Association of Alix with Late Endosomal Lysobisphosphatidic Acid Is Important for Dengue Virus Infection in Human Endothelial Cells Sa-nga Pattanakitsakul,† Jesdaporn Poungsawai,‡ Rattiyaporn Kanlaya,§ Supachok Sinchaikul,| Shui-Tein Chen,|,⊥ and Visith Thongboonkerd*,§,@ Medical Molecular Biology Unit, Office for Research and Development, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand, Department of Immunology and Immunology Graduate Program, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand, Medical Proteomics Unit, Office for Research and Development, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand, Institute of Biological Chemistry and Genomic Research Center, Academia Sinica, Taipei, Taiwan, Institute of Biochemical Sciences, College of Life Science, National Taiwan University, Taipei, Taiwan, and Center for Research in Complex Systems Sciences, Mahidol University, Bangkok, Thailand Received April 21, 2010
The most severe form of dengue virus (DENV) infection is dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), which is accompanied by increased vascular permeability indicating that endothelial cells are the targets of DENV infection. However, molecular mechanisms underlying DENV replication in endothelial cells remained poorly understood. We therefore examined changes in subcellular proteomes of different cellular compartments (including cytosolic, membrane/ organelle, nucleus, and cytoskeleton) of human endothelial (EA.hy926) cells upon DENV2 infection using a 2-DE-based proteomics approach followed by Q-TOF MS and MS/MS. A total of 35 altered proteins were identified in these subcellular locales, including an increase in the level of Alix (apoptosis-linked gene-2-interacting protein X) in the cytosolic fraction of DENV2-infected cells compared to mock control cells. Double immunofluorescence staining revealed colocalization of Alix with late endosomal lysobisphosphatidic acid (LBPA). This complex has been proposed to be involved in the export of DENV proteins from late endosomes to the cytoplasm. Subsequent functional study revealed that pretreatment with an anti-LBPA antibody prior to DENV challenge significantly reduced the level of viral envelope protein synthesis and DENV replication. Our data indicate that Alix plays a pivotal role in the early phase of DENV replication, particularly when it arrives at the late endosome stage. Blocking this step may lead to a novel therapeutic approach to reducing the level of DENV replication in vivo. Keywords: Alix • dengue • endothelial cells • host responses • LBPA • proteome • proteomics
Introduction DENV infection is an epidemic disease in tropical and subtropical regions with an incidence of approximately 100 million cases annually.1,2 Among these, ∼500000 cases exhibit severe DENV infection and are diagnosed as DHF or DSS.1,2 Clinical manifestations of DHF/DSS included thrombocytopenia, vascular leakage, and hemorrhagic diatheses. * To whom correspondence should be addressed: Office for Research and Development, Siriraj Hospital, Mahidol University, 12th Floor, Adulyadej Vikrom Building, 2 Prannok Rd., Bangkoknoi, Bangkok 10700, Thailand. Phone and fax: +66-2-4184793. E-mail:
[email protected] (or)
[email protected]. † Medical Molecular Biology Unit, Office for Research and Development, Faculty of Medicine, Siriraj Hospital, Mahidol University. ‡ Department of Immunology and Immunology Graduate Program, Faculty of Medicine, Siriraj Hospital, Mahidol University. § Medical Proteomics Unit, Office for Research and Development, Faculty of Medicine, Siriraj Hospital, Mahidol University. | Academia Sinica. ⊥ National Taiwan University. @ Center for Research in Complex Systems Sciences, Mahidol University.
4640 Journal of Proteome Research 2010, 9, 4640–4648 Published on Web 07/29/2010
DENV infection is transmitted to humans through the biting of infected mosquitoes. Upon infection, DENV adheres to the target cells using molecules that act as receptors on surfaces of the cells.3–5 Thereafter, the virus is endocytosed into the cells, and the virion enriched with envelope glycoproteins is then fused with the endosomal membrane. The maturation process from early to late endosomes occurs prior to uncoating of nucleocapsid for virus replication and translation. However, mechanisms underlying intracellular processes in host cells that lead to virus replication remain poorly understood. In this study, we therefore identified changes in subcellular proteomes in different cellular compartments (including cytosolic, membrane/organelle, nucleus, and cytoskeleton) of human endothelial (EA.hy926) cells upon DENV2 infection using a 2-DE gel-based proteomics approach followed by quadrupole time-of-flight (Q-TOF) mass spectrometry (MS) and tandem MS (MS/MS). These data offer many opportunities for subsequent highly focused studies such as in this work; a 10.1021/pr100357f
2010 American Chemical Society
Alix and LBPA in Dengue Virus Infection functional study was performed to highlight the role of Alix protein, which was upregulated in DENV2-infected endothelial cells, and late endosomal LBPA, which was its partner, in DENV replication.
