Phosphoproteomic analysis of dengue virus infected U937 cells and identification of pyruvate kinase M2 as a differentially phosphorylated phosphoprotein

Dengue virus (DENV) is an arthropod-borne Flavivirus that can cause a range of symptomatic disease in humans. There are four dengue viruses (DENV 1 to 4) and infection with one DENV only provides transient protection against a heterotypic virus. Second infections are often more severe as the disease is potentiated by antibodies from the first infection through a process known as antibody dependent enhancement (ADE) of infection. Phosphorylation is a major post-translational modification that can have marked effects on a number of processes. To date there has been little information on the phosphorylation changes induced by DENV infection. This study aimed to determine global phosphoproteome changes induced by DENV 2 in U937 cells infected under an ADE protocol. A 2-dimensional electrophoretic approach coupled with a phosphoprotein-specific dye and mass spectroscopic analysis identified 15 statistically significant differentially phosphorylated proteins upon DENV 2 infection. One protein identified as significantly differentially phosphorylated, pyruvate kinase M2 (PKM2) was validated. Treatment with a PKM2 inhibitor modestly reduced levels of infection and viral output, but no change was seen in cellular viral protein levels, suggesting that PKM2 acts on exocytic virus release. While the effect of inhibition of PKM2 was relatively modest, the results highlight the need for a greater understanding of the role of phosphoproteins in DENV infection.

4 strains circulating in Thailand need enhancement of infection to cause DHF 5 . An additional study reported that increased DENV disease severity correlated with high viremia titer, secondary DENV infection and DENV 2 virus serotype 6 . In addition, secondary infections caused by DENV 2 was associated with more cases of DHF than were DENV 4 secondary infections 7 .
Preexisting heterologous antibodies have an important role in the development of severe DENV disease. Antibody-dependent enhancement (ADE) of DENV infection has been proposed as the mechanism underlying DHF/DSS 8,9 . DENV cross-reactive antibodies raised following a primary infection combine with a secondary infecting virus to form infectious immune complexes that enter Fc-receptor bearing cells such as monocytes and macrophages as well as immature and mature dendritic cells 8 . ADE of infection is believed to be driven by two main elements. Firstly, there is an increased number of infected cells due to increased antibody-mediated cell binding and entry of both mature and (partially) immature DENV particles which is also known as extrinsic ADE. Another form called intrinsic ADE occurs through increased virus production per infected cell due to suppression of the innate antiviral response 10 . Intrinsic ADE of DENV infection is believed to involve suppression of the toll-like receptor (TLR) and retinoic acid inducible protein I/melanoma differentiation-associated gene5 (RIG-I/MDA5) signaling pathways thereby decreasing the production of type I interferon and interferonactivated antiviral molecules 11 .
While there is an approved commercially available tetravalent vaccine to protect against DENV, its introduction has been controversial due to the occurrence of more severe disease in flavivirus naïve individuals who received vaccination 12 . Currently, there is no specific drug to treat DENV infection. Kinase inhibitors are of particular interest to the development of antiviral agents, since DENV infection can directly activate signal transducer and activator proteins in the MAP kinase pathway e.g. JNK, p38, NTRK1, MAPKAPK5 and c-src/ FYN kinases [13][14][15][16][17][18] . Therefore, kinase inhibitors that affect host cell factors required for virus replication but have no effect on host cells could be an alternative therapy for DENV infection. At present, there are many kinase inhibitors available in the market 19 . JNK and p38 kinase inhibitors were reported to significantly reduced DENV protein synthesis and viral yield 14 . Several DENV-induced pro-inflammatory mediators such as TNF-α, IL-8, and RANTES were also suppressed by a p38 MAPK inhibitor tested in human peripheral blood mononuclear cells (PBMCs), monocytic THP-1 cells, and the granulocyte KU812 cell line. In addition, oral treatment of DENVinfected AG129 mice with SB203580 prevented a rising hematocrit, lymphopenia, inflammation development, intestinal leakage and significantly improved survival 17 . Another kinase inhibitor, SFV785, has selective effects on NTRK1 and MAPKAPK5 kinase activity, and shows anti-viral activity towards hepatitis C, DENV and yellow fever viruses by inhibiting the production of infectious virus particles 13 . Two pharmacological inhibitors of host kinases AZD0530 and dasatinib, have been shown to inhibit the DENV 2 infectious cycle at the step of steady-state RNA replication, and Fyn kinase was identified as the cellular target mediating the effect 16 . Another advantage of some kinase inhibitors is reducing drug resistance caused by the lack of proofreading of RNA virus polymerases, as compound 16i was reported to act as a DENV inhibitor through targeting both the DENV NS5 polymerase and the host kinases c-Src/Fyn 18 . The compound was demonstrated to inhibit DENV replication at low micromolar concentrations with no significant toxicity to the host cell 18 . Additional evidence of a kinase pathway activated by dengue infection is Janus kinase/activator of transcription 3 (JAK/STAT3). JAK2 and JAK3 inhibitors reduced DENV-induced cell migration and production of chemokines such as IL-8 and RANTES 20 .
