Enteric coronavirus PDCoV evokes a non-Warburg effect by hijacking pyruvic acid as a metabolic hub

Abstract

The Warburg effect, also referred as aerobic glycolysis, is a common metabolic program during viral infection. Through targeted metabolomics combined with biochemical experiments and various cell models, we investigated the central carbon metabolism (CCM) profiles of cells infected with porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus with zoonotic potential. We found that PDCoV infection required glycolysis but decreased glycolytic flux, exhibiting a non-Warburg effect characterized by pyruvic acid accumulation. Mechanistically, PDCoV enhanced pyruvate kinase activity to promote pyruvic acid anabolism, a process that generates pyruvic acid with concomitant ATP production. PDCoV also hijacked pyruvic acid catabolism to increase biosynthesis of non-essential amino acids (NEAAs), suggesting that pyruvic acid is an essential hub for PDCoV to scavenge host energy and metabolites. Furthermore, PDCoV facilitated glutaminolysis to promote the synthesis of NEAA and pyrimidines for optimal proliferation. Our work supports a novel CCM model after viral infection and provides potential anti-PDCoV drug targets.

1. Introduction

Porcine deltacoronavirus (PDCoV) is an emerging porcine enteropathogenic coronavirus that belongs to the newly identified genus Deltacoronavirus within the family Coronaviridae [1]. Clinical symptoms of PDCoV infection mainly include acute diarrhea, vomiting, dehydration, and death in piglets [2]. PDCoV was first detected in porcine rectal swab samples in Hong Kong in 2012, followed by an initial outbreak involving several pig farms in Ohio (United States) in 2014 [3]. Thus far, it has been detected in many countries and regions (e.g., mainland China, South Korea, Thailand, Japan, and the Lao People's Democratic Republic); it has caused significant economic loss within the swine industry [3]. In addition to pigs, PDCoV reportedly can infect calves, turkeys, chickens, and mice [4]. A recent study showed that PDCoV was present in plasma samples of three Haitian children with acute undifferentiated febrile illness, suggesting that it has cross-species transmission and zoonotic potential [5]. Thus, PDCoV may be the eighth coronavirus with the ability to infect humans [6]. The serious threat that PDCoV poses to human and animal health has attracted substantial attention. However, its mechanisms of infection and pathogenesis remain largely unclear.

Viruses are specialized cellular parasites that rely on host cells for the energy and metabolite precursors necessary to produce progeny viruses [7]. Thus, it is not surprising that viruses have evolved multiple mechanisms to remodel host metabolic systems for optimal infection [8]. An understanding of the mechanisms by which viruses regulate and/or hijack host metabolism can provide insights to support the prevention and control of viral diseases. Central carbon metabolism (CCM)—typically regarded as glycolysis, the pentose phosphate pathway (PPP), and the tricarboxylic acid (TCA) cycle—is an essential source of energy and precursors for metabolite synthesis (e.g., amino acids, nucleotides, and lipids). Many viruses, such as dengue virus (DENV) [9], hepatitis C virus (HCV) [10], influenza viruses [11], and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), cause an increase in glycolytic flux, indicated by enhanced glucose uptake and increased lactic acid secretion despite sufficient energy and oxygen [12]. This process is known as aerobic glycolysis or the Warburg effect. The Warburg effect arises from the rapid conversion of pyruvic acid to lactic acid via lactate dehydrogenase A (LDHA) during mitochondrial injury. Through this mechanism, cells preferentially acquire adenosine triphosphate (ATP) via glycolysis instead of oxidative phosphorylation (OXPHOS) when the TCA cycle is disrupted [13,14]. However, some viruses (e.g., human cytomegalovirus [15] and African swine fever virus [16]) enhance glycolysis while increasing TCA cycle flux. Thus, viruses display diverse modes of CCM regulation.

Glutamine (Gln), the second major carbon source after glucose, also serves as an important carbon source for CCM. In addition to its role in the TCA cycle, Gln is an important donor for amino acid synthesis; it also provides a direct source of nitrogen for nucleotide biosynthesis. Many viruses utilize Gln to promote infection through various mechanisms [8]. For example, both porcine reproductive and respiratory syndrome virus [17] and nervous necrosis virus [18] promote glutaminolysis to replenish the TCA cycle for proliferation. Kaposi's sarcoma-associated herpesvirus (KSHV) utilizes glutaminolysis to promote the synthesis of non-essential amino acids (NEAAs), thereby facilitating viral proliferation [19]. Additionally, KSHV [19] and enterovirus 71 (EV71) [20] promote nucleotide de novo synthesis by accelerating glutaminolysis, which enhances their proliferation.

To our knowledge, interactions between PDCoV and host metabolic systems have not been reported. Here, we conducted a central carbon-targeted metabolomic analysis of PDCoV-infected cells, which for the first time revealed that PDCoV infection caused a non-Warburg effect characterized by pyruvic acid accumulation. Notably, PDCoV accelerated pyruvate kinase (PK)-mediated pyruvic acid anabolism to induce an energy transfer from OXPHOS to glycolysis; it also increased pyruvate carboxylase (PC)-catalyzed pyruvic acid catabolism to provide metabolite precursors. Moreover, PDCoV promoted glutaminolysis to enhance the synthesis of NEAAs and pyrimidines. Overall, we identified unique CCM signatures in PDCoV-infected cells, and our results will contribute to the development of metabolism-focused anti-PDCoV strategies.

2. Methods

2.1. Reagents and antibodies

The 2-(7-nitro-2,1,3-benzoxadiazol-4-yl)-D-glucosamine (2-NBDG; HY-116215), 2-Deoxy-d-glucose (2-DG; HY-13966), pyruvic acid (HY-Y0781), Gln (HY-N0390), BAY-876 (HY-100017), PKM2-IN-1 (HY-103617), UK5099 (HY-15475), galloflavin (HY-W040118), rotenone (HY-B1756), oligomycin (HY-16589), oxoglutaric acid (αKG; HY-W013636), CB839 (HY-12248), aspartic acid (Asp; HY-N0666), 6-TG (HY-13765), Brequinar (HY-108325), Aminooxyacetic acid (AOA; HY-107994), glutamic acid (Glu; HY-14608), serine (Ser; HY-N0650), and glycine (Gly; HY-Y0966) were purchased from MedChemExpress (MCE; China). The fetal bovine serum was purchased from OPCEL (China). The galactose (G0750), CPI-613 (SML0404), and nucleosides (ES-008-D) were obtained from Sigma-Aldrich (USA). The glucose (T0887) and oxamate (T19831) were purchased from Topscience (China). The PAA (GC40414) and NEAA (11140050) were purchased from GLPBIO (USA) and Gibco (USA), respectively.