Materials and Methods Cell Culture and Propagation of DENV. Human endothelial cells (EA.hy926) were cultured in Dulbecco’s minimum essential medium/F-12 (DMEM/F-12) (Gibco, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco), 100 units/mL penicillin G, and 100 mg/mL streptomycin (Sigma, St. Louis, MO). Cells were maintained at 37 °C in a humidified incubator with a 5% CO2 atmosphere. DENV serotype 2 (DENV2), strain 16681, was propagated in C6/ 36 cells derived from Aedes albopictus to produce a viral stock according to the protocols described previously.6 The culture supernatant was collected by centrifugation at 1500 rpm for 5 min, and the virus titer was determined by a focus forming assay in pig fibroblast cells (PS clone D) according to the method described by Avirutnan et al.7 The DENV2 stock was then kept at -70 °C until it was used. All the experiments dealing with DENV2 were conducted in the Biosafety Level 2 (BSL-2) laboratory. Infection of EA.hy926 Cells with DENV2. After becoming confluent, EA.hy926 cells were trypsinized with a 0.1% trypsin/ 0.25 mM EDTA mixture in PBS and resuspended in the growth medium. Cells were seeded at the appropriate density into each flask and incubated for 24 h prior to addition of DENV2 from the stock with a multiplicity of infection (MOI) of 10. After incubation at 37 °C for 2 h, the supernatant was removed and fresh complete DMEM medium supplemented with 2% FBS, 100 units/mL penicillin, 100 mg/mL streptomycin, and 2 mM L-glutamine was added to each flask. As described in our previous study, the optimal condition for the study of host response to DENV was the infection with an MOI of 10 and a 24 h postinfection period at 37 °C in a 5% CO2 incubator.8 Mock EA.hy926 cells cultured in parallel with DENV2-infected cells but without DENV infection served as the controls. To confirm the effective DENV infection and determination of infectivity, mock control and DENV2-infected EA.hy926 cells were fixed with 2% formaldehyde in PBS at room temperature (RT) for 1 h, washed with PBS, and permeabilized with 0.2% Triton X-100 in PBS at RT for 10 min. The permeabilized cells were incubated with a mouse monoclonal antibody against DENV2 envelope protein (clone 3H5) at RT for 1 h, washed twice with 0.1% Triton X-100 in PBS, and incubated with rabbit anti-mouse IgG conjugated with FITC (Dako, Glostrup, Denmark) at RT for 30 min in the dark. Thereafter, the cells were washed once with PBS before analysis by flow cytometry using FACScan equipped with CellQuest (BD Biosciences, Palo Alto, CA). The percentage of cell infection was calculated using the formula % infection ) [(number of DENV envelope-positive cells)/(total number of all cells)] × 100%. Analysis of Cell Death by Flow Cytometry and Propidium Iodide Labeling. After infection with an MOI of 10 for 24 h, the mock control and DENV2-infected EA.hy926 cells were collected using low-speed centrifugation at 1500 rpm for 5 min and washed twice with ice-cold DMEM. After the cells had been washed, 1 ng of propidium iodide (BD Biosciences) was added and the mixture incubated with the cells for 5 min in the dark before analysis by flow cytometry using FACScan equipped with CellQuest (BD Biosciences). The cells fixed with 2% formaldehyde and permeabilized with 0.2% Triton X-100 were run in
research articles parallel and served as the positive control, while the untreated EA.hy926 cells served as the negative control. The amount of cell death was calculated using the formula % cell death ) [(number of propidium iodide-positive cells)/(total number of all cells)] × 100%. Subcellular Fractionation. A monolayer of EA.hy926 cells was sequentially extracted with the ProteoExtract Subcellular Proteome Extraction Kit (Mini sPEK) (Calbiochem, EMD Biosciences, Darmstadt, Germany) according to the manufacturer’s instructions. The strategy for subcellular fractionation was based on differential properties of different buffers for solubilizing and fractionating cellular compartments in a stepwise manner into four fractions, including cytosolic, membrane/ organelle, nucleus, and cytoskeleton fractions. Sequential extraction was performed using four different buffers, including buffers I, II, II, and IV, for solubilizing proteins from individual subcellular fractions. Compositions of these buffers are provided as Supporting Information. Proteins derived from these four fractions of mock control and DENV2-infected cells were then precipitated with the ProteoExtract Protein Precipitation Kit (Calbiochem, EMD Biosciences) to concentrate proteins and to remove interfering substances. Protein concentrations of individual samples were finally determined with a Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA) based on the Bradford method. Confirmation of the Purity of Proteins Isolated via Subcellular Fractionation. In total, 15 µg of proteins derived from each of four fractions was resolved via 12% SDS-PAGE in an SE260 mini-vertical Electrophoresis Unit (GE Healthcare, Uppsala, Sweden) at 150 V for 2 h and then transferred onto nitrocellulose membranes. Nonspecific binding was blocked with 5% skim milk in PBS for 1 h. Thereafter, the membranes were incubated at 4 °C overnight with a rabbit polyclonal anticalpain (marker for cytosol), mouse monoclonal anti-pancadherin (marker for membrane/organelle), mouse monoclonal anti-c-jun (marker for nucleus), or mouse monoclonal antipan-cytokeratin (marker for cytoskeleton) antibody (all at dilution of 1:200 in 5% skim milk and PBS) (all were purchased from Santa Cruz Biotechnology, Santa Cruz, CA). After being washed with PBS, the membranes were further incubated with the corresponding secondary antibody conjugated with horseradish peroxidase (1:1000 in 5% skim milk and PBS) at RT for 1 h. Protein bands were then visualized with SuperSignal West Pico chemiluminescence substrate (Pierce Biotechnology, Inc., Rockford, IL) and autoradiography. Two-Dimensional PAGE. A set of five gels derived from each of four subcellular fractions of both mock control and DENV2infected cells (total of 40 gels derived from individual samples) were analyzed in this study. An equal amount of 100 µg of proteins extracted from each sample was mixed with a buffer containing 7 M urea, 2 M thiourea, 20 mg/mL 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 120 mM dithiothreitol (DTT), 2% ampholytes (pH 3-10), 40 mM Tris-HCl, and a trace amount of bromophenol blue, yielding a total volume of 150 µL per strip. The protein mixture was then rehydrated on an immobilized pH gradient (IPG) strip, nonlinear pH 3-10 (GE Healthcare) at RT for 16 h. Subsequently, the first dimensional separation or isoelectric focusing (IEF) was performed using an Ettan IPGphor III IEF System (GE Healthcare) with a step-and-hold mode until a total of 9083 Vh was achieved. The IPG strip was then equilibrated with equilibration buffer I (6 M urea, 130 mM DTT, 112 mM Tris base, 4% SDS, 30% glycerol, and 0.002% bromophenol blue) Journal of Proteome Research • Vol. 9, No. 9, 2010 4641
