The compound packaged in virions is the key to trigger host glycolysis machinery for virus life cycle in the cytoplasm

Abstract

Viruses depend on the host metabolic machinery to complete their life cycle in the host cytoplasm. However, the key viral factors initiating the host machinery after the virus enters the cytoplasm remain unclear. Here, we found that compounds packaged in the virions of white spot syndrome virus, such as palmitic amide, could trigger the viral life cycle in the host cytoplasm. Palmitic amide promoted virus infection by enhancing host glycolysis by binding to triosephosphate isomerase to enhance its enzymatic activity. The glycolysis enhancement resulted in lactate accumulation, thereby promoting hypoxia-inducible factor 1 (HIF-1) expression. HIF-1 upregulation further enhanced glycolysis, which in turn promoted virus infection. Therefore, our study presented novel insight into the initiation of the virus life cycle in host cells.

Keywords: Human Metabolism; Molecular Biology; Virology.

Graphical abstract

Figure 1

Metabolites packaged in virions (A) WSSV virions purified from WSSV-infected shrimp. The purified virions were observed under a transmission electron microscope. Scale bar, 100 nm. (B) LC-MS profiles of metabolites from the WSSV virions. The images were representatives of three biological repeats. The virus supernatant subjected to metabolite extraction and LC-MS analysis served as “blank”. (C) Confirmation of compounds packaged in WSSV virions by LC-MS analysis. (D) Structures of palmitic amide and oleamide.

Figure 2

Effects of the metabolites packaged in virions on virus infection (A) Content of compounds in shrimp hemocytes at different times after the injection of compounds. Palmitic amide and oleamide were injected into WSSV-infected shrimp. At different times after the compound injection, the content of compounds in the shrimp hemocytes was examined by LC-MS. The data represented the results of three independent assays. (B) Influence of palmitic amide and oleamide on WSSV infection. The compounds at various concentrations were injected into the WSSV-infected shrimp. At different times post-infection, the shrimp were subjected to quantitative real-time PCR to quantify the WSSV copies in the shrimp hemocytes. The experiments were biologically repeated for three times (∗p < 0.05; ∗∗p < 0.01). (C) Impact of constant existence of palmitic amide on WSSV content in shrimp. Shrimp were injected with palmitic amide and/or WSSV. WSSV alone was used as a positive control. Palmitic amide (100 μM) and DMSO (dimethyl sulfoxide) without WSSV were included in the injections as negative controls. For the treatment WSSV+100 μM palmitic amide, shrimp were injected three times to ensure the constant presence of palmitic amide. Firstly, shrimp were injected with 100 μM of palmitic amide. Twenty four hours later, the same shrimp were injected with WSSV and palmitic amide (100 μM). Finally, the shrimp were injected with palmitic amide (100 μM) at 24 hr after infection. At different times post-infection, the WSSV copies were quantified by quantitative real-time PCR (∗∗p < 0.01). (D) Shrimp cumulative mortality analysis. The treatments were indicated on the top. The numbers on the horizontal axis represented the days post-infection. Data represented the mean ± standard deviation of triplicate assays (∗p < 0.05; ∗∗p < 0.01).

Figure 3

Activation of the host HIF-1 signaling pathway and glycolysis by palmitic amide (A) Heatmap of differentially expressed genes of shrimp in response to palmitic amide challenge. Shrimp were injected with DMSO or palmitic amide. Twenty-four hours later, the mRNAs of shrimp hemocytes were subjected to sequencing. (B) KEGG classification of the differentially expressed genes. (C) Examination of the mRNA level of differentially expressed genes by quantitative real-time PCR. The error bars represent the means ± standard deviations of three independent experiments (∗p < 0.05; ∗∗p < 0.01). (D) Influence of palmitic amide on the expression of HIF-1. Shrimp were injected with DMSO or palmitic amide. Twenty-four hours later, Western blot analysis was conducted to examine the expression level of HIF-1 in shrimp hemocytes. β-actin was used as a control. (E) Impact of palmitic amide on glycolysis of shrimp. Shrimp were injected with palmitic amide. As a control, DMSO was included in the injection. At 24 hr after injection, the contents of glucose and lactate in the hemocytes of shrimp were examined. The error bars represented the means ± standard deviations of three independent experiments (∗∗p < 0.01). (F) Role of palmitic amide in TCA cycle. Shrimp were injected with palmitic amide or DMSO. At 24 hr after injection, the contents of acetyl CoA and mitochondrial citric acid in the hemocytes of shrimp were examined.

