Introduction The Warburg effect, which was first described by Warburg in the 1930s, is a metabolic rerouting used by tumor cells and cancer cells to support their high energy requirements and high rates of macromolecular synthesis [1], [2]. In cancer cells, the main hallmark of the Warburg effect is aerobic glycolysis, in which glucose consumption and lactate production are both increased even in the presence of oxygen [3]. Several other metabolic pathways are also enhanced, including the pentose phosphate pathway (PPP), amino acid metabolism and lipid homeostasis. The Warburg effect can also be induced in vitro by some vertebrate viruses, including human papillomavirus (HPV) [4]; human cytomegalovirus (HCMV) [5], [6], Kaposi's sarcoma herpesvirus (KSHV) [7] and hepatitis C virus (HCV) [8], and recently we reported an in vivo Warburg-like effect that was induced in shrimp hemocytes by the white spot syndrome virus (WSSV; genus Whispovirus, family Nimaviridae) [9]. WSSV is a large unique, complex, dsDNA virus, and in shrimp hemocytes, its complete in vivo replication cycle takes 22–24 h [9], [10]. Although over 90% of WSSV viral genes show no sequence homology to any other known genes, some of its genes are known to express at different times in its replication cycle, including the immediate early gene ie1, the early gene DNA polymerase (dna pol), the late structural protein gene vp28 and the very late DNA mimic protein gene icp11. Our previous study showed that WSSV induced the hallmarks of metabolic changes associated with the mammalian Warburg effect at the beginning of its genome replication stage (12 hours post injection [hpi]) [9]. However, since this was the first time that an invertebrate virus had been shown to produce this kind of effect, in the present paper, we look more closely at the global metabolomic and proteomic changes induced by WSSV in order to confirm that all of the interrelated metabolic effects seen in vertebrate cells are also found in the invertebrate Warburg effect. For the metabolomic study, we used liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) to identify and measure the levels of intracellular metabolites in shrimp hemocytes at 12 and 24 hpi, i.e. at the beginning and end of WSSV's genome replication cycle. For the proteomic profiling, we used label-free proteomics at the same time points. Since activation of the PI3K-Akt-mTOR signaling pathway is used by cancer cells and viruses to trigger the Warburg effect [11]–[13], and it is also required for the effective replication of vertebrate viruses [14]–[17], in the second part of this study, we use in vivo drug treatments to investigate whether WSSV also uses this signal pathway to trigger the Warburg effect. Results Global proteomic analysis of shrimp hemocytes during acute WSSV infection To understand the global changes triggered by WSSV infection, hemocytes were collected from PBS- and WSSV-injected shrimp at the genome replication stage (12 hpi) and the late stage (24 hpi) of the first WSSV replication cycle [9]. Using a label-free proteomic approach, 868 proteins were identified and quantified. Using a hierarchical clustering algorithm that grouped the shrimp samples by their protein abundance (Fig. S1), we found that WSSV-infected shrimp hemocytes had different proteomic expression patterns at 12 hpi and 24 hpi compared to the corresponding shrimp hemocytes collected from PBS-injected shrimp (Fig. S1A & S1B). No such proteomic clusters were formed by the hemocyte samples collected from PBS-injected shrimp at different time points (Fig. S1C), while two main clusters were formed by the WSSV 12 hpi and WSSV 24 hpi groups (Fig. S1D). Two of the samples, 12-WSSV#1 and 24-WSSV#2, were not assigned to the corresponding cluster, and we therefore excluded these two mis-assigned samples from our subsequent analysis. (We note, however, that even when these two samples are included, the overall protein changes are only very slightly different. Please see Table S1 to compare the results obtained with and without the inclusion of these two anomalous samples). Global metabolomic analysis of shrimp hemocytes during acute WSSV infection To further understand the cellular responses after WSSV infection, we also used a global metabolomic platform to measure the metabolic changes in shrimp during WSSV infection. In this study, LC-ESI-MS data on over 100 metabolites were collected at 12 and 24 h after WSSV- or PBS-injection. However, since we were interested primarily in host processes that are involved in the Warburg effect, we focused particularly on a limited number of important host pathways, including glycolysis, the PPP, nucleotide metabolism and the TCA cycle. Our metabolomic and proteomic data are given in Supplementary Tables S1 and S2. Changes in these pathways at 12 and 24 hpi are shown in Figure 1 and are described in more detail below. 