The Ubiquitin-Proteasome Pathway Is Important for Dengue Virus Infection in Primary Human Endothelial Cells Rattiyaporn Kanlaya,† Sa-nga Pattanakitsakul,‡ Supachok Sinchaikul,§ Shui-Tein Chen,§,| and Visith Thongboonkerd*,†,⊥ Medical Proteomics Unit, Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand, Medical Molecular Biology 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 March 9, 2010
Dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) are the most severe forms of dengue virus infection with hemorrhage and plasma leakage. However, pathogenic mechanisms of DHF and DSS remain poorly understood. We therefore investigated host responses as determined by changes in the cellular proteome of primary human endothelial cells upon infection with dengue virus serotype 2 (DEN-2) at a multiplicity of infection (MOI) of 10 for 24 h. Two-dimensional PAGE and quantitative intensity analysis revealed 38 significantly altered protein spots (16 upregulated and 22 downregulated) in DEN-2-infected cells compared to mock controls. These altered proteins were successfully identified by mass spectrometry, including those involved in oxidative stress response, transcription and translation, cytoskeleton assembly, protein degradation, cell growth regulation, apoptosis, cellular metabolism, and antiviral response. The proteomic data were validated by Western blot analyses [upregulated ubiquitin-activating enzyme E1 (UBE1) and downregulated annexin A2] and an immunofluorescence study (upregulated MxA). Interestingly, we found that MxA was colocalized with DEN-2 viral capsid protein, strengthening its role as an antiviral protein. Moreover, we also identified upregulation of a proteasome subunit. Our functional study revealed the significant role of ubiquitination in dengue infection and UBE1 inhibition by its specific inhibitor (UBEI-41) caused a significant reduction in the level of viral protein synthesis and its infectivity. Our findings suggest that various biological processesweretriggeredinresponsetodengueinfection,particularlyantiviralIFNandubiquitin-proteasome pathways. Keywords: Dengue • proteasome • proteomics • therapeutic targets • ubiquitin
Introduction Dengue virus infection causes a broad spectrum of clinical syndromes ranging from mild febrile illness (dengue fever) to life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Little is known about target cells of dengue infection in vivo, as well as subcellular mechanisms of DHF and DSS, because of a lack of an appropriate animal model. Because the hallmarks of DHF and DSS are vascular * To whom correspondence should be addressed: Medical Proteomics Unit, Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, 12th Floor Adulyadejvikrom Building, 2 Prannok Rd., Bangkoknoi, Bangkok 10700, Thailand. Phone and fax: +662-4184793. E-mail:
[email protected] or
[email protected]. † Medical Proteomics Unit, Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University. ‡ Medical Molecular Biology 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.
4960 Journal of Proteome Research 2010, 9, 4960–4971 Published on Web 08/19/2010
leakage and hemorrhage, an emphasis on the host response of endothelial cells to dengue virus infection has been raised. A recent study has provided strong evidence to support the possibility that endothelial cells are important target cells for dengue virus infection by demonstrating dengue viral antigens in Kuffer and sinusoidal endothelial cells of liver and lung, respectively, obtained by both biopsy and autopsy.1 Various tissue sections from fatal DSS cases during an epidemic outbreak in Santiago de Cuba showed TUNEL-positive reaction and apoptotic cell death in intestinal serosa and alveolar capillary endothelial cells.2 The established mouse model of DHF revealed that endothelial damage occurred as a result of both virus and tumor necrosis factor-R (TNF-R) to induce apoptosis.3 Additionally, in response to dengue virus infection, the infected human endothelial cells selectively secreted chemokines RANTES (regulated upon activation, normal T-cellexpressed and -secreted), interleukin-8 (IL-8), and IL-6 into the culture supernatant, similar to the phenomenon found in plasma, sera, and pleural fluid of DHF and DSS patients.4-6 10.1021/pr100219y
2010 American Chemical Society
Ubiquitin-Proteasome Pathway and Dengue Infection
research articles
Changes in vascular permeability and rearrangement of the cytoskeleton induced by dengue virus infection were also demonstrated in human endothelial cells in vitro.7,8 Dengue virus infection and treatment with TNF-R synergistically affected (increased) vascular permeability as demonstrated by the decreased transendothelial electrical resistance.7 Infected microvascular endothelial cells (HMEC-1) produced IL-8, which in turn contributed to reorganization of the cytoskeleton and modified transendothelial permeability.8 Most previous studies of global cellular responses of endothelial cells to dengue virus infection were conducted at the transcriptional level. Differential display RT-PCR and an Affymetrix oligonucleotide microarray were employed to analyze altered transcriptome profiles of DEN-2-infected human umbilical vein endothelial cells (HUVECs). The results showed that the majority of differentially expressed transcripts were involved in host defense mechanisms, including stress response, wounding, inflammatory modulation, and antiviral pathways. At least three signaling pathways, including TNF-R, IL-1β, and IFN-R/β pathways, have been suggested to be involved in the inflammatory response.9 Novel sets of altered transcripts of human endothelial-like cells (ECV304) infected with DEN-2 were also identified by differential display RT-PCR10 and microarray technology.11 These altered transcripts were involved in signal transduction, protein translation and modification, cytoskeleton assembly, and the cell cycle. While many studies have focused on transcriptional changes, we have recently reported a set of altered proteins in DEN-2-infected EA.hy926 cells using a proteomics approach and demonstrated that changes in actin cytoskeleton and junctional proteins might be a molecular basis underlying vascular permeability in severe cases.12 However, EA.hy926 human endothelial cells originated from fusion of HUVECs with lung carcinoma cell line A549.13 Thus, the data obtained from our previous study might not reflect changes in patients. Hence, the information about alterations in the proteome in primary human endothelial cells is needed to link in vitro findings to clinical events. This study aimed to characterize early host response as determined by changes in the cellular proteome in HUVECs upon dengue virus infection. A two-dimensional electrophoresis-based proteomics approach was applied to identify such changes, which were then validated by Western blot analyses and immunofluorescence study. Furthermore, a functional study of coordinately altered proteins involved in the ubiquitin-proteasome pathway was performed to highlight their significant role in dengue virus infection.
After a 24 h incubation, nonadherent cells were washed out with M199 medium twice, whereas the adhered cells were cultured with fresh endothelial growth medium (Cambrex Bio Science Walkersville Inc., Walkersville, MD). The culture medium was changed every 48 h until the cells were confluent. Only HUVECs derived from five or fewer passages were used in all experiments throughout this study. Surface Expression of CD31 on HUVECs. The isolated HUVECs were characterized for surface expression of CD31, which is the marker for endothelial lineage. Briefly, HUVECs were collected in a 2.5 mM EDTA/PBS mixture and washed once with plain M199 medium followed by centrifugation at 400g for 3 min at 4 °C. All incubation steps were performed on ice. Fc receptors that might be available on HUVEC surfaces were blocked by incubation with 10% human AB serum diluted with 1% bovine serum albumin (BSA) in PBS for 30 min. After being washed with PBS, the cells were incubated with the mouse monoclonal anti-CD31 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) (1:50 in a 1% BSA/PBS mixture) at room temperature (RT, 25 °C) for 1 h. The cells were then washed once and further incubated with rabbit anti-mouse IgG conjugated with FITC (Dako, Glostrup, Denmark) at RT for 30 min. The cells were then washed twice and analyzed by flow cytometry using FACScan equipped with CellQuest (Becton Dickinson, Franklin Lake, NJ). EA.hy926 cells (endothelial lineage) and THP-1 (monocytic lineage) were processed in parallel and served as the positive controls, whereas a mouse isotype control antibody served as the negative control. Production of Dengue Virus Stock and Titration. C6/36, a cell line from Aedes albopictus (ATCC CRL-1660), and PscloneD, a swine fibroblast cell line, were cultured at 28 and 37 °C, respectively, in L-15 medium (Gibco) containing 10% heatinactivated FBS (Gibco), 10% tryptose phosphate broth (TPB) (Sigma), 100 units/mL penicillin, and 100 µg/mL streptomycin (Sigma). DEN-2 (strain 16681) was propagated in C6/36 cells. Briefly, a confluent monolayer of C6/36 cells was incubated with DEN-2 at a multiplicity of infection (MOI) of 0.1 in maintenance medium (L-15 medium containing 1% heatinactivated FBS, 10% TPB, 100 units/mL penicillin, and 100 µg/ mL streptomycin) at 28 °C for 3 h with gentle shaking. Subsequently, the supernatant was removed and replaced with fresh maintenance medium and further incubated at 28 °C until an approximately 50% cytopathic effect (CPE) was observed. The culture supernatant was finally collected by centrifugation at 200g and 4 °C for 5 min. Virus stock was kept as aliquots at -70 °C until use. Virus titer was determined by a focus forming assay using a swine fibroblast cell line, PscloneD. The stained foci were used for calculation of virus titer [focus forming units per milliliter (FFU/mL)] in the culture supernatant as described previously.15 Infection of HUVECs with DEN-2. HUVEC monolayers were infected with DEN-2 at various MOIs (1, 5, and 10) and incubated at 37 °C for 2 h. The supernatant was then removed and replaced with fresh maintenance medium [M199 supplemented with 5% (v/v) heat-inactivated FBS, 100 units/mL penicillin, and 100 µg/mL streptomycin (Sigma)]. The cells were further incubated at 37 °C in 5% CO2 for 12, 24, and 48 h postinfection. The parallel uninfected cells served as the mock control.