Rabbit polyclonal antibody (pAb) against Pyruvate kinase muscle isozyme 1 (PKM1; AB116271) and mouse monoclonal antibody (mAb) against β-actin (AB8226) were purchased from Abcam (UK). Rabbit mAbs against Carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD; T58022) and Pyruvate kinase muscle isozyme 2 (PKM2; T55764S) were acquired from Abmart (China). Rabbit pAbs against PC (PA1857S), pyruvate dehydrogenase A1 (PDHA1; PK77675S), and pyruvate dehydrogenase X (PDHX; PK42678S), and mouse mAb against VDAC1 (MK90173) were purchased from Abmart. Rabbit pAb against phosphoribosyl pyrophosphate synthetase 2 (PRPS2; A12645) was purchased from ABclonal (China). Mouse mAb against PDCoV N protein has been described previously. Alexa Fluor 488 (A23210) and Alexa Fluor 594 (A23410) conjugated goat anti-mouse IgG were purchased from Abbkine (China). HRP-conjugated goat anti-mouse IgG (A0209) and goat anti-rabbit IgG (A0208) were purchased from Beyotime (China).

2.2. Quantification of cellular metabolites by LC-MS/MS

PDCoV-infected or mock-infected IPI-2I cells were sonicated in 80% methanol aqueous solution, and supernatants were collected by centrifugation (18000 g, 15 min) at 4 °C. Next, 40 μL of supernatant, 20 μL of 3-NPH (200 mM), and 20 μL of 1-Ethyl-3(3-(dimethylamino)propyl)carbodiimide (EDC) (120 mM) were added to each well of a 96-well plate; the plate was incubated at 30 °C with a rotation speed of 1000 g for 60 min (Allsheng, China; MSC-100). Subsequently, 350 μL of iced methanol were added to each well. After centrifugation (4000 g) at 4 °C for an additional 20 min, 150 μL of supernatant were transferred to each well in a new 96-well plate. Then, sample metabolite levels were evaluated using a liquid chromatograph−mass spectrometer (LC-MS/MS; Acquity-I Xevo TQ-S, Waters Corp., USA). After metabolite extraction, samples were homogenized on ice for 5 min with radioimmunoprecipitation assay lysis buffer (Beyotime; P0013), then sonicated at 300 W (30%) for 10 min on ice. A bicinchoninic acid (BCA) protein assay kit (Beyotime; P0010) was used to measure protein concentrations. Metabolite levels were normalized according to protein concentration.

2.3. Glucose uptake assay

Cells grown to 80% confluence were infected with PDCoV. At the indicated time points, the cell culture medium was replaced with glucose-free medium. After incubation for 1 h, cells were treated for 15 min with 2-NBDG at a final concentration of 100 μM. Next, cells were collected and washed twice with phosphate-buffered saline, then analyzed via flow cytometry (Beckman, USA) using FlowJo software.

2.4. Mitochondria isolation

IPI-2I cells grown to 80% confluence were infected with PDCoV for the indicated times. After two washes with phosphate-buffered saline, cells were dispersed with trypsin and collected by centrifugation at 850g for 2 min. Approximately 1 × 10⁷ cells per sample were used for mitochondria isolation with kits purchased from Invent (China; MP-007), in accordance with the manufacturer's instructions.

2.5. Seahorse XF real-time ATP rate assay

The Seahorse XF Real-Time ATP rate assay kit (Seahorse Bioscience, USA; 103592–100) was used to examine the rates of mitochondrial ATP production and glycolytic ATP production in PDCoV-infected cells. Cells were seeded in XFe24 Cell Culture Microplates at a concentration of 20,000 cells/100 μL in each well. Seahorse XFe24 Sensor Cartridges were hydrated and placed in a non-CO₂ 37 °C incubator overnight. Cells were washed twice using XF Real-Time ATP rate Assay Medium (XF Base Medium [Seahorse Bioscience; 103575–100] with 25 mM glucose [Seahorse Bioscience; 103577–100], 1 mM pyruvic acid [Seahorse Bioscience; 103578–100], and 4 mM l-glutamine [Seahorse Bioscience; 103579–100]; pH was adjusted to 7.4 at 37 °C). Cells were covered with assay medium for a final volume of 500 μL/well, then placed in a 37 °C incubator without CO₂ for 1 h prior to the assay. During incubation, test compounds were loaded into XFe24 Sensor Cartridges: port A, oligomycin (final concentration of 1.5 μM); port B, rotenone (final concentration of 0.5 μM) and antimycin A (final concentration of 0.5 μM). Subsequently, the assay was performed on an Agilent Seahorse XFe24 device (Seahorse Bioscience); results were visualized and analyzed using Wave Desktop and Report Generator software.

2.6. Seahorse cell mitochondrial/glycolysis stress test

Seahorse XF cell mitochondrial/glycolysis stress test kits (Seahorse Bioscience; 103015–100/103020–100) were used to detect the mitochondrial respiration capacity and glycolysis capacity of IPI-2I, LLC-PK1 and Huh-7 cells. Briefly, cells were seeded in XFe24 Cell Culture Microplates and cultured overnight. In mitochondrial stress test, port A (oligomycin; final concentration of 1 μM), port B (FCCP; final concentration of 2 μM), and port C (rotenone; final concentration of 0.5 μM) were added in sequential order. In glycolysis stress test, port A (glucose; final concentration of 10 mM), port B (oligomycin; final concentration of 1 μM), and port C (2-DG; final concentration of 50 mM) were added in sequential order. Subsequently, the assays were performed on an Agilent Seahorse XFe24 device; results were visualized and analyzed using Wave Desktop and Report Generator software. Homogenization was performed by assaying the protein concentration of samples using a BCA protein assay kit.

2.7. ADP/ATP, NAD⁺/NADH ratio, and ATP amount assays

ADP/ATP ratio, NAD⁺/NADH ratio, and ATP amount were analyzed using ADP/ATP Ratio Assay Kits (Sigma-Aldrich; MAK135), NAD⁺/NADH Ratio Assay Kits (Sigma-Aldrich; MAK037), and ATP determination Kits (Invitrogen, USA; A22066) in accordance with the manufacturer's instructions.

2.8. Statistical analysis

Data are represented as mean ± standard deviation (SD). Differences between different groups were assessed using Student's t-test, one-way or two-way ANOVA. All statistical analysis were performed using the GraphPad Software (GraphPad Inc., USA).