10.1371/journal.ppat.1004196.g001 Figure 1 WSSV induces the Warburg effect in the cellular proteome and metabolome of shrimp hemocytes at the replication stage (12 hpi) but not at the late stage (24 hpi). Changes in the levels of enzymes and proteins (ellipses) and metabolites (rectangles) relative to PBS-injected controls are color-coded to represent up- (red) or down- (green) regulation. Yellow represents no change. Colorless boxes and ellipses indicate that no data was detected. Protein data were collected from 3–5 pooled samples of 5 shrimp using quantitative label-free proteomics and expressed on a logarithmic scale. Metabolomic data were collected from 5–6 pooled samples of 10 shrimp using LC-ESI/MS. Numeric values for the proteomic and metabolomic data are given in Tables S1 and S2, respectively. WSSV infection enhances glycolysis at the WSSV genome replication stage (12 hpi) In mammalian cells, the two main pathways of carbon metabolism, glycolysis and the TCA cycle, oxidize hexose sugars to form ATP and NADPH, or else convert the same sugars to precursors of nucleotides, amino acids, and lipids. In shrimp hemocytes, WSSV infection at 12 hpi has previously been shown to increase glucose consumption and lactate production in ways that resemble the Warburg effect, but details of the intracellular changes in the carbon metabolism have not yet been investigated. In WSSV-injected shrimp hemocytes at 12 hpi, there was a significant increase (p 1 and isotopic pattern> = 3) were used for the extraction of ion intensity data from the MS1 spectra. To adjust for system errors such as sample loading and intensity shift across LC-MS/MS runs, the peptide ion intensity was normalized using the Progenesis LC-MS robust mean, which was derived from the peptide log2 ratio distributions between a reference and the targeted LC-MS/MS run. The peak list generated from the qualified peptide features was used to search against a combined database that consisted of an in-house white shrimp database, the shrimp white spot syndrome virus database from NCBI, and the common Repository of Adventitious Proteins database downloaded from the Global Proteome Machine in the MASCOT 2.3 server (Matrix Science). To keep the false discovery rate below than 5% (as estimated from the target-decoy database), only peptides with a Mascot ion score greater than 17 were included for subsequent protein analysis. Proteins were automatically assigned to functional group, and only these proteins that met both of the following criteria were reported: 1) the protein had the most peptide hits within its group, and 2) the protein included at least one unique, quantifiable peptide. Protein quantitation was based on the sum of the total ion intensity of the unique peptides. Lastly, to map the results into the biological network, MetaCore network software (GeneGO) was used for pathway analysis of the expressed proteins. Using liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) to monitor the effect of Torin 1 on the metabolome of WSSV-infected shrimp In this experiment, 2 h before shrimp were challenged with WSSV or PBS, they were pretreated with PEG or Torin 1 (20 µg/g shrimp) by intramuscular injection to produce a total of four experimental groups: the PEG-PBS group, the PEG-WSSV group, the Torin 1-PBS group and the Torin 1-WSSV group. At 12 and 24 hpi, 5–6 pooled hemocyte samples (10 shrimp in each sample) were collected from each group using anticoagulant as described above. After centrifugation at 800×g for 1 min followed by washing twice with 1× PBS, the hemocytes were resuspended with 0.33× PBS and kept on ice for 10 min. The