research articles for 15 min, followed by another equilibration step in buffer II (6 M urea, 135 mM iodoacetamide, 112 mM Tris base, 4% SDS, 30% glycerol, and 0.002% bromophenol blue) for 15 min. Thereafter, the strip was placed onto a 12% polyacrylamide slab gel (8 cm × 9.5 cm), and the second dimensional separation was performed in an SE260 mini-Vertical Electrophoresis Unit (GE Healthcare) at 150 V for approximately 2 h. Separated proteins were then visualized with Deep Purple fluorescence dye (GE Healthcare), and the two-dimensional gel image was obtained with a Typhoon 9200 laser scanner (GE Healthcare). Matching and Quantitative Analysis of Protein Spots. Matching and analysis of protein spots were performed using Image Master 2D Platinum (GE Healthcare). Parameters used for spot detection were as follows: (i) minimal area, 10 pixels; (ii) smooth factor, 2.0; and (iii) saliency, 2.0. A reference gel was created from an artificial gel combining all of the spots presenting in different gels into one image. The reference gel was used for determination of both the existence of and the difference in protein expression among gels. Intensity volumes of individual spots were obtained and subjected to statistical analysis. Differentially expressed protein spots were subjected to in-gel tryptic digestion and identification by mass spectrometry. Statistical Analysis. An unpaired Student’s t test was performed to compare intensity volumes of corresponding spots between mock control and DENV2-infected groups, using SPSS version 13.0 (SPSS, Chicago, IL). The criteria for defining spots with significant differences (either increase or decrease) included (i) p values of 10 counts ( include/exclude list) were selected for CID MS/MS using argon as the collision gas and a mass-dependent (5 V rolling collision energy until the end of the probe pattern was reached. The MS and MS/MS data were extracted and output as the searchable .txt and .pkl files, respectively, for independent searches using the MASCOT search engine (http://www.matrixscience.com), assuming that peptides were monoisotopic. Fixed modification was carbamidomethylation at cysteine residues, whereas variable modification was oxidation at methionine residues. Only one missed trypsin cleavage was allowed, and peptide mass tolerances of 100 and 50 ppm were allowed for peptide mass fingerprinting and MS/MS ion searching, respectively. Confirmation of Mass Spectrometric Data by Western Blot Analysis. The identity and upregulation of programmed cell death 6 interacting protein or apoptosis-linked gene-2interacting protein X (Alix) were confirmed by Western blot analysis. In total, 15 µg of proteins derived from the cytosolic fraction of mock control and DENV2-infected cells was resolved via 12% SDS-PAGE (as described above) and transferred onto a nitrocellulose membrane. Western blot procedures were conducted as mentioned above but with mouse monoclonal anti-Alix (Santa Cruz Biotechnology) as a primary antibody (1: 1000 in 5% skim milk and PBS). After the Alix band was detected, the membrane was treated with 0.2 mM NaOH for 5 min at RT. After being stripped and washed, the same membrane was reprobed with mouse monoclonal anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (Santa Cruz Biotechnology) (1:1000 in 5% skim milk and PBS) to confirm equal loading. Double Immunofluorescence Staining for Alix and LBPA. EA.hy926 cells were grown on a glass coverslip. After a 24 h postinfection with DENV2 as mentioned above, the cells were washed with PBS, fixed with 3.7% formaldehyde in PBS for 10 min, and permeabilized with 1% Triton X-100 in PBS for 10 min. After the samples had been washed with PBS three times, nonspecific binding was blocked with 5% BSA in PBS at RT for 30 min. The cells were then incubated overnight at 4 °C with rabbit polyclonal anti-Alix (Santa Cruz Biotechnology) and mouse monoclonal anti-LBPA antibody (Echelon Biosciences Inc., Salt Lake City, UT) (both at a dilution of 1:200 in 1% BSA and PBS). After three additional washing steps with PBS, the cells were incubated with goat anti-rabbit IgG conjugated with Cy3 and goat anti-mouse IgG conjugated with Alexa 488 (Invitrogen, Paisley, U.K.) (both at a dilution of 1:10000 in 1% BSA and PBS) at RT for 1 h in the dark. Colocalization of Alix and LBPA was then examined using a laser scanning confocal microscope (LSM 510 Meta, Carl Zeiss, Jena, Germany). Pretreatment of EA.hy926 Cells with an Anti-LBPA Antibody. Prior to DENV2 infection, EA.hy926 cells were preincubated with 50 µg/mL monoclonal anti-LBPA (lysobisphosphatidic acid) (Echelon Biosciences Inc.) at 37 °C for 24 h. This antibody can be internalized into late endosomes and accumulated with LBPA by fluid phase endocytosis.9 Thereafter, the cells with or without anti-LBPA pretreatment were infected with DENV2 at an MOI of 10 for 2 h. Thereafter, the culture supernatant was replaced with fresh medium (DMEM/F-12 containing 5% FBS), and the cells were further incubated at 37 °C with 5% CO2 for 24 h. The cells were then collected and processed for immunofluorescence staining to confirm the internalization of anti-LBPA into the cells (at late endosomes) using goat anti-mouse IgG conjugated with Cy3 (Jackson
Alix and LBPA in Dengue Virus Infection
research articles
Figure 1. Western blot analysis of markers for cytosol (A, calpain), membrane/organelle (B, pan-cadherin), nucleus (C, c-jun), and cytoskeleton (D, pan-cytokeratin) to confirm the purity of subcellular fractionation. WC, F1, F2, F3, and F4 represent proteins derived from whole cell lysate, cytosol, membrane/organelle, nucleus, and cytoskeleton fractions, respectively.