3. Results

3.1. PDCoV infection induces significant changes in CCM

To evaluate the effects of PDCoV infection on CCM, we initially conducted viral proliferation assays. Porcine ileum epithelial (IPI–2I) cells were infected with PDCoV at a multiplicity of infection (MOI) of 0.25, 0.5, or 1. At 6, 12, 18, and 24 h post-infection (hpi), cells were harvested for TCID₅₀ assays. The results showed that both the 0.5 and 1 MOI groups exhibited higher viral titers compared to the 0.25 MOI group at 6, 12, and 18 hpi. Moreover, there was no significant difference in viral titers between 0.5 and 1 MOI groups at each time point after infection. Consequently, cells were infected with PDCoV at a MOI of 0.5 and harvested at 6, 12, and 18 hpi for targeted metabolomic analysis using liquid chromatography–mass spectrometry (LC-MS/MS). Metabolomic data stability was confirmed by partial least squares discriminant analysis (Supplementary Figs. 1A–C). Changes in CCM-associated metabolites were summarized using a heatmap (Fig. 1B) and a metabolic pathway diagram (Fig. 1C). In terms of glycolysis, fructose-1-phosphate, dihydroxyacetone phosphate, lactic acid, and 3-phosphoglyceric acid were significantly downregulated; only pyruvic acid was significantly upregulated at 12 and/or 18 hpi, suggesting that PDCoV infection disrupted glycolysis (Fig. 1D). With respect to the TCA cycle, αKG and malic acid were significantly downregulated, whereas oxalacetic acid (OAA), isocitric acid, and fumaric acid were slightly downregulated at 12 and/or 18 hpi; thus, we suspected that PDCoV infection disrupted the TCA cycle (Fig. 1E). As for the PPP, no significant changes were observed. However, the levels of Gln, Glu, and Asp were significantly reduced at 12 and/or 18 hpi, indicating that PDCoV infection altered glutamine metabolism (Fig. 1F). Collectively, the findings suggested that PDCoV infection induces broad changes in CCM.

Fig. 1.

Central carbon-targeted metabolomic analysis of PDCoV-infected IPI-2I cells. (A) IPI-2I cells were infected with PDCoV (MOI = 0.25, 0.5, or 1), then harvested at 6, 12, 18, and 24 hpi for TCID₅₀ assays (mean ± SD; n = 3). (B–F) IPI-2I cells infected or mock-infected with PDCoV (MOI = 0.5) were harvested at 6, 12, and 18 hpi and processed for metabolomic analysis using LC-MS/MS (mean ± SD; n = 6). (B) Heatmap visualization of differential cellular metabolites. (C) Schematic diagram of the central carbon metabolic network. (D–F) Significant differential metabolites in glycolysis (D), the TCA cycle (E), and Gln metabolism (F). Values are shown as mean ± SD. The experiments in (A) were repeated three times, and the experiments in (B–F) were repeated six times. Statistical analyses for (D–F) were performed using the two-way ANOVA test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

3.2. PDCoV infection decreases TCA cycle-derived energy production

Because CCM is a major source of energy, instances of CCM disruption generally are accompanied by altered energy metabolism. The upregulation of adenosine diphosphate (ADP)/ATP and oxidized vs. reduced nicotinamide adenine dinucleotide (i.e., NAD⁺/NADH) ratios indicates energy metabolism impairment and TCA cycle disruption. We found that PDCoV infection significantly decreased ATP amount and increased the ADP/ATP ratio in IPI-2I cells (Fig. 2A and B and Supplementary Figs. 2A and B). We also found that PDCoV infection upregulated the NAD⁺/NADH ratio by decreasing NADH levels without modifying NAD⁺ (Fig. 2C–E and Supplementary Figs. 2C–E). To further explore the effects of PDCoV infection on energy metabolism, we performed mitochondrial stress tests to measure real-time oxygen consumption rates (OCRs). The results showed that the OCRs of PDCoV-infected IPI-2I cells were significantly lower than the OCRs of mock-infected cells at 12 and 18 hpi, but no significant change was observed at 6 hpi (Fig. 2F and Supplementary Figs. 2F–I). Specifically, PDCoV infection led to a significant reduction in basal respiration and ATP production at 6, 12, and 18 hpi, as well as a decrease in maximal respiration at 12 and 18 hpi (Fig. 2G and Supplementary Fig. 2J). These results suggested that PDCoV infection induces mitochondrial impairment, leading to TCA cycle dysfunction accompanied by decreased energy production.

Fig. 2.

PDCoV infection causes impaired energy metabolism. IPI-2I cells were infected or mock-infected with PDCoV (MOI = 0.5). At the indicated time points after infection, cells were harvested for analyses of ATP amount (% of mock) (A), ADP/ATP ratio (B), NADH level (C), NAD⁺ level (D), ratio of NAD⁺/NADH (E), or real-time oxygen consumption rate (OCR) (F). (F) Protein concentration for homogenization was quantified using the BCA protein assay kit. (G) Analyses of basal respiration, maximal respiration, and ATP production based on OCR. Values are shown as mean ± SD. All experiments were repeated at least three times. Statistical analyses for (A–E) and (G) were performed by the two-way ANOVA test. **, p < 0.01; ***, p < 0.001; NS., not significant.

3.3. PDCoV hijacks glycolysis by increasing the proportion of glycolysis-derived ATP

Because TCA cycle impairment is usually accompanied by active glycolysis during viral infection [14,21], we investigated whether PDCoV infection enhances glycolysis. Glucose is the substrate utilized in glycolysis; and 2-NBDG, a fluorescent glucose analog, is commonly used to monitor glucose uptake [22]. Flow cytometry analysis using 2-NBDG showed that PDCoV infection did not significantly affect glucose uptake at 6 hpi, but it decreased glucose uptake at 12 and 18 hpi (Fig. 3A and B and Supplementary Fig. 3A). Lactic acid is the end product of glycolysis. We found that although there was no significant difference in the secretion of lactic acid between mock-infected and PDCoV-infected cells (Supplementary Figs. 3B and C), the metabolomic results in Fig. 1D showed a significant decrease in intracellular lactic acid levels after PDCoV infection. Based on these results, we speculated that PDCoV infection might reduce glycolytic fluxes. To further confirm our speculation, we measured the extracellular acidification rate (ECAR) in PDCoV-infected cells. The results showed that PDCoV infection downregulated ECAR (Fig. 3C and Supplementary Figs. 3D–G), as evidenced by decreases in glycolysis, glycolytic capacity, and glycolytic reserve (Fig. 3D and Supplementary Fig. 3H). Taken together, our findings indicated that PDCoV infection reduces glycolysis.

Fig. 3.

PDCoV utilizes glycolysis by increasing the proportion of glycoATP without enhancing glycolysis. (A–D) IPI-2I cells were infected or mock-infected with PDCoV (MOI = 0.5) for 6, 12, and 18 hpi. (A) Glucose uptake was determined by flow cytometry using 2-(7-nitro-2,1,3-benzoxadiazol-4-yl)-d-glucosamine (2-NBDG). (B) Histograms show quantification of glucose uptake via median fluorescence intensity. (C) Cells were harvested for real-time ECAR analysis. (D) Analyses of glycolysis, glycolytic capacity, and glycolytic reserve based on the ECAR data in (C). (E) IPI-2I cells were infected or mock-infected with PDCoV (MOI = 0.5 or 0.1) in medium containing different concentrations of glucose (0, 6.25, 12.5, or 25 mM) for 18 h. Whole-cell extracts were prepared, and viral titers were analyzed by TCID₅₀ assays. (F) Effects of 2-Deoxy-d-glucose (2-DG) treatment or galactose substitution on PDCoV titers. IPI-2I cells were pretreated with 2-DG (20 mM) for 3 h, then infected with PDCoV (MOI = 0.5). Glucose in DMEM was replaced or not replaced with galactose (25 mM). At 6, 12, and 18 hpi, cells were collected for TCID₅₀ assays. Ctrl: Control. (G–I) IPI-2I cells were infected with PDCoV (MOI = 0.5), then harvested at 6, 12, and 18 hpi. (G) Seahorse XF Real-time ATP rate analysis. (H) Energy map of mitochondrial ATP production rate (mitoATP) versus glycolytic ATP production rate (glycoATP). (I) Basal metabolism percentages of OXPHOS-derived and glycolysis-derived energy (total 100%). (C and G–I) Protein concentration for homogenization was quantified using the BCA protein assay kit. Values are shown as mean ± SD. All experiments were repeated at least three times. Statistical analyses for (B, D) and (F, G, and I) were performed by the two-way ANOVA test, and statistical analyses for (E) were performed by the one-way ANOVA test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; NS., not significant.