Materials and Methods Isolation and Cultivation of HUVECs. HUVECs were isolated from umbilical cords as described previously by Marin et al.14 with slight modifications. Briefly, umbilical cords were cut at the edges with an extension tube inserted into the vein. The remaining blood was washed out with PBS, whereas endothelial cells were trypsinized with 0.05% trypsin in 0.53 mM EDTA at 37 °C for 20 min and eluted with M199 medium supplemented with 5% (v/v) heat-inactivated fetal bovine serum (FBS) (Gibco, Grand Island, NY). Isolated cells were collected by centrifugation at 100g for 10 min at 4 °C. The cell pellet was washed once with M199 medium. Finally, the isolated cells were propagated in a culture flask coated with 1% gelatin in growth medium [M199 medium supplemented with 10% (v/v) heat-inactivated FBS, 100 units/mL penicillin, and 100 µg/mL streptomycin (Sigma, St.Louis, MO)] at 37 °C with 5% CO2 and 95% humidity.
Detection of the DEN-2 Antigen by Cytoplasmic Staining and Quantitative Analysis of DEN-2 Infectivity. To determine an optimal condition for infection and DEN-2 infectivity in HUVECs, mock control and DEN-2-infected cells were detected Journal of Proteome Research • Vol. 9, No. 10, 2010 4961
research articles for dengue viral antigen. The cells were harvested 12, 24, and 48 h after the infection. The harvested cells were washed with plain M199 medium, fixed with 2% formaldehyde (BDH, Poole, U.K.) in PBS at RT for 1 h, and then permeabilized with 0.1% Triton X-100 (Fluka, Buchs, Switzerland) in PBS. Permeabilized cells were incubated with the 3H5 monoclonal antibody (culture supernatant collected from hybridoma cell clone 3H5) specific to DEN-2 envelope (E) protein at RT for 1 h. Subsequently, the cells were washed with 0.1% Triton X-100 in PBS and incubated with rabbit anti-mouse IgG conjugated with FITC (DAKO) at RT in the dark for 30 min. After being washed with 0.1% Triton X-100 in PBS, the cells were resuspended in PBS and analyzed by flow cytometry using FACScan equipped with CellQuest (Becton Dickinson). The percentage of DEN-2 infection (%infection) was calculated according to the formula %infection ) [(number of infected cells)/(number of total cells)] × 100%. Flow Cytometric Analysis of Cell Death. Mock control and DEN-2-infected cells were harvested as described above and resuspended in M199 medium supplemented with 10% (v/v) heat-inactivated FBS. The cells were pelleted by centrifugation at 400g for 5 min and washed once in ice-cold annexin V buffer [10 mM HEPES (pH 7.4) containing 140 mM NaCl and 2.5 mM CaCl2 · 2H2O]. After being washed, the cells were resuspended with annexin V buffer at a density of 5 × 105 cells/mL and then incubated with FITC-conjugated annexin V (BD Biosciences, San Diego, CA) on ice in the dark for 15 min. Propidium iodide (BD Biosciences) at a final concentration of 0.2 µg/mL was added to the cell suspension prior to analysis by flow cytometry. The fixed and permeabilized cells were used as the positive control, whereas the untreated cells were used as the negative control. The percentage of cell death (%cell death) was calculated according to the formula %cell death ) [(number of annexin V and/or propidium iodide-positive cells)/(number of total cells)] × 100%. Protein Extraction for Two-Dimensional (2D) PAGE. Mock control and DEN-2-infected cells were harvested from five individual culture flasks for each group. The condition chosen for proteome analysis was infection at an MOI of 10 and a postinfection incubation period of 24 h, at which the majority of the cells were infected by the virus but the percentage of cell death did not increase. Cell pellets were collected by centrifugation at 400g for 5 min and washed three times with PBS. Cellular proteins were extracted using a lysis buffer containing 7 M urea, 2 M thiourea, 40 mg/mL 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 120 mM dithiothreitol (DTT), 2% (v/v) ampholytes (pH 3-10), and 40 mM Tris base at 4 °C for 30 min. Unsolubilized materials were then removed by centrifugation at 13000g and 4 °C for 5 min, and protein concentrations in the supernatants were measured using a Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA) based on the Bradford method. 2D PAGE, Staining, and Visualization of Protein Spots. A total of 150 µg of protein was mixed with a rehydration buffer containing 7 M urea, 2 M thiourea, 20 mg/mL CHAPS, 120 mM DTT, 40 mM Tris base, 2% ampholytes (pH 3-10), and a trace amount of bromophenol blue to make a total volume of 150 µL per strip. The mixture was loaded into the Immobiline DryStrip (nonlinear pH 3-10) (GE Healthcare, Uppsala, Sweden) at RT for 16 h. The one-dimensional separation or isoelectric focusing (IEF) was performed at 20 °C using an Ettan IPGphor II IEF System (GE Healthcare) with a step-and-hold gradient to reach 9083 V h. The strips were equilibrated with 4962