The majority of previous studies have modeled primary infection, and there is little information of kinases and their cellular targets in secondary DENV infection. Therefore, to explore the differential regulation of kinases in a secondary DENV infection model, phosphoproteomics was employed. Identifying differentially phosphorylated proteins may help in the understanding of host cell factors and cell signaling pathways involved in secondary DENV infection. Therefore, this study focused on a secondary infection model of DENV 2 infection and a phosphoproteome analysis of U937 monocytic cells infected with DENV 2 under conditions of ADE was undertaken using 2-DE gel electrophoresis followed by LC-MS/MS for protein identification. The study identified pyruvate kinase M2 as differentially phosphorylated and the role of this protein in DENV 2 infection was analyzed.

Results
ADe infection. ADE infection of U937 cells was optimized using varying concentrations of monoclonal antibody HB-114 21 and comparing between mock and DENV 2 infected cells. The optimization included a direct infection with no antibody using MOI = 20. After 48 h of infection, it was found that in the absence of antibodies, the percentage of infected cells was 16.340 ± 2.286% ( Supplementary Fig. 1), while an antibody dilution of 1:200 resulted in the highest percentage of infection of 69.780 ± 0.710% which was significantly different from the direct infection (P < 0.05), confirming the cells were infected under a condition of ADE. A higher concentration of the antibody (1:20 dilution), resulted in a reduced level of infection of 30.173 ± 0.418%, which was still significantly higher than direct infection P < 0.05. Consistent with our previous study, no neutralization was observed 22 . Therefore, an antibody dilution of 1:200 was selected for further large scale preparation of DENV 2 infected U937 cells.
Large scale preparation of infected U937 cells. U937 cells were propagated in T-175 cm 2 tissue culture flask for 3 days after which cells were collected and the cell density was adjusted to 1.82 × 10 6 cells/ml. For the preparation of the antibody-virus complex, DENV 2 strain 16681 (multiplicity of infection (MOI) of 20) was mixed with monoclonal antibody HB-114 21 at a final dilution of 1:200 and incubated for 1 h at 4 °C. Then the complex mixture was added to 3 × 10 7 U937 cells which were incubated at 37 °C, 5% CO 2 for 2 h. Finally, culture media was added to the cells to give a final cell density of 3 × 10 5 cells/ml. Fresh culture media was added every 24 h, and the culture was incubated for 2 days. For mock infected cells, the culture media was mixed with only antibody HB-114 and the same procedure as the protocol of virus infection was followed. The morphology of Phosphoprotein enrichment and 2-D gel electrophoresis. The total protein obtained from the sonication of approximately 7.5-8.0 × 10 7 mock or DENV 2 infected cells ranged from 3,700-5,000 μg per sample. For phosphoprotein enrichment, approximately 3,700-4,000 μg protein was loaded onto a pre-equilibrated phosphoprotein affinity column for 30 min at 4 °C. Columns were washed three times to remove non-specific binding proteins, and phosphoproteins were eluted in 5 fractions. Aliquots of each purified fraction were examined by SDS-PAGE ( Supplementary Fig. 4). The five elution fractions from each sample were pooled and concentrated using a 10 kDa cut-off concentrator. The yield of phosphoproteins ranged from 9-15 percent of the total amount of protein loaded onto the column, and the concentrated enriched phosphoproteins were also analyzed by SDS-PAGE ( Supplementary Fig. 4). All purifications were performed independently in triplicate. Samples were then separated by 2D PAGE using 300 µg enriched phosphoproteins, and gels were stained with ProQ Diamond to detect phosphoproteins and subsequently with SYPRO Ruby to detect total proteins. Representative dual view 2-D gels are shown