Next, we investigated whether glycolysis affects PDCoV proliferation. Surprisingly, we found that a decrease in glucose (the substrate utilized in glycolysis) led to reduced titers, RNA copy number, and viral protein levels of PDCoV (Fig. 3E, Supplementary Figs. 3I and 3J). Furthermore, we observed that disruption of glycolysis by treatment with 2-DG (a competitive inhibitor of Hexokinase 2 [HK2]) or the substitution of glucose with galactose suppressed PDCoV proliferation in IPI-2I cells (Fig. 3F). These results indicated that glycolysis is required for PDCoV proliferation.

Therefore, we explored how PDCoV utilizes glycolysis without upregulating this pathway. When the TCA cycle is impaired, glycolysis usually compensates for the lack of ATP production during OXPHOS [23]. Because PDCoV infection causes TCA cycle dysfunction, we investigated whether PDCoV infection increases the production of glycolysis-derived ATP (i.e., glycoATP). Real-time ATP synthesis rate assays revealed that PDCoV infection did not enhance glycoATP production in IPI-2I cells (Fig. 3G). However, an energy map comparing the mitochondrial and glycolytic ATP production rates showed a metabolic switch from OXPHOS to glycolysis (Fig. 3H). Specifically, PDCoV infection decreased the proportion of OXPHOS-derived ATP but increased the proportion of glycoATP at 12 and 18 hpi (Fig. 3I). These results indicated that PDCoV utilizes glycolysis to compensate for the ATP deficit during OXPHOS, thereby supporting viral proliferation.

We also confirmed this energy metabolism pattern using two other PDCoV-susceptible cell lines with different types of metabolic energy: LLC-PK1 (porcine kidney cells; non-transformed cells with a balance between OXPHOS and glycolysis) and Huh-7 (human hepatocellular cancer cells; tumor cells independent of glycolysis) [24]. Based on the infection kinetics of PDCoV in LLC-PK1 and Huh-7 cells (Supplementary Figs. 4A and B), changes in energy metabolism were detected in PDCoV-infected cells at 6, 12, and 24 hpi. Consistent with the findings in IPI-2I cells, analyses of LLC-PK1 and Huh-7 cells showed that PDCoV infection had no impact on lactic acid release. However, it decreased glucose uptake and ECAR (Supplementary Fig. 5 A–F); however, it downregulated both OXPHOS-derived ATP production and glycoATP production while increasing the proportion of glycoATP relative to total ATP (Supplementary Figs. 5G–J). Additionally, glycolysis disruption via glucose deficiency, galactose substitution, or 2-DG treatment inhibited PDCoV proliferation in LLC-PK1 and Huh7 cells (Supplementary Figs. 5K and L). These results indicated that there is no cellular specificity in terms of the requirement for energy metabolism by PDCoV.

3.4. PDCoV enhances PK-mediated pyruvic acid anabolism to promote infection

Pyruvic acid was the only CCM metabolite upregulated after PDCoV infection in IPI-2I cells (Fig. 1D), and PDCoV infection increased the production of pyruvic acid in both LLC-PK1 and Huh-7 cells (Supplementary Figs. 6A and B). Glycolysis is the metabolic pathway by which glucose is converted into pyruvic acid. However, PDCoV infection did not induce an increase in glucose uptake (Fig. 3A and B), suggesting that PDCoV infection-induced accumulation of pyruvic acid is not caused by an increase in intracellular glucose content. To confirm this suspicion, we utilized BAY-876, an inhibitor of glucose transporter [25], and glucose-free medium to decrease intracellular glucose content. The results showed that, regardless of reductions in intracellular glucose content, PDCoV infection induced significant upregulation of pyruvic acid without influencing lactic acid production (Fig. 4A and Supplementary Figs. 6C–E).

Fig. 4.

PDCoV activates PK to promote viral replication. (A) IPI-2I cells were pretreated with BAY-876 (50 nM) for 24 h. DMSO was used as a control. Subsequently, the cells were infected or mock-infected with PDCoV (MOI = 0.5) in the presence or absence of glucose; cells were harvested at 6, 12, and 18 hpi for the detection of pyruvic acid concentration using a kit purchased from Abbkine (KTB1121). (B) IPI-2I cells were infected or mock-infected with PDCoV (MOI = 0.5), then harvested for the detection of pyruvate kinase (PK) activity at 6, 12, and 18 hpi using a kit purchased from Abbkine (KTB1120). Protein concentration for homogenization was quantified using the BCA protein assay kit. (C) IPI-2I cells were transfected with PKM-specific siRNA (siPKM) or negative siRNA (siNC). After 24 h, cells were infected with PDCoV (MOI = 0.5 or 0.1). At 18 hpi, cells were collected for the detection of PDCoV titers by TCID₅₀ assays. (D) IPI-2I cells were pretreated with PKM2-IN-1 for 48 h, then infected with PDCoV (MOI = 0.5 or 0.1) for 18 h in the presence of PKM2-IN-1. PDCoV titers were analyzed by TCID₅₀ assays. Values are shown as mean ± SD and all data represent at least three independent experiments. Statistical analyses were performed by the two-way ANOVA test (A, B) or one-way ANOVA test (C, D). *, p < 0.05; **, p < 0.01; ***, p < 0.001; NS., not significant.

PK is a rate-limiting enzyme that catalyzes the conversion of phosphoenolpyruvate and ADP to pyruvic acid and ATP [26]. To determine how PDCoV induces pyruvic acid accumulation, we examined the effect of PDCoV infection on PK. We found that PDCoV infection caused significant upregulation of PK activity at 12 and 18 hpi (Fig. 4B), leading to accumulation of pyruvic acid and increased production of ATP. We performed further analyses of the effect of PK on PDCoV proliferation. PKM, consisting of PKM1 and PKM2, is a critical active component of PK [27]. Using PKM-specific small interfering RNA (siPKM) or a PKM inhibitor (PKM2-IN-1) [28], we demonstrated that downregulation of PKM expression and activity significantly inhibited PDCoV proliferation (Fig. 4C and D and Supplementary Fig. 6F). Taken together, our results indicated that PDCoV infection enhances PK activity to facilitate pyruvic acid anabolism, thereby promoting viral proliferation.

