibody incubation. The signal was visualized by SuperSignal chemiluminescent substrate (Pierce, IL). The concentration of PI4P was determined by correlating the intensity of samples to a standard curve generated from known concentrations of PI4P.
RNA Polymerase Lipid-Binding Assay
Recombinant PV polymerase (3Dpol
) in pET26Ub-3D was purified as described (Gohara et al., 1999
). Lipid dot-blot strips (PIP micro Strips and membrane lipid Strips) were purchased (Echelon Biosciences, UT). The nitrocellulose lipid blots were incubated in blocking buffer [3% (w/v) fatty acid-free BSA fraction V (EMD, NJ) in TBST-50 (50 mM Tris, 150 mM NaCl, 0.1% Tween20 at pH 7.4)] for 1 hr at RT and then incubated in the same buffer with purified PV 3Dpol
overnight at 4°C. The lipid blots were then washed in TBST-50 (5 times, 10 min each). To detect lipid protein interactions, strips were then incubated with an anti-3Dpol
antibody (1:5000 in blocking buffer) for 1 hr at RT. Blots were washed as before and incubated with anti-rabbit horseradish peroxidase (1:10000 in blocking buffer) (Pierce, IL) for 1 hr at RT. 3Dpol
bound to the lipids immobilized on the membrane were visualized by incubating with chemiluminescent substrate (Pierce, IL). To ensure lipid-binding specificity, several different concentrations of a protein in range from 1 to 4 μg/ml were tested. The lowest concentration giving specific binding and a detectable signal on a western blot was chosen for the lipid dot-blot experiments. Control experiments included parallel blots using no 3Dpol
protein and no lipid spotted membranes.
Infected (CVB3; 4 hr) and noninfected HeLa cells were scraped and suspended in hypotonic buffer (50 mM KCl, 25 mM HEPES, 1 mM DTT, 1 mM EDTA). Suspended sells were broken up using cell homogenizer (Isobiotec, Germany) to preserve the replication organelle membranes. The nucleus and unbroken cells were removed by centrifugation, and the supernatant cell lysates were incubated with 1ug of monoclonal PI4KIIIβ antibody (Millipore Corp., MA) at 4° C overnight. Protein A/G magnetic beads (New England Biolabs, MA) were added and incubated at 4° C for additional 2 hr. The pull down fractions were resolved by SDS-PAGE and transferred to nitrocellulose membranes which are then blocked with 5% Nonfat Milk (3A, 3AB, 3C, 3CD blot) or 5%FCS 1%BSA (PI4KIIIβ blot) in TBST solution (10 mM Tris pH = 7.4, 150 mM NaCl, 0.1%Tween20), incubated anti-3A, anti-3Dpol or anti-PI4KIIIβ antibody and washed 5 times with TBST before incubating with horseradish peroxidase-conjugated secondary antibodies. Bound antibody complexes were detected by chemiluminescence.
Pearson Correlation Coefficient Calculations We statistically assessed the degree of colocalization between two components using a classical Pearson correlation coefficient for the image intensity in the red and green channels using the following formulae (Bolte and Cordelieres 2006):
Bolte, S., and Cordelières, F.P. (2006). A guided tour into subcellular colocalization analysis in light microscopy. J. Microsc. 224, 213–232.
Dowler, S., Kular, G., and Alessi, D.R. (2002). Protein lipid overlay assay. Sci. STKE 129, PL6.
Gohara, D.W., Ha, C.S., Kumar, S., Ghosh, B., Arnold, J.J., Wisniewski, T.J., and Cameron, C.E. (1999). Production of “authentic” poliovirus RNA-dependent RNA polymerase (3D(pol)) by ubiquitin-protease-mediated cleavage in Escherichia coli. Protein Expr. Purif. 17, 128–138.
Jović, M., Kieken, F., Naslavsky, N., Sorgen, P.L., and Caplan, S. (2009). Eps15 homology domain1-associated tubules contain phosphatidylinositol-4-phosphate and phosphatidylinositol- (4,5)-bisphosphate and are required for efficient recycling. Mol. Biol. Cell 20, 2731–2743.