in Fig. 1, and individual replicate gels are provided in Supplemental materials. The majority of phosphoproteins from both mock infected and DENV 2 infected cells were focused between pI 4-7 with the protein molecular weight ranging from 17-175 kDa, while the Sypro Ruby stained proteins focused evenly between pI 3-10 ( Fig. 1), suggesting that U937 possesses more acidic phosphoproteins than basic phosphoproteins. In the mock and DENV 2 samples there was a mean of 349 and 362 phosphoprotein spots and a mean of 817 and 787 total protein spots respectively, based on the triplicated gels. Analysis of the ProQ Diamond stained gels revealed fifteen phosphoprotein spots that were differentially phosphorylated. Seven phosphoprotein spots showed increased phosphorylation, while eight phosphoprotein spots showed reduced phosphorylation. The mean percent gel volumes of the 15 differentially phosphorylated proteins are shown in Supplementary Fig. 5. In addition, the ratio between total protein and the phosphorylation signal was determined. With this analysis it was found that 14 of the phosphoprotein spots retained statistical significance ( Supplementary Fig. 6). The ratio of spot 319 was the only one that showed no significant difference, possibly due to some variation in the mock spot intensity. However, there was no phosphoprotein signal for this spot in the infected samples, suggesting significant downregulation of phosphorylation as seen in the first analysis. Total protein analysis from the SYPRO Ruby stained gels identified 11 total protein spots that were differentially expressed, with seven proteins being up-regulated, two proteins being down-regulated and two spots were found only in DENV 2 infected samples (data not shown). None of the 15 phosphoprotein spots were located at the same position as the 11 differentially expressed total protein spots indicating that the intensity differences noted in the phosphoproteins were due to differential phosphorylation alone, and not due to alterations in expression levels.
Identification of differentially expressed phosphoproteins. All 15 differentially phosphorylated phosphoprotein spots were identified by LC/MS-MS. Table 1 summarizes the phosphoproteins identified with their accession numbers, molecular weight, pI, MOWSE score, sequence coverage and their biological process. The comparison between DENV 2 infection and mock infection showed that 7 proteins, namely albumin, endoplasmic reticulum resident protein 29, mitochondrial import receptor subunit TOM34, elongation factor 1-delta, glyceraldehyde-3-phosphate dehydrogenase, protein disulfide isomerase A1 and pyruvate kinase M2 showed increased phosphorylation (more than 1.5-fold compared to the mock), while immunoglobulin light chain variable region, 14-3-3 protein gamma, cytochrome c oxidase subunit 5A, UTP-glucose-1-phosphate uridylyltransferase, profilin-2, nascent polypeptide associated complex subunit alpha, nucleophosmin and methylthioadenosine phosphorylase showed significantly reduced levels of phosphorylation in DENV 2 infected samples.

Analysis of differentially expressed phosphoproteins and validation.
To determine the association between the differentially phosphorylated phosphoproteins, the 15 identified phosphoproteins from Table 1 were subjected to a pathway analysis using the STRING (Search Tool for the Retrieval of Interacting Genes/ Proteins) database ( Fig. 2 and Supplementary Table 1). Sixty-two biological process pathways were identified, including generation of precursor metabolites and energy (11 proteins; false discovery rate 8.45e −10 ), mitochondrial electron transport, cytochrome c to oxygen (5 proteins; false discovery rate 1.90e −08 ) and ATP metabolic process (8 proteins; false discovery rate 2.48e −08 ). The overall protein-protein interaction (PPI) enrichment p-value was 1.48e −06 . In summary STRING identified phosphoproteins involved in glycolysis including mitochondrial processes.