Immunoresearch Laboratories, Inc., West Grove, PA) at a 1:10000 dilution and RT for 1 h in the dark. Finally, the cells were washed several times and mounted on a glass slide with 50% (v/v) glycerol in PBS and visualized with a laser scanning confocal microscope (LSM 510 Meta, Carl Zeiss). Detection of DENV2 Infection, DENV2 Envelope Protein, and Viral Titer after Neutralization by Anti-LBPA. The EA.hy926 cells with or without pretreatment with anti-LBPA for 24 h were infected with DENV2 at the MOI of 10 as mentioned above. Thereafter, the cells were serially collected for detection of DENV2 envelope protein by immunofluorescence staining with a monoclonal antibody against DENV2 envelope protein (clone 3H5) at 4 °C overnight, followed by goat anti-mouse IgG conjugated with Cy3 (Jackson Immunoresearch Laboratories) at a 1:10000 dilution and RT for 1 h in the dark together with Hoechst dye (Invitrogen Corp., Carlsbad, CA) for nuclear stain at a 1:1000 dilution and RT for 1 h in the dark. The images were taken and analyzed with a laser scanning confocal microscope (LSM 510 Meta, Carl Zeiss). Moreover, the culture supernatants of these cells (with vs without anti-LBPA pretreatment) at the 24 h postinfection time point were examined to determine the virus titer by a focus forming assay in pig fibroblast cells (PS clone D) as described previously.7
Results Infectivity and Cell Death. At an MOI of 10 and with a postinfection incubation period of 24 h, 92.31 ( 2.62% of EA.hy926 cells were successfully infected with DENV2 (as demonstrated by positive DENV2 envelope protein by flow cytometric analysis). Analysis of cell death by flow cytometry using propidium iodide staining revealed a comparable percent cell death in mock control and DENV2-infected cells [12.77 ( 1.84 and 13.16 ( 0.46%, respectively (p ) 0.738)]. These results indicated that changes in subcellular proteomes to be determined in the next section would reflect the early host response to DENV infection, not late changes due to cell death or cytotoxicity induced by virus. Purity of Subcellular Fractionation. Subcellular fractionation was performed by using the Subcellular Proteome Extraction Kit (Mini sPEK) (Calbiochem, EMD Biosciences). The purity of subcellular fractionation was examined by Western blot analyses of markers for individual subcellular compartments, including cytosol (calpain), membrane/organelle (pan-cadherin), nucleus (c-jun), and cytoskeleton (pan-cytokeratin). The results showed satisfactory purity as all these markers were present in respective subcellular fractions and there were
almost none of contaminations observed, except only for a tiny amount of pan-cadherin that was also observed in the nucleus (F3) fraction (Figure 1). Moreover, band intensities of these marker proteins were much more intense than those in the whole cell lysate, indicating successful enrichment of proteins in individual subcellular compartments. Alterations in Subcellular Proteomes of EA.hy926 Human Endothelial Cells upon DENV2 Infection. Five individual experiments with each of four subcellular fractions of both mock control and DENV2-infected groups were performed to examine changes in subcellular proteomes of human endothelial cells upon DENV2 infection. In total, 40 samples were examined by two-dimensional PAGE and quantitative spot intensity analyses. Statistical analysis revealed significant changes in abundance levels for a total of 35 protein spots in all four subcellular fractions (14 in cytosol, five in membrane/organelle, eight in nucleus, and eight in cytoskeleton) (Figure 2 and Table 1). All of these differentially expressed spots were successfully identified by Q-TOF MS and/or MS/MS analyses (Table 1). These altered proteins were categorized on the basis of their functions as summarized in Table S1 of the Supporting Information. Most of the upregulated proteins were involved in the endocytosis system (programmed cell death 6 interacting protein, valosin-containing protein, and vacuolar protein sorting 13C protein isoform 1B) and cellular metabolism (transglutaminase 2 isoform a, spermidine synthase, and purine nucleoside phosphorylase), whereas the downregulated proteins were mainly involved in molecular chaperoning (heat shock 70 kDa protein isoforms and heat shock 90 kDa protein isoforms). Moreover, two DENV viral proteins (NS1 and NS5; spots B3 and B2, respectively) were found exclusively in the membrane/organelle fraction (Table 1). Validation of the Increase in the Level of Cytosolic Alix in DENV2-Infected Endothelial Cells. Because of its role in endocytosis, we focused our attention on the increased level of programmed cell death 6 interacting protein (also known as apoptosis-linked gene-2-interacting protein X or Alix) (spots A2 and A3 in Figure 2). Western blot analysis confirmed the upregulation of Alix in DENV2-infected EA.hy926 endothelial cells, whereas the GAPDH band showed equal loading (Figure 3). Colocalization of Alix and the Late Endosome Marker. Program cell death 6 interacting protein or Alix has been described to play an important role in HIV1 budding10 and transport of the nucleocapsid of vesicular stomatitis virus from endosome to cytosol for viral replication.9 In this study, we Journal of Proteome Research • Vol. 9, No. 9, 2010 4643
research articles
Pattanakitsakul et al.
Figure 2. Two-dimensional proteome map of significantly altered proteins in individual subcellular compartments of EA.hy926 cells upon DENV2 infection. Five independent samples were analyzed using each subcellular compartment of both mock control and DENV2infected EA.hy926 cells (in total, 40 gels were analyzed). Differentially expressed protein spots are labeled with numbers following capital letters (A-D for cytosol, membrane/organelle, nucleus, and cytoskeleton fractions, respectively).