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Kanlaya et al. equilibration buffer I [6 M urea, 130 mM DTT, 112 mM Tris base, 4% (w/v) SDS, 30% glycerol, and 0.002% bromophenol blue] for 15 min, followed by equilibration in buffer II [6 M urea, 135 mM iodoacetamide, 112 mM Tris base, 4% (w/v) SDS, 30% glycerol, and 0.002% bromophenol blue] for 15 min. Thereafter, the two-dimensional separation was performed by placing the strips on top of a 12% polyacrylamide slab gel using the SE260 mini-Vertical Electrophoresis Unit (GE Healthcare) at 150 V for approximately 2 h. 2D gels were fixed in a solution containing 10% (v/v) methanol and 7% (v/v) acetic acid for 30 min and stained with SYPRO Ruby (Invitrogen-Molecular Probes, Eugene, OR) for 16 h with gentle shaking. Image capture of 2D gels was performed using a Typhoon 9200 laser scanner (GE Healthcare). A total of 10 gels (five gels derived from individual culture flasks for each group) were obtained. Matching and Quantitative Analysis of Protein Spots. Image Master 2D Platinum version 6.0 (GE Healthcare) was used for matching and analysis of protein spots visualized in individual gels. 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 appearing in different gels into one image. The reference gel was used for the determination of the existence and difference in protein expression between 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. In-Gel Tryptic Digestion. The protein spots whose intensity levels significantly differed between groups were excised from 2D gels, washed twice with 200 µL of 50% acetonitrile (ACN)/ 25 mM NH4HCO3 buffer (pH 8.0) at room temperature for 15 min, and then washed once with 200 µL of 100% ACN. After the samples had been washed, the solvent was removed, and the gel pieces were dried with a SpeedVac concentrator (Savant, Holbrook, NY) and rehydrated with 10 µL of 1% (w/v) trypsin (Promega, Madison, WI) in 25 mM NH4HCO3. After rehydration, the gel pieces were crushed and incubated at 37 °C for at least 16 h. Peptides were subsequently extracted twice with 50 µL of a 50% ACN/5% trifluoroacetic acid (TFA) mixture; the extracted solutions were then combined and dried with a SpeedVac concentrator. The peptide pellets were resuspended with 10 µL of 0.1% TFA and purified using ZipTipC18 (Millipore, Bedford, MA). The peptide solution was drawn up and down in the ZipTipC18 10 times and then washed with 10 µL of 0.1% formic acid by drawing up and expelling the washing solution three times. The peptides were finally eluted with 5 µL of a 75% ACN/0.1% formic acid mixture. Protein Identification by Quadrupole Time-of-Flight (Q-TOF) Mass Spectrometry (MS) and/or Tandem MS (MS/ MS). The proteolytic samples were premixed 1:1 with a matrix solution [5 mg/mL R-cyano-4-hydroxycinnamic acid (CHCA) in 50% ACN, 0.1% (v/v) TFA, and 2% (w/v) ammonium citrate] and spotted onto the 96-well sample stage. The samples were analyzed with the Q-TOF Ultima mass spectrometer (Micromass, Manchester, U.K.), which was fully automated with a predefined probe motion pattern and the peak intensity threshold for switching over from MS survey scanning to MS/ MS, and from one MS/MS to another. Within each sample well, parent ions that met the predefined criteria (any peak within the m/z 800-3000 range with an intensity 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
Ubiquitin-Proteasome Pathway and Dengue Infection
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energy until the end of the probe pattern was reached. The LM and HM resolutions of the quadrupole were both set at 10 to give a precursor selection window that was ∼4 Da wide. The instrument was externally calibrated to