To validate the phosphoproteomic analysis, PKM2 was selected for validation. U937 cells were mock infected or DENV 2 infected, and the levels of phosphoryation of PKM2 at Tyr 105 and Ser 37 were determined, together with the expression levels of PKM2 and GAPDH. Results (Fig. 3) showed an increase in PKM2 phosphorylation at both amino acids in DENV 2 infected samples as compared to mock infected samples, consistent with the original phosphoproteomic analysis. Similarly, consistent with the phosphoproteomic analysis, no differences were seen in expression levels of PKM2 when comparing between mock and DENV 2 infected samples.  Fig. 9) showed that neither compound had a direct virucidal activity.

Effect of PKM2 kinase inhibitor/activator on DENV 2 infection and virion production.
A timeof addition analysis was performed using the PKM2 inhibitor or the PKM2 activator to post-treat DENV 2 infected cells at 0-, 3-and 24 h p.i., in parallel with mock and vehicle treated cells. Cells were analysed by flow cytometry to determine the percentage of infection, and additionally cell viability was determined. Results ( Fig. 4A) showed that cells treated with PKM2 inhibitor at 0 and 3 h p.i., showed a significant reduction in levels of infection, with a maximal effect of a reduction of 33% being seen with treatment at 3 h p.i. Markedly, no effect was observed upon cell viability (Fig. 4A). Analysis of the supernatants of DENV 2 infected cells by plaque assay showed that the PKM2 inhibitor significantly decreased DENV 2 production by 0.331-0.442 log 10 at 0 and 3 h (Fig. 4B). In contrast, the same experiment using the PKM2 activator showed no effect on the level of DENV 2 infection or on DENV 2 production ( Supplementary Fig. 10).

Discussion
DENV infections are a significant public health problem in many parts of the world 23 , and evidence has suggested that severe manifestations are associated with second infections, particularly where the second infection is with DENV 2 or 4 4-7 . There have been several proteomic analysis of DENV infection in cell culture systems [24][25][26][27][28][29][30][31][32] , and at least one combined proteome and phosphoproteome analysis 33 , but all of these studies have been undertaken modeling primary infections. In this study, U937 cells were infected under conditions of ADE with DENV 2 to model secondary infection, and based on a phosphoproteome analysis 15 phosphoproteins were identified as significantly differentially phosphorylated. Seven proteins showed increased phosphorylation, two showed reduced phosphorylation and six proteins showed no evidence of phosphorylation in DENV 2 infected cells (highly down-regulated phosphorylation).   www.nature.com/scientificreports/ Four of the 15 identified phosphoproteins (COX5A, UGP2, GAPDH and PKM2) are involved in the generation of precursor metabolites and energy processes, suggesting a significant role for these processes in DENV infection. Two of these proteins (COX5A and UGP2) showed a loss of phosphorylation as compared to mock infected cells, while two (GAPDH and PKM2) showed increased phosphorylation in DENV 2 infected cells as compared to mock infected cells. In terms of protein selection for further verification and evaluation, PKM2 was selected as this had the highest protein score, suggesting that this protein was identified with high confidence and, in addition, commercially available antibodies to both PKM2 and phospho-PKM2 were readily available.