found upregulation of Alix in DENV2-infected EA.hy926 cells. However, its role in DENV infection remains unknown. We therefore explored the functional significance of Alix in DENV replication in endothelial host cells. During the important process of initiation of viral replication and infection, the virus must uncoat or denude its nucleocapsid from the late endosome to the cytoplasm. We thus hypothesized that this step may require interaction of Alix with the late endosome in human endothelial cells. Our hypothesis was addressed by double immunofluorescence staining of Alix and the late endosomal marker, lysobisphosphatidic acid (LBPA). Figure 4 clearly demonstrates the colocalization of Alix and LBPA. Successful Introduction of the Anti-LBPA Antibody into Endothelial Cells before DENV2 Infection. To further explore the functional significance of interaction between Alix and the late endosome, we sought to block binding of Alix with LBPA by preincubating the cells with monoclonal anti-LBPA prior to DENV2 infection. Figure 5 illustrates that we successfully introduced anti-LBPA into the EA.hy926 cells prior to DENV2 infection. We then hypothesized that this neutralization or blockage would affect DENV2 viral replication. Effects of Neutralization by Anti-LBPA Pretreatment on DENV2 Viral Replication and Infection. EA.hy926 cells with or without anti-LBPA pretreatment were infected with DENV2 at an MOI of 10 for 2 h as described for earlier experiments. Thereafter, the cells were serially collected 1, 2, 6, 12, and 24 h postinfection for detection of DENV envelope protein by immunofluorescence staining with the monoclonal antibody against DENV2 envelope protein (clone 3H5). The results showed that pretreatment with anti-LBPA could significantly delay DENV2 replication as demonstrated by delayed expres4644
Journal of Proteome Research • Vol. 9, No. 9, 2010
sion of DENV2 envelope protein at all time points, from 1 h postinfection through the end of the study (Figure 6). This effect was also quantitatively analyzed by performing a focus forming assay to measure the virus titer in DENV2-infected cells with and without pretreatment with anti-LBPA. At the 24 h postinfection time point, the cells pretreated with anti-LBPA had much less virus titer as compared to those without anti-LBPA pretreatment (Figure 7).
Discussion The advantage of subcellular proteome analysis of differential protein expression revealed more defined proteins in their locales as well as translocation between intracellular compartments. Most of the altered proteins in DENV2-infected EA.hy926 cells were observed in cytosol rather than in cytoskeleton, nucleus, and membrane/organelle. Interestingly, some of the adjacent spots that were differentially expressed in DENV2infected versus mock control cells were identified as the same proteins within the same series (i.e., A2 and A3 as Alix, C7 and C8 as heterogeneous nuclear ribonucleoprotein M isoform a, and D1 and D2 as transferrin). These spots were present with similar molecular masses and with a small difference in isoelectric points (pI); thus, they were most likely to be posttranslationally modified proteins. However, some spots were identified as the same protein but with marked differences in molecular mass and pI (i.e., D7 and D8 as serum albumin); thus, they were most likely fragments or different forms of the same protein. DENV2 infection induced downregulation of mainly molecular chaperones, whereas most of the upregulated proteins
A1 A2
D1 D2
C8
C7
C6
C4 C5
C3
C2
C1
B5
B4
B2 B3
B1
A13 A14
A12
A11
A10
A9
A7 A8
A6
A5
A4
A3
protein name
alanyl-tRNA synthetase programmed cell death 6 interacting protein programmed cell death 6 interacting protein valosin-containing protein ATP-dependent DNA helicase II transglutaminase 2 isoform a moesin heat shock 70 kDa protein 1B EH domain containing 1, isoform CRA_a glucose-6-phosphate dehydrogenase spermidine synthase purine nucleoside phosphorylase ALB protein damage-specific DNA binding protein 1 (127 kDa) minichromosome maintenance complex component 4 NS5 protein nonstructural protein NS1 transcription termination factor-like protein eukaryotic translation elongation factor 1R1 70 kDa heat shock protein 4 major vault protein 90 kDa heat shock protein β, member 1 R-actinin 4 Ca2+-dependent activator protein for secretion 2 90 kDa heat shock protein 1, R heterogeneous nuclear ribonucleoprotein M isoform a heterogeneous nuclear ribonucleoprotein M isoform a transferrin transferrin
spot
gi|114326282 gi|114326282
gi|14141152
gi|14141152
gi|83699649
gi|2804273 gi|119603981
gi|4507677
gi|197099612
gi|38327039
gi|4503471
gi|149637937
gi|2731529 gi|159024813
gi|33469917
gi|74267962 gi|119594342
gi|387033
gi|531202