samples were then centrifuged at 10,000×g for 10 min, and 100% MeOH was added to the supernatant at a ratio 1∶ 2. After being centrifuged again at 10,000×g for 10 min, the supernatants were lyophilized, dissolved in 35 µl ddH2O and subjected to LC-ESI/MS metabolomic analysis as follows: To enhance the detection of the carboxylic acid and organic phosphate signals, 5 µl aniline/HCl reaction buffer (0.3 M aniline [Sigma-Aldrich, USA] in 60 mM HCl) and 5 µl of 20 mg/ml N - (3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC; Sigma-Aldrich, USA) were added to each sample of the hemocyte residue. Each mixture was vortexed and incubated at 25°C for 2 h, after which the reaction was stopped by adding 5 µl of 10% ammonium hydroxide. The aniline derivatized samples were then analyzed using an LC-ESI-MS system consisting of an ultra-performance liquid chromatography (UPLC) system (Ultimate3000 RSLC, Dionex) and a quadrupole time-of-flight (TOF) mass spectrometer with an the electrospray ionization (ESI) source (maXis UHR-QToF system, Bruker Daltonics). The shrimp metabolites were separated by reversed-phase liquid chromatography (RPLC) on a BEH C18 column (2.1×100 mm, Walters). The LC parameters were as follows: autosampler temperature, 4°C; injection volume, 10 µl; and flow rate, 0.4 ml/min. After pre-starting with 1% mobile phase B (0.1% formic acid in ACN) for 4 min, the elution started from 99% mobile phase A (0.1% formic acid in ddH2O) and 1% mobile phase B (0.1% formic acid in ACN). After holding at 1% for 0.5 min and raising to 60% over 5 min, mobile phase B was further raised to 90% in another 0.5 min, held at 90% for 1.5 min, and then lowered back to 1% in 0.5 min. The column was then equilibrated by pumping 99% B for 4 min. The acquisition parameters for LC-ESI-MS chromatograms were as follows: dry gas temperature, 190°C; dry gas flow rate, 8 L/min; nebulizer gas, 1.4 bar and capillary voltage, 3,500 V. Mass spectra were recorded from m/z 100–1000 in the negative ion mode. Data were acquired by HyStar and micrOTOF control software (Bruker Daltonics) and processed by DataAnalysis and TargetAnalysis software (Bruker Daltonics). Each metabolite was identified by matching with its theoretical m/z value and with the isotope pattern derived from its chemical formula. The identified metabolites were quantified by summing the corresponding area of the extracted ion chromatogram, and metabolite signal levels were presented as the mean of the 5–6 pooled hemocyte samples from each experimental group at each time point. To investigate the WSSV-induced metabolic changes in shrimp hemocytes, the fold changes in the PEG-WSSV group were calculated relative to the PBS injection group (PEG-PBS group). To investigate the WSSV-induced metabolic changes in the mTOR-inactivated shrimp, the fold changes in the Torin 1-WSSV group were calculated relative to the PBS injection group (Torin 1-PBS group). Lastly, the effect of Torin 1 pretreatment was shown by calculating the fold changes of the Torin 1-PBS group relative to the PEG pretreatment group (PEG-PBS group). Student's t-test was used to identify statistically significant changes. In vivo knock-down of LvRheb expression by dsRNA-mediated RNA interference Preparation of the dsRNA was done following Wang et al [44]. Briefly, first, the partial sequences (approximately 300–400 bp) of LvRheb and EGFP were generated and amplified by PCR for use as linearizing DNA templates. Next, the T7 promoter sequence was incorporated into these linearized DNA templates by using PCR with the following specific primer sets: Experimental group: LvRheb-dsT7F531/LvRheb-R882 and LvRheb-F531/LvRheb-dsT7R882; Control group: EGFP-dsT7F/EGFP-dsR and EGFP-dsT7R/EGFP-dsF (see Table S3 for details). The T7 RiboMAX Express large-scale RNA production system (Promega) was then used to synthesize the ssRNAs according to the manufacturer's instructions. The corresponding ssRNAs were mixed and annealed to become dsRNA by incubation at 70°C for 20 min, followed by slowly cooling to room temperature for 30 min. After purification and precipitation of the dsRNA by phenol/chloroform/isoamyl alcohol extraction, the dsRNA were quantified by UV spectrophotometer and verified by agarose gel electrophoresis. The