Two previous studies have implicated PKM2 as having a role in DENV infection. Firstly Pando-Robles and colleagues 29 identified PKM2 as being down-regulated in DENV infected hepatocyte Huh-7 cells, while it was subsequently identified as up-regulated in U937 cells infected with DENV 2 through a direct infection protocol 34 . In this study, the increased phosphorylation of PKM2 detected was not associated with a spot that showed differential expression levels. Thus, it is possible that expression of PKM2 is modulated in DENV infection in a cell type specific manner. Pyruvate kinase (PK) is a rate-limiting glycolytic enzyme that catalyze the transphosphorylation between phosphoenolpyruvate (PEP) and adenosine diphosphate, which produces pyruvate and ATP and plays a role in regulating cell metabolism 35 . There are four mammalian pyruvate kinase isoforms: liver-type PK (PKL); red blood cell PK (PKR); and PK muscle isozyme M1 and M2 (PKM1 and PKM2, respectively). Most adult tissues express PKM2, and expression of the other three isoforms is tissue-specific and regulated by various promoters and alternative splicing 36 . PKM2 has been reported to be modified by phosphorylation at many positions e.g. tyrosine, serine and threonine in response to various stimuli thereby modulating its structure and function properties 37 , and both Tyr105 and Ser37 were phosphorylated at significantly higher levels in DENV 2 infected cells than in mock infected cells. Phosphorylation of PKM2 at Y105 results in inhibition of its catalytic activity, and diversion of glycolytic flux into biosynthetic metabolism promoting the Warburg effect, a mechanism commonly found in tumor cells 38 . In DENV infection, it has been shown that glycolysis is induced and is necessary for efficient DENV replication 39 and thus it is likely that the effects of PKM2 inhibition seen here are unrelated to the glycolytic functions of PKM2.
Inhibition of PKM2 resulted in a small but significant decrease in DENV infection levels and virus output. The inhibitor used in this study functions through the inhibition of the fructose-1,6-bisphosphate-(FBP-) dependent activation of pyruvate kinase PKM2 which gives rise to an inactive tetramer and inhibits pyruvate kinase activity. This results in decreased aerobic glycolysis and PKM2 phosphorylation 40,41 . As noted, while it is possible that the inhibition of PKM2 results in the decrease of DENV infection and virus production, the fact that cellular protein levels were apparently unaffected would argue against this as a mechanism of action. While the primary function of PKM2 is involved in regulation of glycolysis in the cytosol, it is known that PKM2 can also be found in the mitochondria and nucleus. In the mitochondria it is believed that PKM2 acts to limit ROS-induced apoptosis in cancer cells 42 . DENV infection has been shown to promote increased ROS levels 43,44 , and cellular ROS levels have been shown to control antiviral processes and cell death in DENV infected cells 45 . In the nucleus PKM2 can act as a transcription factor, inducing glycolysis gene expression through c-MYC 46 as well as acting as a co-activator of the STAT5A transcription factor 47 . However while West Nile Virus and Zika virus have been shown to block STAT5 phosphorylation, DENV and yellow fever virus were shown not to block this phosphorylation 48 , and so it is unlikely that PKM2 is exerting its effect through this pathway.
PKM2 has also been shown to play a role in exosome release through phosphorylation of synaptosomalassociated protein 23 (SNAP-23) 49 . SNAP-23 is known to control the dock and release of secretory granules and exosomes. The release of DENV from host cells remains comparatively under-investigated. Studies using electron microscopy have suggested that virions are released by exocytosis 50,51 and a more recent study using correlative scanning-transmission electron microscopy suggested that chimeric flavivirus virus particles were released as individual particles in small exocytosis vesicles 52 . The studies using electron microscopy are supported by a study that identified exocyst complex component 7 (EXOC7 or EX070), a part of the exocyst complex that regulates vesicular trafficking and the late stages of exocytosis, as necessary for virus egress 53 . Thus it is possible that inhibition of PKM2 reduces virus egress, resulting in a reduction in the number of infected cells and reduced titer. Given that this does not affect virus translation, this would be consistent with the results of the western blotting in which no reduction of viral protein expression was observed. Although the number of infected cells is reduced as egress is diminished, the level of protein per infected cell would be higher, resulting in no net overall change.
Overall, this project has identified a number of phosphoproteins that are differentially phosphorylated in response to DENV 2 infection. These proteins are involved in a number of processes including generation of precursor metabolites and energy. One protein, PKM2 was validated and inhibition of phosphorylation was shown to affect level of infection and virus titer. It is possible that this effect results from modulation of virus egress. While the effect of inhibition was relatively modest, the results highlight the need for a greater understanding of the role of phosphoproteins in DENV infection, and that studies on the exocytic release of DENV are particularly required. cells were cultured and DENV 2 (strain16681) propagated and virus titer determined exactly as previously described 22 .

Materials and methods cells and viruses.