gi|26224790
gi|119594723
gi|4505257 gi|167466173
gi|39777597
gi|10863945
gi|6005942
gi|6755002
gi|1015321 gi|22027538
NCBI ID
b
MS MS
MS, MS/MS
MS/MS
MS, MS/MS
MS, MS/MS MS
MS
MS
MS
MS, MS/MS
MS
MS MS
MS
MS MS
MS
MS, MS/MS
MS
MS
MS, MS/MS MS, MS/MS
MS, MS/MS
MS
MS
MS, MS/MS
MS MS
method of identification
104, NA 104, NA
80, 74
NA, 25
91, 71
162, 40 78, NA
92, NA
76, NA
71, NA
50, 38
72, NA
70, NA 93, NA
104, NA
105, NA 146, NA
116, NA
61, 98
113, NA
116, NA
105, 48 111, 59
181, 10
71, NA
173, NA
51, 41
93, NA 119, NA
identification scores (MS, MS/MS)
30, NA 30, NA
25, 3
NA, 3
21, 1
31, 1 20, NA
26, NA
19, NA
22, NA
28, 5
25, NA
39, NA 33, NA
23, NA
21, NA 29, NA
52, NA
23, 6
40, NA
32, NA
27, 1 32, 4
36, 1
21, NA
37, NA
32, 1
26, NA 26, NA
%Covc (MS, MS/MS)
17, NA 17, NA
9, 1
NA, 1
14, 1
23, 1 13, NA
18, NA
12, NA
12, NA
9, 2
7, NA
9, NA 13, NA
17, NA
14, NA 21, NA
16, NA
8, 3
17, NA
16, NA
18, 1 15, 2
19, 1
15, NA
24, NA
21, 1
16, NA 16, NA
no. of matched peptides (MS, MS/MS) pI
6.75 6.75
8.84
8.84
5.07
5.27 6.30
4.76
5.36
5.11
9.10
8.95
6.15 6.41
6.28
5.88 5.16
7.09
5.21
6.91
6.25
6.08 5.48
5.11
5.55
5.14
6.13
5.31 6.13
79.85 79.86
77.46
77.46
98.65
102.66 111.59
92.69
99.45
95.12
50.45
45.56
38.42 40.59
97.07
71.18 128.91
32.38
34.36
55.18
61.94
67.89 70.29
78.42
83.22
89.95
96.59
107.47 96.59
molecular mass (kDa)
0.0239 ( 0.0031 0.1090 ( 0.0146 0.0416 ( 0.0036 0.1285 ( 0.0145 0.0564 ( 0.0063 0.0474 ( 0.0034 0.0615 ( 0.0066 0.0615 ( 0.0067 0.0303 ( 0.0042 0.0226 ( 0.0065 0.0563 ( 0.0042 0.1365 ( 0.0125 0.3012 ( 0.0326 0.0336 ( 0.0020
0.0154 ( 0.0013 0.0652 ( 0.0029 0.0287 ( 0.0012 0.2132 ( 0.0270 0.0817 ( 0.0065 0.0762 ( 0.0098 0.0434 ( 0.0021 0.0473 ( 0.0081 0.0060 ( 0.0006 0.0452 ( 0.0040 0.0770 ( 0.0074 0.0000 ( 0.0000 0.0000 ( 0.0000 0.0501 ( 0.0038
0.1006 ( 0.0116 0.1917 ( 0.0111 0.3614 ( 0.0170 0.0763 ( 0.0860 0.4191 ( 0.0374 0.3865 ( 0.0591 0.3491 ( 0.0459 0.0915 ( 0.0123 0.1360 ( 0.0219
0.1625 ( 0.0069 0.2277 ( 0.0090 0.3633 ( 0.0354 0.0382 ( 0.0039 0.5768 ( 0.0402 0.1968 ( 0.3436 0.2176 ( 0.0244 0.0398 ( 0.0103 0.0784 ( 0.0080
0.0618 ( 0.0053
0.0798 ( 0.0082
0.0562 ( 0.0042
0.0897 ( 0.0070
0.1424 ( 0.0040
0.1214 ( 0.0067
0.4464 ( 0.0483
0.0567 ( 0.0048 0.0652 ( 0.0018
0.0883 ( 0.0085 0.0497 ( 0.0031
0.2960 ( 0.0392
DENV2infected
mock control
intensity level (mean ( standard error of the mean)
Table 1. Quantitative Data and Identities of Significantly Altered Proteins in Individual Subcellular Compartments of DENV2-Infected EA.hy926 Cellsa
2.29 1.73
1.60
1.96
0.72
0.71 1.99
0.84
0.61
0.68
1.50
0.67
DIV/0d DIV/0d
0.73
5.09 0.50
1.61
1.42
0.62
0.69
1.45 0.60
1.67
1.55
1.42
1.17
0.64 1.31
ratio (DENV2-infected/ mock control)
0.006 0.023
0.024
0.024
0.021
0.032 0.004
0.036
0.001
0.013
0.042
0.004
0.001 0.001
0.040
0.010 0.018
0.016
0.032
0.025
0.023
0.009 0.025
0.019
0.036
0.033
0.027
0.012 0.003
p value
Alix and LBPA in Dengue Virus Infection
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Journal of Proteome Research • Vol. 9, No. 9, 2010 4645
24.81
5.52 9.10
9.00
5.82 5.82
20, 1 11, 1
9, NA
11, NA 11, NA
34, 2 33, 2
46, NA
25, NA 21, NA
%Cov is the percent sequence coverage c
0.1542 ( 0.0076 0.2588 ( 0.0659 0.1231 ( 0.0085 0.0000 ( 0.0000
53.52 50.42
5.98 23, NA 10, NA
Figure 3. Confirmation of the proteomic and mass spectrometric data. The increased level of program cell death 6 interacting protein or Alix in the cytosolic fraction of DENV2-infected cells was nicely confirmed by Western blot analysis (A), whereas GAPDH was examined to confirm equal loading of total protein (B). M1 and M2 represent two independent samples derived from the cytosolic fraction of mock control cells, whereas I1 and I2 represent those derived from DENV2-infected cells.
4646
D7 D8
D6
D4 D5
a A1-A14, cytosol; B1-B5, membrane/organelle; C1-C8, nucleus; D1-D8, cytoskeleton. NA means not applicable. {[(number of matched residues)/(total number of residues in the entire sequence)] × 100%}. d DIV/0 ) divide by zero.
88, NA 92, NA MS MS gi|1351907 gi|1351908
87, NA MS gi|4507513
139, 36 72, 8 MS, MS/MS MS, MS/MS gi|181573 gi|62897525
79, NA MS
vacuolar protein sorting 13C protein isoform 1B cytokeratin 8 eukaryotic translation elongation factor 1R1 tissue inhibitor of metalloproteinase 3 precursor serum albumin serum albumin D3
gi|66348091
protein name spot
Table 1. Continued
NCBIb ID
method of identification
identification scores (MS, MS/MS)
b
71.24 71.24
404.82
pI no. of matched peptides (MS, MS/MS)
National Center for Biotechnology Information.
1.25 DIV/0d
0.4178 ( 0.0575 0.6808 ( 0.0569
0.014 0.00
0.61
0.3273 ( 0.0106 1.9941 ( 0.1294 0.2868 ( 0.0124 1.5414 ( 0.1577
0.006
1.14 1.29
0.0654 ( 0.0071 0.0381 ( 0.0099
0.023 0.036
1.71
DENV2infected mock control
0.036
Pattanakitsakul et al.