final dsRNA products were stored at −80°C before being used in the following in vivo experiments. For the gene silencing experiments, the experimental group was injected with LvRheb dsRNA (1 µg/g shrimp), while the control groups were injected with EGFP dsRNA or PBS only. To determine the efficiency of the gene silencing for pooled hemocytes samples (3 shrimp in each pool sample) were collected from each group at the indicated time points. Total RNA was extracted from these samples, and cDNA was synthesized using Superscriptase II Reverse Transcriptase (Invitrogen) with Anchor-dTv primer (Table S3). Real-time PCRs were then performed to measure the expression levels of LvRheb and EF1-α with the following specific primer sets: LvRheb-qF/LvRheb-qR and EF1-α-qF/EF1-α-qR. In uninfected shrimp, gene silencing was maximally effective at 3 days after dsRNA injection (data not shown). In subsequent experiments, the shrimp were therefore challenged at 3 days post dsRNA injection. Quantification of WSSV gene (IE1 and VP28) expression in LvRheb-knockdown shrimp For this knockdown experiment, shrimp were randomly divided into 3 groups and injected with LvRheb dsRNA, EFGP dsRNA, or PBS. At 3 days post dsRNA injection, shrimp were then challenged with WSSV. Four pooled hemocyte samples were collected from each group at various time points (12, 24, 36, and 48 hpi), with each pooled sample taken from 3 shrimp. Total cDNA was then prepared from each sample as described above. To quantify the relative expression of the WSSV ie1 and vp28 genes, real-time PCR was performed with the specific primers IE1-qF/IE1-qR, VP28-qF/VP28-qR, and EF1-α-qF/EF1-α-qR (Table S3) using the Bio-Rad detection system with Brilliant SYBR Green QPCR master mix (Applied Biosystems). Data values were calculated by the 2−ΔΔCT method. Statistically significant differences between groups were analyzed by Student's t-test. Quantification of the WSSV genome copy number in LvRheb-knockdown shrimp Four pleopod samples (3 shrimp in each sample) were also collected from of the above experimental groups at the same time points. The samples were subjected to genomic DNA extraction using a DTAB/CTAB DNA extraction kit (GeneReach Biotechnology Corp.). WSSV genomic DNA copies were quantified using IQ Real WSSV quantitative system (GeneReach Biotechnology Corp.), which is a commercial real-time PCR based on the TaqMan assay. Determination of the concentration of hemolymph lactate in shrimp pretreated with Rapamycin, Torin 1, LY294002 and MK2206 after WSSV infection At 12 and 24 h post WSSV injection, 4–5 hemolymph samples (3 shrimp in each sample) were collected from groups of shrimp pretreated with LY294002, MK2206, Rapamycin, Torin 1 or PEG/PBS (control) without using anticoagulant. After being kept at 4°C for 12–16 hours, the samples were centrifuged at 13000×g for 15 min at 4°C, and the supernatants were transferred to new tubes. The concentration of glucose and lactate in the supernatants was then determined using enzymatic colorimetric test kits (Fortress Diagnostics Limited). Relative quantification of the expression of WSSV genes in shrimp pretreated with Rapamycin, Torin 1, LY294002, MK2206 and BKM120 After total hemocyte cDNA was prepared from all samples as described above, real-time PCR was performed with the specific primer sets IE1-qF/IE1-qR, DNApol-qF/DNApol-qR, VP28-qF/VP28-qR, ICP11-qF/ICP11-qR and EF1-α-qF/EF1-α-qR (Table S3) using the Bio-Rad detection system with Brilliant SYBR Green QPCR master mix (Applied Biosystems). Data values were calculated and presented as described above. Student's t-test was used to statistically analyze the Rapamycin and Torin 1 results. The LY294002 experiments used Tukey's multiple-comparison test (SPSS computer software) to evaluate statitiscally significant differences between experiemtnal groups. Quantification of WSSV genome copy number in shrimp pretreated with Rapamycin, Torin 1, LY294002 and MK2206 Genomic DNA was extracted from pleopod samples, and the number of WSSV genomic DNA copies was quantified by the IQ Real WSSV quantitative system (GeneReach Biotechnology Corp.) as described above. Data values were calculated, presented and statistically analyzed as described above. Protein extraction and western