Figure S1. Arf1 and Enteroviral Replication Protein Colocalization, Related to
(A) Native Arf1 distribution in uninfected and CVB3-infected cells mimics that of Arf1-GFP.
(B) Confocal image (optical slice 2 μm) of a single HeLa cell expressing Arf1-CFP immunostained with antibodies to viral 2C protein and CFP, at 4 hr infection with PV.
Figure S2. siRNA-Mediated Reduction of Host Factors and Impact on Enterovirus Replication, Related to and
(A) Western blots of HeLa cells treated with nontargeting (lane 1) or Arf1, GBF1, PI4KIIIβ siRNAs (lane 2); equal amounts of protein (confirmed by actin blotting) for nontarget/Arf1, nontarget/GBF1, and nontarget/PI4KIIIβ conditions were loaded on SDS-PAGE gels. Bar graph shows quantification of representative blot, where the extent of siRNA depletion has been normalized to nontargeting siRNA condition.
(B and C) CVB3 replicon assays in HeLa cells treated with nontargeting siRNA and Arf1 siRNA (B) or GBF1 siRNA (C). Eight replicate samples were assayed for nontargeting, Arf1, and GBF1 siRNA treatment conditions.
Figure S3. Host Cell Secretory Machinery Distribution and Dynamics in Uninfected and Enterovius-Infected Cells, Related to
(A–G) Colocalization in HeLa cells of Arf1-GFP and secretory pathway components in uninfected cells. Antibodies to GFP were utilized to localize Arf1.
(H and I) Disruption of secretory trafficking in CVB3 infected cells. HeLa cells expressing ts045VSVG-GFP were infected with CVB3 or mock for 2 hr prior to 32°C switch. Image showing Golgi accumulation of ts045VSVG-GFP 30 min after switch from non-permissive 40°C to permissive 32°C in mock-infected cells (H). Note the lack of ts045VSVG-GFP accumulation at the Golgi after 30 min in CVB3-infected cells (I).
(J) Transferrin Receptor is not localized to replication organelles. HeLa cells at 0 hr and 4 hr post CVB3 infection were fixed and coimmunostained with antibodies to native GBF1 to label the replication organelles and Transferrin Receptor. All images unless indicated are confocal sections 1 mm optical slice thickness. Scale bar, 10 μm.
Figure S4. Pearson Correlation Coefficients of Arf1 and Host Secretory Components Pre- and Post-Enterovirus Infection, Related to and
Pearson Correlation Coefficients were calculated as a measure of the degree of colocalization between Arf1-labeled membranes and 3COP, GalT, GGA, TGN46, GM130, ERGIC53, PI4KIIIβ proteins. The coefficients were calculated at 0 hr post-infection when Arf1 is localized to the Golgi apparatus and at 4 hr post-infection when Arf1 is localized at the replication organelles. In general small Pearson coefficients (<0.5) indicate low or no colocalization.
Figure S5. High-Level Transient Ectopic Enteroviral 3A Expression, Related to
Secretory machinery distribution in cells expressing high levels of 3A-myc. All cells were transfected with 3A-myc and then coimmunostained with antibodies against –myc and (A) bCOP, (B) GalT, (C) sec31, (D) PI4KIIIβ. Scale bar, 5 μm.
Figure S6. PIK93 Impact on Enterovirus RNA Replication and Cell Viability, Related to
(A) Impact on PV replication of different concentrations of PIK93 (added at time 0 hr).
(B) Impact on replication of PIK93 added at 1 hr and 3 hr post replicon transfection.
(C) PIK93 effects on cell viability. Cells were treated with the indicated PIK93 concentrations for 24 hr.
Figure S7. Impact of PI4KIIIα and PI4KIIIβ Reduction on ER Exit Sites in HeLa Cells, Related to
HeLa cells were treated with nontargeting, PI4KIIIβ, or PI4KIIIα siRNA's for 72 hr; fixed and immunostained with antibodies to sec31 to label ER exit sites. Note the decrease in the number of ER exit sites in PI4KIIIα siRNA-treated cells whereas it is unaffected in PI4KIIIβ siRNA-treated cells.