%Covc (MS, MS/MS)
molecular mass (kDa)
intensity level (mean ( standard error of the mean)
ratio (DENV2-infected/ mock control)
p value
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Journal of Proteome Research • Vol. 9, No. 9, 2010
Figure 4. Association between Alix and the late endosome in DENV2-infected EA.hy926 cells. Double immunofluorescence staining revealed colocalization of Alix (in red) and LBPA (in green) which served as a marker for the late endosome. The original magnification power was 630× in all panels.
were those involved in endocytosis and cellular metabolism (Figure 2, Table 1, and Table S1 of the Supporting Information). In addition, the increased levels of proteins in the cytoskeleton compartment might be also related to several features, including the endocytosis system, cell structure, and translation. Moreover, the identification of DENV proteins (NS1 and NS5), which were found exclusively in the membrane/organelle compartment of DENV2-infected EA.hy926 cells, indicated that the DENV replication complex was associated with the membrane-bound organelles.11,12 These viral proteins may cooperate with other host proteins to play an important role in the synthesis of viral and host proteins. Interestingly, we identified upregulation of two isoforms of Alix that was upregulated in the cytosolic compartment of DENV2-infected cells (spots A2 and A3 in Figure 2). Its upregulation was confirmed by Western blot analysis (Figure 3). This protein has been shown to play an important role in HIV1 and vesicular stomatitis virus replication, particularly during viral budding and transport of the viral nucleocapsid into the cytoplasm.9,10 However, its precise role in DENV infection and replication remains unknown and had not been previously investigated. Alix has dual roles in intraluminal vesicle formation. It can interact with the endosomal sorting complex required for transport (ESCRT) and then regulates ESCRT-dependent formation of intraluminal vesicles. Another role of Alix is controlling the process of intraluminal vesicle formation in the late endosome through the interaction with lysobisphosphatidic
Alix and LBPA in Dengue Virus Infection
research articles
Figure 7. Virus titer of infectious virion released from DENV2infected EA.hy926 cells without or with anti-LBPA pretreatment. At the 24 h postinfection time point, the virus titer in the culture supernatant was measured by a focus forming assay. The data are presented as means ( the standard deviation of three independent experiments.
Figure 5. Internalization of the mouse monoclonal anti-LBPA antibody into EA.hy926 cells. Pretreatment of EA.hy926 cells with the anti-LBPA antibody at 50 µg/mL 24 h prior to DENV2 infection showed successful internalization of the anti-LBPA antibody into the cells as detected by staining with goat anti-mouse IgG conjugated with Alexa 488 (in green). The original magnification power was 630×.
acid (LBPA).13 During the entry of DENV into cells and fusion with the endosome, the significant process of DENV in replicating and initiating infection requires maturation of the late endosome, uncoating of the viral nucleocapsid, and delivery of its nucleocapsid to the cytoplasm.14 Thus, the late endosome should play a critical role in DENV replication. LBPA, a phospholipid primarily found inside intraluminal vesicles of the late endosome,15–18 has been identified with well-supported evidence as a marker for the late endosome.19 We therefore examined the association between Alix and the late endosome using LBPA as its marker. Our results clearly demonstrated that Alix colocalized with LBPA (Figure 4), indicating its association and interaction with the late endosome. The significance of the association between Alix and the
late endosome was also confirmed by a functional study. Pretreatment with anti-LBPA prior to DENV2 infection could efficiently delay the production of DENV2 envelope protein (Figure 6) and significantly reduce the level of viral replication and release (Figure 7). Although we could not observe a complete suppression of DENV replication and infection by blocking the association between Alix and the late endosome using anti-LBPA, our data may also lead to the identification of a novel therapeutic target for controlling DENV infection and preventing its development to DHF/DSS. In addition, DENV may require other host proteins or mechanisms for viral replication and infection. Alix itself has the ability to interact with other proteins, including CIN85 (cbl-interacting protein, 85 kDa), endophilins, Tsg101 (tumor susceptibility gene 101), and CHMP4B (charged multivesicular body protein 4B) of ESCRT-III.16 These proteins have been reported to interact with Alix and to be involved in endosomal membrane vesiculation and transport of proteins into the cytoplasm.20,21 In summary, we report here for the first time the significance of association between Alix and the late endosome for DENV viral replication and infection in human endothelial cells. Our data provide some novel insights into mechanisms of DENV infection and also offer an opportunity to define new therapeutic targets for the treatment of DENV infection and prevention of its severe form (i.e., DHF/DSS).