blot analysis Shrimp gill tissues were lysed in 0.33× PBS with protein inhibitor and phosphatase inhibitor (Roche). Protein concentrations in each lysate were measured by Bio-Rad Protein Assay. Approximately 25 µg of protein lysate per sample were separated by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto polyvinylidene fluoride (PDVF) membranes, blocked with 1–3% skim milk in Tris-buffered saline with 0.1% Tween 20 (TBST) for 1 hour at room temperature, and then incubated overnight in primary antibody in TBST at 4°C. Following three extensive washes with TBST, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz) for 1 hour at room temperature. After three more washes with TBST, the signals were developed by ECL detection agents (Amersham) and detected using chemiluminescence (Image Quant LAS 4000 mini). Supporting Information Figure S1 Hierarchical K-means Clustering of ∼800 hemocyte protein expression profiles obtained from a large-scale, high-throughput, label-free, quantitative LC-MS/MS analysis. (A), (B) At 12 hours and 24 hours post injection, the protein profiles of the PBS and WSSV groups formed two distinct clusters based on their log2 protein abundance, suggesting that the host cell protein pattern was markedly changed after WSSV infection at both time points. (C) Although there was no significant difference between the PBS groups at 12 and 24 hpi, the protein profiles of (D) the WSSV groups at 12 and 24 hpi formed two distinct clades, indicating that the host responses were different after WSSV infection at 12 and 24 hpi. Two of the samples, 12-WSSV#1 and 24-WSSV#2, were not assigned to the corresponding cluster, and we therefore excluded these two mis-assigned samples from our subsequent analysis. (TIF) Click here for additional data file. Figure S2 Proteomic data suggests that the mTOR pathway is activated at the replication stage (12 hpi) of WSSV infection. (A) Changes in the levels of enzymes and proteins (ellipses) relative to PBS-injected controls are color-coded to represent up- (red) or down- (green) regulation. Yellow represents no change. Colorless ellipses indicate that no data was detected. (B) WSSV-induced phosphorylation of 4E-BP1 was still detected even after Rheb was knocked down by Rheb dsRNA. Each lane shows the results for a pooled sample (n = 3) of total protein extracted from gills and probes with antibodies against 4E-BP1-PT37/46, ICP11 and actin. (C) WSSV-induced phosphorylation of 4E-BP1 was suppressed by pretreatment with the inhibitor LY294002. Each lane shows the result for a pooled sample (n = 3) of total protein subjected to Western blotting with antibodies against 4E-BP1-PT37/46 and actin. (D) WSSV replication was significantly reduced by specifically suppressing using pretreatment with 0.625 µg/g shrimp of the selective pan-class I PI3K inhibitor BKM120 [45]. Data represent the mean ± SD of five pooled samples with each sample being taken from three different shrimp. (TIF) Click here for additional data file. Figure S3 In Torin 1-pretreated shrimp, the Warburg effect was not seen either at 24 hpi in WSSV-infected shrimp or at 12∼24 hpi in PBS-injected shrimp. (A) Two hours after treatment with Torin 1, shrimp were injected with PBS or a WSSV inoculum. At 24 hpi, 6 pooled hemocytes samples (10 shrimp per pool) were collected from each group. Changes in the metabolomic levels of the WSSV-infected samples relative to the PBS controls are color-coded as described in Figure 1. Numerical data for 24 hpi is given in Table S2. (B) Effect of Torin 1 pretreatment at 12 and 24 h post PBS injection. The metabolic intermediates in Torin 1-pretreated shrimps injected with PBS were either down-regulated or remained unchanged. Changes in the metabolome for Torin 1-PBS versus PEG-PBS at 12 hpi and 24 hpi are shown in color-coded boxes as described in Figure 1, with numerical data given in Table S2. (TIF) Click here for additional data file. Table S1 Global changes in the shrimp hemocyte proteome after WSSV infection. (DOCX) Click here for additional data file. Table S2 Global changes in the shrimp hemocyte metabolome after WSSV infection. (DOCX) Click here for additional data file. Table S3 PCR primers used in this study. (DOCX) Click here for additional data file.