3.5. PDCoV enhances pyruvic acid catabolism to OAA to promote proliferation

We further investigated whether pyruvic acid catabolism is also regulated and utilized by PDCoV. Pyruvic acid catabolism occurs in the cytoplasm and mitochondria. In the cytoplasm outside of the mitochondria, pyruvic acid can be converted to lactic acid via LDHA catalysis [29] or to alanine via glutamic-pyruvate transaminase 2 (GPT2) catalysis [30]. To antagonize pyruvic acid catabolism in the cytoplasm, IPI-2I cells were treated with the LDHA inhibitors galloflavin and oxamate or transfected with GPT2-specific siRNA (siGPT2). The results showed that both galloflavin and oxamate significantly reduced PDCoV titer (Fig. 5A and B), whereas siGPT2-mediated GPT2 knockdown did not have a significant inhibitory effect on PDCoV titer (Fig. 5C and Supplementary Fig. 7A); therefore, pyruvic acid catabolism to lactic acid (rather than alanine) promoted the proliferation of PDCoV. However, PDCoV infection did not enhance lactic acid secretion (Fig. 3B), indicating that PDCoV could not augment pyruvic acid catabolism to lactic acid to increase viral proliferation.

Fig. 5.

PDCoV enhances mitochondrial pyruvic acid catabolism to OAA to promote viral proliferation. (A–G) IPI-2I cells were pretreated with galloflavin (A) for 6 h, oxamate (B) for 2 h, UK5099 (D) for 12 h, phenylacetic acid (PAA) (E) for 24 h, CPI-613 (G) for 12 h, or transfected with GPT2-specific siRNA (siGPT2) (C) or pyruvate carboxylase (PC)-specific siRNA (siPC) (F) for 24 h. The cells were subsequently infected with PDCoV (MOI = 0.5 or 0.1) for 18 h. Whole-cell extracts were prepared, and viral titers were detected by TCID₅₀ assays. (H) Schematic diagram of mitochondrial pyruvic acid catabolism (left) and the effects of PDCoV infection on expression of mitochondrial PC, PDHA1, and PDHX (right). Mitochondria were isolated from IPI-2I cells infected or mock-infected with PDCoV (MOI = 0.5) at 6, 12, and 18 hpi for western blotting. The band intensities were analyzed using Image J software. VDAC1 and β-actin were used as loading controls for mitochondria and cytoplasm, respectively. The quantification of the PC/β-actin ratio, PDHA1/β-actin ratio, and PDHX/β-actin ratio is presented. (I, J) IPI-2I cells were infected or mock-infected with PDCoV (MOI = 0.5). At 12 hpi, mitochondria and cytoplasm were separated for the detection of pyruvate dehydrogenase (PDH) (I) and PC (J) enzyme activities using assay kits purchased from Abbkine (KTB1270) and Solarbio (BC0735), respectively. Protein concentration for homogenization was quantified using the BCA protein assay kit. Values in (A–G, I, J) are shown as mean ± SD and all data represent three independent experiments. Statistical analyses were performed by the one-way ANOVA test (A–G) or unpaired two-tailed Student's t-test (I, J). *, p < 0.05; **, p < 0.01; ***, p < 0.001; NS., not significant. (K) Schematic diagram illustrating the impact of PDCoV on pyruvic acid metabolism.

In mitochondria, pyruvic acid mainly participates in the PDH-catalyzed synthesis of acetyl coenzyme A (Ac-CoA) and the PC-catalyzed synthesis of OAA [31]. To investigate whether mitochondrial pyruvic acid catabolism modulates PDCoV infection, we used UK5099 (an inhibitor of pyruvic acid transport from cytoplasm to mitochondria), CPI-613 (a PDH inhibitor), phenylacetic acid (PAA; a PC inhibitor), and PC-specific siRNA (siPC) to disrupt mitochondrial pyruvic acid catabolism. The results showed that UK5099, CPI-613, PAA, and siPC inhibited PDCoV proliferation (Fig. 5D–G and Supplementary Fig. 7B), indicating that mitochondrial pyruvic acid catabolism has a beneficial effect on PDCoV infection.

Subsequently, we examined how PDCoV regulates mitochondrial pyruvic acid catabolism. PDH is a multienzyme complex composed of PDHA1, PDHB, and PDHX [32,33]. We found that in mitochondria, PDCoV infection exerted no obvious effects on the protein expression levels of PC, PDHA1, and PDHX (Fig. 5H), but significantly reduced PDH enzyme activity (Fig. 5I) and upregulated PC enzyme activity (Fig. 5J). Considering that in mitochondria, PDH mainly catalyzes the conversion of pyruvic acid to Ac-CoA, and PC primarily catalyzes the conversion of pyruvic acid to OAA, we proposed that PDCoV infection promoted the catabolism of pyruvic acid to OAA, rather than Ac-CoA, to support viral proliferation (Fig. 5K).

3.6. PDCoV utilizes glutaminolysis to acquire Glu and Asp for viral proliferation

Along with glucose, Gln is a key carbon source in CCM that provides energy and participates in biosynthesis [34]. Our study has demonstrated that PDCoV infection led to a downregulation of Gln (Fig. 1F), but whether PDCoV infection promotes Gln catabolism (i.e., glutaminolysis) remains unclear. Therefore, we first detected the effects of PDCoV infection on key enzymes involved in glutaminolysis. The results showed that PDCoV infection upregulated glutaminase (GLS), glutamate dehydrogenase (GLUD1), asparagine synthase (ASNS), and glutamic oxaloacetic transaminase 1/2 (GOT1/2) at 6, 12, and/or 18 hpi (Fig. 6A). Furthermore, we measured the changes in enzyme activity of GLS, the first enzyme in glutaminolysis, after PDCoV infection. Our findings revealed that PDCoV infection significantly increased the enzyme activity of GLS at 12 and 18 hpi (Fig. 6B). We also observed a dose-dependent upregulation of GLS enzyme activity at 12 hpi during PDCoV infection (Supplementary Fig. 8A). Collectively, these results suggested that PDCoV infection facilitated glutaminolysis. Additionally, we found that Gln upregulated PDCoV titer in a dose-dependent manner (Fig. 6C), we thus speculated that PDCoV enhances glutaminolysis to promote viral proliferation. Gln is mainly converted to Glu, through a reaction catalyzed by GLS, for participation in CCM. We observed that CB839 (a GLS inhibitor) inhibited PDCoV proliferation in a dose-dependent manner (Fig. 6D), indicating that Gln catabolism to Glu was beneficial for PDCoV infection. Furthermore, PDCoV infection led to Glu downregulation (Fig. 1F), implying that PDCoV also promotes Glu catabolism to facilitate viral proliferation. Glu participates in αKG biosynthesis via GLUD1 or in NEAA biosynthesis through aminotransferase. Although αKG did not reverse the decrease in PDCoV titer caused by Gln deprivation (Fig. 6E), AOA (an aminotransferase inhibitor) inhibited PDCoV infection (Fig. 6F), suggesting that the conversion of Glu to NEAA (rather than αKG) was beneficial for PDCoV proliferation.