Standard plaque assay. For quantification of virus titer standard plaque assay was undertaken in, LLC-MK2 cells exactly as previously described 22  was tenfold serially diluted from 10 -1 -10 -6 with RPMI 1640 in triplicate. A control tube without the presence of the antibody was also set up. DENV 2 virus was prepared at an MOI of 20 in RPMI and placed on ice. To form virus-antibody complexes, each antibody dilution was incubated with DENV 2 in 0.5 ml tubes at 4 °C for 1 h by gently inverting the tubes every 20 min. The final dilution of antibodies ranged from 1/20 to 1/2,000,000. U937 cells in RPMI medium containing no FBS were seeded as 5 × 10 5 cells/well in 6-well plates. ADE infection was induced by adding the DENV 2-immune complex to U937 cells and incubating for 2 h at 37 °C with 5% CO 2 with rocking of the plates every 30 min. After 2 h incubation, complete RPMI 1640 with FBS was added to the cells to give a final cell density of 3.

Quantification of DENV 2 infected U937 cells by flow cytometry. Quantification of infection was
undertaken by flow cytometry exactly as previously described 22 , except that analysis was undertaken on a Cyan ADP 9-color flow cytometer (Beckman Coulter, Brea, CA) and analysis was performed using Kaluza software (Beckman Coulter, Brea, CA). All experiments were undertaken independently in duplicate. Infected cells were gated as M2.

Phosphoprotein preparation for 2-D electrophoresis. A Pierce phosphoprotein enrichment kit
(Thermo Fisher Scientific Inc., Waltham, MA) was employed for the enrichment of phosphoproteins. Briefly, U937 cell pellets were resuspended in lysis/binding/wash buffer with CHAPS, 1X Halt protease inhibitor EDTA free (Thermo Fisher Scientific Inc., Waltham, MA) and 1X Halt phosphatase inhibitor cocktail (Thermo Fisher Scientific Inc., Waltham, MA). The cell suspensions were sonicated intermittently, centrifuged and the soluble protein fractions were collected. Approximately 3.7-4.0 mg of protein from each U937 cell lysate was applied to the phosphoprotein affinity column. The samples were incubated on the column for 30 min at 4 °C and washed with lysis/binding/wash buffer to remove non-specific binding proteins. Phosphoproteins were eluted with 5 ml of elution buffer (75 mM sodium phosphate, 500 mM sodium chloride; pH 7.5) and concentrated. The protein concentration for each sample was determined using the Bradford Protein assay (Bio-Rad Laboratories, Hercules, CA). Phosphoprotein-enriched samples were stored at − 80 °C until required.

2-D electrophoresis.
Two-dimensional gel electrophoresis separation of 300 μg of enriched phosphoproteins was performed independently in triplicate as described previously 54 . protein visualization. After separation, the gels were stained with Pro-Q Diamond phosphoprotein gel stain (Molecular Probes, Inc., Eugene, OR). Briefly, the gels were fixed in 50% methanol and 10% acetic acid and then washed with ultrapure water. The gels were stained in Pro-Q Diamond phosphoprotein gel stain with gentle agitation in the dark for 90 min. After staining, the gels were destained in 20% acetronitrile, 50 mM sodium acetate, pH4.0 for 1.5 h at room temperature and protected from light. The fluorescent spot images were acquired using a Typhoon Trio (GE Healthcare, Buckinghamshire, UK) with a 532 nm laser for excitation and a 580 nm filter for emission. Subsequently, the gels were washed and stained with SYPRO Ruby Protein gel stains (Bio-Rad Laboratories, Hercules, CA) overnight in the dark. The gels were washed with 10% methanol and 7% acetic acid. Fluorescence-stained proteins were then visualized using the scanner with a 532 nm laser and a 610 nm band pass filter. To cut out putative phosphoproteins for protein identification, all gels were stained with Coomasie G-250. The resulting spot pattern coincided with that of SYPRO Ruby staining therefore, the information from the Pro-Q Diamond/ SYPRO Ruby superimposed views were used to define the positions of the phosphoproteins in the Coomasie-stained gels.