Figure 6. Immunofluorescence staining of DENV2 envelope protein in DENV2-infected EA.hy926 cells without or with anti-LBPA pretreatment. After DENV2 infection at an MOI of 10 for 2 h, the cells were serially harvested for immunodetection of DENV2 envelope protein (red) (using a monoclonal antibody against DENV2 envelope protein; clone 3H5) 1, 2, 6, 12, and 24 h postinfection. Journal of Proteome Research • Vol. 9, No. 9, 2010 4647
research articles Acknowledgment. We are grateful to Dr. Chunya Puttikhunt for providing cultured supernatant collected from a hybridoma clone. This work was supported by the National Center for Genetic Engineering and Biotechnology (BIOTEC), the National Science and Technology Development Agency, Thailand (BT-B-02-MG-B4-5003) (to S.P. and V.T.), a Siriraj Graduate Thesis Scholarship (to J.P.), and a research grant from Mahidol University, Commission on Higher Education (to V.T., R.K., and S.P.). S.P. and V.T. are also supported by a “Chalermphrakiat” Grant, Faculty of Medicine, Siriraj Hospital. Supporting Information Available: Compositions of buffers used in subcellular fractionation and functional classification of altered proteins in DENV2-infected EA.hy926 cells (Table S1). This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Halstead, S. B. Dengue. Lancet 2007, 370, 1644–1652. (2) Kurane, I. Dengue hemorrhagic fever with special emphasis on immunopathogenesis. Comp. Immunol. Microbiol. Infect. Dis. 2007, 30, 329–340. (3) Moi, M. L.; Lim, C. K.; Takasaki, T.; Kurane, I. Involvement of the Fcγ receptor IIA cytoplasmic domain in antibody-dependent enhancement of dengue virus infection. J. Gen. Virol. 2010, 91, 103–111. (4) Miller, J. L.; de Wet, B. J.; Martinez-Pomares, L.; Radcliffe, C. M.; Dwek, R. A.; Rudd, P. M.; Gordon, S. The mannose receptor mediates dengue virus infection of macrophages. PLoS Pathog. 2008, 4, e17. (5) Reyes-Del Valle, J.; Chavez-Salinas, S.; Medina, F.; Del Angel, R. M. Heat shock protein 90 and heat shock protein 70 are components of dengue virus receptor complex in human cells. J. Virol. 2005, 79, 4557–4567. (6) Pattanakitsakul, S. N.; Rungrojcharoenkit, K.; Kanlaya, R.; Sinchaikul, S.; Noisakran, S.; Chen, S. T.; Malasit, P.; Thongboonkerd, V. Proteomic analysis of host responses in HepG2 cells during dengue virus infection. J. Proteome Res. 2007, 6, 4592–4600. (7) Avirutnan, P.; Punyadee, N.; Noisakran, S.; Komoltri, C.; Thiemmeca, S.; Auethavornanan, K.; Jairungsri, A.; Kanlaya, R.; Tangthawornchaikul, N.; Puttikhunt, C.; Pattanakitsakul, S. N.; Yenchitsomanus, P. T.; Mongkolsapaya, J.; Kasinrerk, W.; Sittisombut, N.; Husmann, M.; Blettner, M.; Vasanawathana, S.; Bhakdi, S.; Malasit, P. Vascular leakage in severe dengue virus infections: A potential role for the nonstructural viral protein NS1 and complement. J. Infect. Dis. 2006, 193, 1078–1088.
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Pattanakitsakul et al. (8) Kanlaya, R.; Pattanakitsakul, S. N.; Sinchaikul, S.; Chen, S. T.; Thongboonkerd, V. Alterations in actin cytoskeletal assembly and junctional protein complexes in human endothelial cells induced by dengue virus infection and mimicry of leukocyte transendothelial migration. J. Proteome Res. 2009, 8, 2551–2562. (9) Le, B. I.; Luyet, P. P.; Pons, V.; Ferguson, C.; Emans, N.; Petiot, A.; Mayran, N.; Demaurex, N.; Faure, J.; Sadoul, R.; Parton, R. G.; Gruenberg, J. Endosome-to-cytosol transport of viral nucleocapsids. Nat. Cell Biol. 2005, 7, 653–664. (10) Munshi, U. M.; Kim, J.; Nagashima, K.; Hurley, J. H.; Freed, E. O. An Alix fragment potently inhibits HIV-1 budding: Characterization of binding to retroviral YPXL late domains. J. Biol. Chem. 2007, 282, 3847–3855. (11) Kushner, D. B.; Lindenbach, B. D.; Grdzelishvili, V. Z.; Noueiry, A. O.; Paul, S. M.; Ahlquist, P. Systematic, genome-wide identification of host genes affecting replication of a positive-strand RNA virus. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 15764–15769. (12) Mackenzie, J. Wrapping things up about virus RNA replication. Traffic 2005, 6, 967–977. (13) Williams, R. L.; Urbe, S. The emerging shape of the ESCRT machinery. Nat. Rev. Mol. Cell Biol. 2007, 8, 355–368. (14) Mukhopadhyay, S.; Kuhn, R. J.; Rossmann, M. G. A structural perspective of the flavivirus life cycle. Nat. Rev. Microbiol. 2005, 3, 13–22. (15) Mahul-Mellier, A. L.; Strappazzon, F.; Chatellard-Causse, C.; Blot, B.; Beal, D.; Torch, S.; Hemming, F.; Petiot, A.; Verna, J. M.; Fraboulet, S.; Sadoul, R. Alix and ALG-2 make a link between endosomes and neuronal death. Biochem. Soc. Trans. 2009, 37, 200–203. (16) Mahul-Mellier, A. L.; Strappazzon, F.; Petiot, A.; Chatellard-Causse, C.; Torch, S.; Blot, B.; Freeman, K.; Kuhn, L.; Garin, J.; Verna, J. M.; Fraboulet, S.; Sadoul, R. Alix and ALG-2 are involved in tumor necrosis factor receptor 1-induced cell death. J. Biol. Chem. 2008, 283, 34954–34965. (17) Gottlinger, H. G. How HIV-1 hijacks ALIX. Nat. Struct. Mol. Biol. 2007, 14, 254–256. (18) Sadoul, R. Do Alix and ALG-2 really control endosomes for better or for worse. Biol. Cell 2006, 98, 69–77. (19) Kobayashi, T.; Stang, E.; Fang, K. S.; de Moerloose, P.; Parton, R. G.; Gruenberg, J. A lipid associated with the antiphospholipid syndrome regulates endosome structure and function. Nature 1998, 392, 193–197. (20) Chatellard-Causse, C.; Blot, B.; Cristina, N.; Torch, S.; Missotten, M.; Sadoul, R. Alix (ALG-2-interacting protein X), a protein involved in apoptosis, binds to endophilins and induces cytoplasmic vacuolization. J. Biol. Chem. 2002, 277, 29108–29115. (21) Chen, B.; Borinstein, S. C.; Gillis, J.; Sykes, V. W.; Bogler, O. The glioma-associated protein SETA interacts with AIP1/Alix and ALG-2 and modulates apoptosis in astrocytes. J. Biol. Chem. 2000, 275, 19275–19281.
PR100357F