Fig. 6.

Gln catabolism to Glu and Asp is beneficial for PDCoV infection. (A–B) IPI-2I cells were infected with PDCoV (MOI = 0.5), then harvested at 6, 12, and 18 hpi. The mRNA levels of key enzymes involved in glutaminolysis (GLS, GLUD1, ASNS, GOT1, and GOT2) and the enzyme activity of GLS were detected by RT-qPCR (A) and GLS enzyme activity assay kits (Solarbio; BC1455) (B). Protein concentration for homogenization in (B) was quantified using the BCA protein assay kit. (C–F) IPI-2I cells were infected with PDCoV (MOI = 0.5 or 0.1) for 18 h in the indicated media and PDCoV titers were detected by TCID₅₀ assays. (C) Cells were infected with PDCoV in media containing different concentrations of glutamine (0, 1, 2, or 4 mM). (D) Cells were pretreated with CB839 (0, 0.5, or 2 μM) for 12 h, then infected with PDCoV (MOI = 0.5). (E) Cells were infected with PDCoV in Gln-containing medium, Gln-free medium, or Gln-free medium supplemented with oxoglutaric acid (αKG) (1 or 4 mM). (F) Cells were pretreated with Aminooxyacetic acid (AOA) (0, 0.5, or 2 mM) for 24 h, then infected with PDCoV (MOI = 0.5) in the presence of AOA. (G) IPI-2I cells were transfected with vector or the eukaryotic expression plasmid pCAGGS-Flag-SLC1A3, then infected with PDCoV (MOI = 0.5) in Gln-containing medium, Gln-free medium, or Gln-free medium supplemented with Asp (4 mM) or Glu (4 mM). Values are shown as mean ± SD and all data represent three independent experiments. Statistical analyses were performed by the two-way ANOVA test (A, B, and G) or one-way ANOVA test (C–F). *, p < 0.05; **, p < 0.01; ***, p < 0.001; NS., not significant. (H) Schematic diagram illustrating the impact of PDCoV on glutaminolysis.

To confirm that the products of glutaminolysis during PDCoV infection were utilized for NEAA biosynthesis, we exogenously added an NEAA mixture or single NEAAs (Asp, Glu, Ser, and Gly) to Gln-free medium. Surprisingly, the result showed that Gln deprivation significantly decreased the titer of PDCoV; however, neither the NEAA mixture nor single NEAAs could rescue the decrease in PDCoV titer caused by Gln deprivation (Supplementary Fig. 8B). Next, we investigated why the inhibition of NEAA biosynthesis by AOA antagonized PDCoV proliferation, but exogenous addition of the NEAA did not promote PDCoV infection. Acidic amino acid transporters are inert, and their efficiencies are considerably lower than the efficiencies of alkaline and neutral amino acid transporters [35]. To exclude the effect of inefficient acidic amino acid transport (i.e., ensure sufficient uptake of acidic amino acids), we constructed an overexpression system for SLC1A3, the main transporter of acidic amino acids [36]. The results showed that two acidic amino acids, Asp and Glu, significantly reversed the decrease in PDCoV titer caused by Gln deprivation in cells overexpressing SLC1A3 (Fig. 6G and Supplementary Fig. 8C), indicating that PDCoV requires Gln-derived Asp and Glu for viral infection (Fig. 6H).

3.7. Disruption of Gln-derived pyrimidine synthesis inhibits PDCoV infection

Because Gln and Asp are important sources of substrates for nucleotide synthesis [37] (Fig. 7A), and PDCoV utilizes Gln catabolism to Asp to promote viral proliferation (Fig. 6E), we speculated that PDCoV promotes Gln catabolism to support nucleotide synthesis. The results showed that the exogenous addition of a nucleoside mixture to Gln-free medium partially restored the decrease in PDCoV titer caused by Gln deprivation (Fig. 7B), suggesting that PDCoV infection could enhance glutaminolysis to promote the nucleoside synthesis necessary for viral proliferation. Next, we investigated the types of nucleotides utilized by PDCoV. CAD and dihydroorotate dehydrogenase (DHODH) are two key enzymes in the de novo synthesis of pyrimidines; PRPS2 and phosphoribosyl pyrophosphate amidotransferase (PPAT) are two key enzymes in the de novo synthesis of purines (Fig. 7A). Pyrimidine synthesis was inhibited by CAD-specific siRNA (siCAD) or a DHODH inhibitor (Brequinar); purine synthesis was inhibited by PRPS2-specific siRNA (siPRPS2) or a PPAT inhibitor (6-TG). We found that both CAD knockdown and Brequinar treatment significantly reduced PDCoV titer (Fig. 7C and D and Supplementary Fig. 9A), whereas PRPS2 knockdown and 6-TG treatment did not have a significant inhibitory effect on PDCoV proliferation (Fig. 7E and F and Supplementary Fig. 9B). Therefore, we concluded that pyrimidines de novo synthesis, rather than purines de novo synthesis, are preferentially utilized during PDCoV infection (Fig. 7G).

Fig. 7.

PDCoV requires pyrimidines for optimal replication. (A) Schematic diagram of metabolic pathways involved in de novo synthesis of pyrimidines and purines. (B–F) IPI-2I cells were infected with PDCoV (MOI = 0.5 or 0.1) for 18 h in the indicated media and PDCoV titers were detected by TCID₅₀ assays. (B) Cells were infected with PDCoV in Gln-containing medium, Gln-free medium, or Gln-free medium supplemented with nucleosides (0.5 × ). (C, E) Cells were transfected with carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD)-specific siRNA (siCAD), PRPS2-specific siRNA (siPRPS2), or siNC for 24 h, then infected with PDCoV. (D, F) Cells were infected with PDCoV in media supplemented with the indicated concentrations of Brequinar (0, 1, or 5 nM) or 6-TG (0, 1, or 5 μM). Values are shown as mean ± SD and all data represent three independent experiments. Statistical analyses for (B–F) were performed by the one-way ANOVA test. **, p < 0.01; ***, p < 0.001; NS., not significant. (G) Schematic diagram illustrating the impact of PDCoV on nucleotide metabolism.