Protein identification and liquid chromatography-mass spectrometry analysis. Protein spots
showing differential phosphorylation were removed and subjected to in-gel tryptic digestion and mass spectroscopic analysis essentially as described previously 54 . The MS/MS spectrometry data were searched against the NCBI database using the MASCOT search engine, as described elsewhere 54  . The cells were then sonicated using a sonicator with at an amplitude level of 6 for 5 s and pulsed twice for 10 s before centrifugation at 12,000 × g for 15 min at 4 °C. Then the cell lysates were transferred to new tubes. Proteins in the lysates were concentrated using a Viva-spin 2 ultrafiltration column with a 10 kDa molecular weight cut-off (GE Healthcare, Buckinghamshire, UK) in accordance with the manufacturer's recommendations. The protein concentration was determined by the Bradford assay using a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA) according to the manufacturers instructions. Approximately 10-20 µg of concentrated proteins were resolved by SDS-PAGE and proteins were transferred to PVDF membranes. Western blot analysis for detection of host phosphoproteins was carried out using the following antibodies: an anti-phospho-PKM2 (Tyr105) rabbit monoclonal antibody (Cell Signaling, USA), an anti-phospho-PKM2 (Ser37) rabbit polyclonal antibody (GeneTex, Irvine, CA), a rabbit polyclonal anti-PKM2 antibody (Abcam, Cambridge, UK) and an anti-GAPDH (14C10) rabbit monoclonal antibody (Cell Signaling Technology, Danvers, MA). For the detection of DENV proteins, antibodies used include an anti-envelope rabbit polyclonal antibody (GeneTex, Irvine, CA), an anti-NS1 mouse monoclonal antibody (R&D systems, Minneapolis, MN) and an anti-NS5 mouse monoclonal antibody (GeneTex, Irvine, CA). The secondary antibodies used were a horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology, Dallas, TX) and an HRP conjugated rabbit anti-mouse IgG (Sigma-Aldrich, St. Louis, MO To determine the IC 50 for PKM2 inhibitor and PKM2 activator, U937 cells were incubated with various concentrations of each compound for 24 h and 48 h. before analysis of cell viability using the CellTiter 96 Aqueous One Solution Cell Proliferation assay (MTS, Promega, Madison, WI) according to the manufacturer's recommendations and measured with spectrophotometer (Spectra MR Microplate spectrophotometer, DYNEX Technologies, Chantilly, VA) at an absorbance value of 490 nm. IC 50 curves were generated using GraphPad Prism version 5.0. Percent survival (Y axis) versus log concentration of inhibitor/activator (X axis) was plotted. The IC 50 was calculated by the software. Each experiment was done independently in triplicate with duplicate analysis.
Assay for virucidal activity. Stock DENV 2 was incubated in medium containing PKM2 inhibitor or PKM2 activator in a 37ºC water bath for 1 h prior to infection of LLC-MK2 cells after which the DENV 2 titer was determined by standard plaque assay. At least three independent measurements were collected to determine the mean and SEM values.
Kinase inhibitor/activator post treatment of infected cells. In p.i. treatment studies, 100 μM PKM2 inhibitor or 20 μM PKM2 activator were added to mock or DENV 2 infected cells at 0, 3 and 24 h after infection. Cells were incubated under standard conditions until analyzed. Supernatant and cells were harvested at appropriate time points, and all experiments were performed independently in triplicate. Control experiments were undertaken using 0.1% and 0.01% DMSO for PKM2 inhibitor and PKM2 activator, respectively. Statistical analysis. The data are expressed as mean ± SEM. Proteome data analysis was undertaken using the Perseus software platform (https ://www.perse us-frame work.org) with One-sample T-test and Two-sample T-test analysis. All IC 50 , standard plaque assay, percent infected cells and western blot data were analyzed using the Graphpad Prism program version 5.0 (GraphPad Software Inc., San Diego, CA). Statistical analysis of significance was undertaken by unpaired t-test or One-Way ANOVA with Dunnett's Multiple Comparison Test including Bonferroni's multiple comparison test. P values less than 0.05 were considered statistically significant.

Data availability
All data generated or analysed during this study are included in this published article (and its Supplementary materials file).