4. Discussion

Because the rate of glycoATP production is more rapid than the rate of OXPHOS-derived ATP production, most viruses enhance aerobic glycolysis (i.e., the Warburg effect) to promote their proliferation [[38], [39], [40]]. The primary characteristics of the Warburg effect are increases in glucose uptake and lactic acid secretion [[41], [42], [43]]. These increases are typically accompanied by increases in the activities of glycolytic enzymes, such as HK2 [44,45], Fructose 6 phosphate kinase (PFKM) [46,47], and LDHA [48]. For example, HCV NS5A activates HK2 to upregulate glucose consumption and lactic acid production [49]; herpes simplex virus 1 (HSV-1) infection elevates PFKM activity, thereby increasing glucose uptake and lactic acid secretion [46]. Moreover, coronaviruses such as SARS-CoV-2, Middle Eastern respiratory syndrome coronavirus, and mouse hepatitis virus (MHV) induce the Warburg effect in various ways [50]. In particular, SARS-CoV-2 and MHV increase the rate of extracellular acidification [12,51,52]. MHV infection also upregulates the protein expression level of HK2, but not PFKM [52]. However, we found that PDCoV infection did not increase glucose uptake and lactic acid release; it exhibited no significant effect on the expression patterns of HK2, PFKM, and LDHA (data not shown). These findings suggested that PDCoV infection did not induce the Warburg effect. Among the viruses examined to date, Vaccinia virus (VACV) is the sole reported virus that does not activate glycolysis, which appears to be a non-Warburg effect; however VACV requires Gln, while not glucose, for efficient replication [53]. In contrast to VACV, PDCoV infection requires glycolysis but decreases glycolytic flux, and the presence of glucose is beneficial for viral proliferation. The key novel finding in the present study was that a glucose-dependent virus could remodel cellular energy metabolism through a non-Warburg effect.

Our data revealed that the levels of pyruvic acid were decreased at 6 hpi, but increased at 12 and 18 hpi by PDCoV infection (Fig. 1, Fig. 4A). However, the mechanism underlying this fluctuation in pyruvic acid levels remains unclear. We observed that the activity of PK was not affected by PDCoV infection at 6 hpi, but was significantly upregulated at 12 and 18 hpi (Fig. 4B). This suggested that the alterations in PK activity might account for the divergent trends in pyruvic acid levels at different time points after PDCoV infection. Additionally, we found that OAA levels were slightly elevated at 6 hpi and decreased at 12 and 18 hpi, suggesting that the catabolism of pyruvic acid was more dominant than its anabolism at 6 hpi, but weaker than its anabolism at 12 and 18 hpi.

Energy transfer from OXPHOS to glycolysis is reportedly caused by the rapid conversion of pyruvic acid to lactic acid [54,55]. However, in PDCoV-infected cells, PK-induced energy transfer was accompanied by an increase in pyruvic acid production, suggesting that energy transfer from OXPHOS to glycolysis can occur in the context of pyruvic acid accumulation. In addition to its role in regulating energy transfer, PK was utilized by PDCoV in pyruvic acid-mediated amino acid biosynthesis. Therefore, it is not surprising that PK activation by PDCoV was beneficial for viral proliferation. PK also has crucial roles in the proliferation of other viruses. For example, DENV upregulates the level of PK phosphorylation to promote viral replication [56]. Reduction of PK activity inhibits the proliferation of HCV and hepatitis B virus by decreasing the production of ATP and other glycolytic intermediates [57]. Taken together, PK may be a potential broad-spectrum antiviral drug target.

NAD⁺ and NADH are critical coenzymes involved in multiple metabolic processes. Herein, we observed that PDCoV infection had no significant effect on NAD⁺ levels, but significantly reduced NADH content at 12 and 18 hpi (Fig. 2C and D). Considering that PDCoV infection caused decreases in the fluxes of both glycolysis and the TCA cycle as revealed by the results of ECAR and OCR (Fig. 2, Fig. 3C), and NADH is primarily generated during the TCA cycle and glycolysis by the reduction of NAD⁺, we surmise that the decrease in NADH production might be mainly attributable to the disruption of glycolysis and TCA cycle during PDCoV infection. Furthermore, the conversion of pyruvic acid to lactic acid consumes NADH, thus the reduction in NADH production might further contribute to the decrease in lactic acid content in PDCoV-infected cells. As for NAD⁺, most other viruses, especially those capable of promoting glycolysis, such as human immunodeficiency virus [58], HSV-1 [59], Zika virus [60], SARS-CoV-2 [61], and MHV [62], reduce NAD⁺ levels. Different from these viruses, in the present study, we found that PDCoV infection did not facilitate glycolysis, and consistently, NAD⁺ levels were not decreased by PDCoV infection. Notably, inhibition of NAD⁺ consumption has been demonstrated to be detrimental to the proliferation of many viruses [52,63,64], we thus speculated that limiting NAD⁺ consumption in PDCoV-infected cells maybe a novel antiviral strategy for the host, which requires investigation in further experiments.

Nucleotide synthesis requires the transfer of carbon or nitrogen from many amino acids, such as Ser, Gly, Asp, Gln, and Glu. Therefore, the high viral demand for nucleotides is usually accompanied by upregulation of amino acid synthesis [8,19,65]. For example, KSHV accelerates the de novo synthesis of nucleotides by promoting synthesis of NEAAs via GLS, GOT1, and GLUD1 [19]. HSV-1 upregulates GOT2, a key enzyme involved in amino acid synthesis, to accelerate the de novo synthesis of pyrimidines [66]. In the present study, we found that PDCoV infection required Asp, Gln, and Glu, rather than Ser and Gly (two donors of one-carbon units). Thus, Asp and Gln/Glu, but not one-carbon units (e.g., Ser and Gly), may contribute to nucleotide synthesis during PDCoV infection. In addition to the roles of amino acids, the PPP participates in nucleotide synthesis by providing ribose 5-phosphate. Some viruses, such as SARS-CoV-2 [67] and Newcastle disease virus (NDV) [68], are fully dependent on the PPP for nucleotide synthesis. However, PDCoV infection did not have a significant effect on the PPP, suggesting that PDCoV does not utilize the PPP for nucleotide synthesis.

Furthermore, de novo synthesis of nucleotides involves many enzymes, some of which are critical for the proliferation of viruses such as EV71, SARS-CoV-2, coxsackievirus A16, and coxsackievirus B3 [20,69,70]. Notably, most of the enzymes affecting viral replication are associated with the de novo synthesis of pyrimidines, but not purine de novo synthesis. In this study, we also confirmed that CAD and DHODH, the pivotal enzymes for the de novo synthesis of pyrimidines, rather than PRPS2 and PPAT, the key enzymes for the de novo synthesis of purines, played important roles in PDCoV proliferation. Based on these results, we conclude that the de novo synthesis of pyrimidines, as opposed to purines, is preferentially utilized during PDCoV infection. This preference maybe attributed to the fact that the processes involved in de novo synthesis of purines are more complex than those of pyrimidines. Purine de novo synthesis begins with 5-phosphoribose-1-pyrophosphate, a product of the PPP pathway, and necessitates folate and one-carbon units, whereas pyrimidine de novo synthesis originates from Asp and Gln, etc [71]. This suggests that purine de novo synthesis is more energy-intensive and intricate compared to pyrimidine de novo synthesis [71]. Therefore, we speculate that it is inefficient for viruses to acquire purines by hijacking the purine de novo synthesis pathway, and instead, they probably primarily utilize other purine synthesis pathways (e.g., salvage pathways) to obtain the purines required for viral replication. This provides a possible explanation as to why inhibiting purine de novo synthesis does not significantly hinder the proliferation of most viruses. Overall, the present and previous findings suggest that the de novo synthesis processes of nucleotides, especially pyrimidines, may be promising antiviral targets.

Several viruses have been shown to regulate cellular metabolism by regulating some critical metabolic enzymes through viral proteins. For instance, HCV NS5A protein utilize its N-terminal region to interact with PC, thereby enhancing fatty acid synthesis, which finally facilitates viral replication [72]; Foot-and-mouth disease virus 2C protein exhibits interactions with CAD, which catalyzes the de novo synthesis of pyrimidines and in turn promotes viral proliferation [73]. Regarding coronaviruses, CAD has also been demonstrated to be activated by SARS-CoV-2 Nsp9 to catalyze pyrimidine de novo synthesis, which ultimately enhances the proliferation of SARS-CoV-2 [74]. Additionally, PK interacts with N protein of SARS-CoV, thus hijacking host glycolysis to augment viral infection [75]. In the present study, we also found that inhibition of PC, PK, and CAD antagonized PDCoV proliferation, suggesting that these three metabolic enzymes are important for PDCoV infection. However, the involved viral protein(s) and underlying regulatory mechanisms remain unclear and require further studies to investigate.

Although many viruses disrupt TCA cycle, they behave differently with respect to mitochondrial ATP demand. For example, whereas a decrease in mitochondrial ATP production does not have an obvious effect on NDV infection [24], it inhibits MHV infection [52]. In this study, we also detected the role of mitochondrial ATP in PDCoV infection. There is considerable evidence that mitochondrial ATP is generated by OXPHOS. The electron transport chain (ETC), which consists of five protein complexes (I, II, III, IV, and V), is a key component of OXPHOS [76]. In the present study, dose-dependent reductions of PDCoV titer were observed during treatment with Rotenone (an inhibitor of ETC complex I) and Oligomycin (an inhibitor of ETC complex V), suggesting that mitochondrial ATP is essential for PDCoV infection (Supplementary Fig. 10).

Viruses are specialized parasitic organisms. Upon infection, virus exert stress on host cells and may cause cell death. In this study, to minimize the potential interference of cellular self-factors in our investigation of the regulation of metabolism by PDCoV infection, we deliberately chose three different time points after infection and set up a mock group as a control at each time point. However, we recognize that even with these measures, the impact of cellular self-factors, such as cell death, on metabolism cannot be entirely rule out. Consequently, there is an urgent need to develop more sensitive metabolomic assays to study virus-metabolism interactions more accurately in the future.

In conclusion, this study showed that PDCoV infection disrupts metabolic homeostasis with respect to glycolysis, the TCA cycle, and glutaminolysis, causing a non-Warburg effect characterized by pyruvic acid accumulation. Intriguingly, PDCoV infection utilizes glycolysis by increasing the proportion of glycoATP, primarily through enhanced pyruvic acid anabolism. Furthermore, we found that pyruvic acid catabolism to OAA and glutaminolysis contribute to both amino acid synthesis and pyrimidine de novo synthesis in PDCoV-infected cells (Fig. 8). These findings provide a novel perspective regarding interactions between PDCoV and host CCM, with implications for anti-PDCoV drug targets.

Fig. 8.

Simplified illustration of the mechanism by which PDCoV hijacks CCM to promote viral proliferation. PDCoV infection disrupts glycolysis, the TCA cycle, and glutaminolysis. Glycolysis is initiated with glucose, which is metabolized to pyruvic acid through sequential enzymatic reactions. PDCoV enhances pyruvic acid synthesis and increases glycoATP proportion by activating PK to promote viral proliferation. Pyruvic acid enters the mitochondria and is converted to Ac-CoA via PDH catalysis or to OAA via PC catalysis. PDCoV activates PC, rather than PDH, to support viral infection. Disruption of the TCA cycle by PDCoV infection leads to decreases in ATP production and NADH level. Glutaminolysis produces Glu and αKG. PDCoV promotes Gln conversion to Glu, thus providing a sufficient source of carbon and nitrogen for amino acid synthesis via transamination, which may be related to Asp synthesis involving OAA. PDCoV-induced glutaminolysis also influences pyrimidine de novo synthesis. Red and blue arrows indicate upregulation and downregulation, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

CRediT authorship contribution statement

Guanning Su: Writing – original draft, Project administration, Formal analysis, Data curation. Jiao Liu: Project administration, Data curation. Chenrui Duan: Project administration, Data curation. Puxian Fang: Funding acquisition. Liurong Fang: Supervision, Funding acquisition. Yanrong Zhou: Writing – review & editing, Writing – original draft, Validation, Supervision, Formal analysis, Data curation, Conceptualization. Shaobo Xiao: Writing – review & editing, Supervision, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare that they have no conflicts of interest with the contents of this article.

Abbreviations

Ac-CoA

Acetyl coenzyme A

AOA

Aminooxyacetic acid

ASNS

Asparagine synthase

Asp

Aspartic acid

ATP

Adenosine triphosphate

CAD

Carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase

CCM

Central carbon metabolism

DENV

Dengue virus

DHODH

Dihydroorotate dehydrogenase

ETC

Electron transport chain

EV71

Enterovirus 71

Gln

Glutamine

GLS

Glutaminase

Glu

Glutamic acid

GLUD1

Glutamate dehydrogenase

Gly

Glycine

GOT1/2

Glutamic oxaloacetic transaminase1/2

GPT2

Glutamic-pyruvate transaminase 2

HCV

Hepatitis C virus

HK2

Hexokinase 2

HSV-1

Herpes simplex virus 1

KSHV

Kaposi's sarcoma-associated herpesvirus

LDHA

Lactate dehydrogenase A

MHV

Mouse hepatitis virus

NDV

Newcastle disease virus

NEAAs

Non-essential amino acids

OAA

Oxalacetic acid

OXPHOS

Oxidative phosphorylation

PC

Pyruvate carboxylase

PDCoV

Porcine deltacoronavirus

PDH

Pyruvate dehydrogenase

PFKM

Fructose 6 phosphate kinase

PK

Pyruvate kinase

PKM1/2

Pyruvate kinase muscle isozyme 1/2

PPAT

Phosphoribosyl pyrophosphate amidotransferase

PPP

Pentose phosphate pathway

PRPS2

Phosphoribosyl pyrophosphate synthetase 2

SARS-CoV-2

Severe acute respiratory syndrome coronavirus 2

Ser

Serine

TCA

Tricarboxylic acid

VACV

Vaccinia virus

αKG

Oxoglutaric acid

2-DG

2-Deoxy-d-glucose

2-NBDG

2-(7-nitro-2,1,3-benzoxadiazol-4-yl)-d-glucosamine

Appendix A. Supplementary data

The following is the supplementary data to this article:

Data availability

Data will be made available on request.