Figure 4

Role of HIF-1 in virus infection and glycolysis (A) Upregulation of HIF-1 in shrimp in response to virus infection. Shrimp were challenged with WSSV. At different times post-infection, the expression level of HIF-1 in shrimp hemocytes was examined by Northern blot (up) and Western blot (down). β-actin was used as a control. (B) Knockdown of HIF-1 by sequence-specific siRNA (HIF-1-siRNA) in WSSV-infected shrimp. Shrimp were co-injected with HIF-1-siRNA and WSSV. As a control, HIF-1-siRNA-scrambled was included in the injection. At different times after injection, the HIF-1 mRNA level and protein level in the hemocytes of shrimp were examined by Northern blot (left) and Western blot (right) separately. β-actin was used as a control. (C) Influence of HIF-1 silencing on WSSV infection in shrimp. The WSSV content was quantified using quantitative real-time PCR at different times post-infection. WSSV alone, PBS, and HIF-1-scrambled were used as controls (∗p < 0.05; ∗∗p < 0.01). (D) Effects of HIF-1 silencing on the mortality of WSSV-infected shrimp. The numbers on the horizontal axis indicated the days post-infection (∗p < 0.05; ∗∗p < 0.01). (E) Role of HIF-1 in glycolysis of shrimp. Shrimp were injected with HIF-1-siRNA. HIF-1-siRNA-scrambled was included in the injection as a control. Thirty-six hours later, the contents of glucose and lactate in the hemocytes of shrimp were examined (∗∗p < 0.01).

Figure 5

Palmitic amide interacted with the TPI protein and increased the TPI activity (A) Proteins interacted with palmitic amide. Shrimp hemocytes were lysed and then incubated with biotin-labeled palmitic amide. The proteins were analyzed by SDS-PAGE with Coomassie blue staining. Dynabeads alone was used as a control. The arrow indicated the differential protein. M, protein marker. (B) Identification of the protein bound to palmitic amide by mass spectrometry. The protein was identified to be triosephosphate isomerase. The matched peptides were indicated with underlines and numbers. (C) Western blot analysis of the proteins interacted with palmitic amide. The arrow indicated the TPI. M, protein marker. (D) Thermodynamic characterization of the interaction between palmitic amide and TPI protein. The purified recombinant TPI protein (100 μM) and incubated with palmitic amide (1 mM), followed by isothermal titration calorimetry (ITC) analysis. DMSO was used as a control. (E) Impact of palmitic amide on TPI activity in shrimp. Shrimp were injected with palmitic amide (100 μM). Twenty-four hours later, the activity of TPI of hemocytes was examined. DMSO was used as a control. The error bars denoted the means ± standard deviations of three independent experiments (∗∗p < 0.01). (F) Influence of palmitic amide on the expression of TPI in shrimp. Shrimp were injected with palmitic amide (100 μM) or DMSO. Twenty-four hours later, the expression level of TPI in the hemocytes of shrimp was examined by Western blot analysis. β-actin was used as a control.

Figure 6

The influence of TPI on virus infection (A) Upregulation of TPI in shrimp in response to virus infection. Shrimp were challenged with WSSV. At different times post-infection, the expression level of TPI in shrimp hemocytes was examined by Northern blot and Western blot. β-actin was used as a control. (B) Knockdown of TPI by sequence-specific siRNA (TPI-siRNA) in WSSV-infected shrimp. Shrimp were co-injected with TPI-siRNA and WSSV. As a control, TPI-siRNA-scrambled was included in the injection. At different times after injection, the TPI mRNA level in the hemocytes of shrimp was examined by Northern blot. β-actin was used as a control. (C) Western blot analysis of TPI protein in siRNA-treated shrimp. β-actin was used as a control. (D) Influence of TPI silencing on WSSV infection in shrimp. The WSSV content was quantified using quantitative real-time PCR at different times post-infection. WSSV alone and PBS were used as controls. The error bars denoted the means ± standard deviations of three independent experiments (∗∗p < 0.01). (E) Effects of TPI silencing on the mortality of WSSV-infected shrimp. The numbers on the horizontal axis indicated the post-infection days (∗p < 0.05; ∗∗p < 0.01).

Figure 7

Relationship between TPI, HIF-1, and glycolysis (A) Influence of TPI inhibitor on TPI activity in shrimp. Shrimp were injected with TPI inhibitor phenazine (100 μM). Twenty-four hours later, the enzymatic activity of TPI in the hemocytes of shrimp was examined. DMSO was used as a control. The error bars denoted the means ± standard deviations of three independent experiments (∗∗p < 0.01). (B) Impact of the inhibition of TPI activity on glycolysis of shrimp. At 24 hr after the injection of TPI inhibitor, the contents of glucose and lactate in the hemocytes of shrimp were examined. The error bars represented the means ± standard deviations of three independent experiments (∗∗p < 0.01). (C) Effects of the suppression of TPI activity on the HIF-1 expression in shrimp. At 24 hr after the injection of TPI inhibitor, Western blot analysis was conducted to examine the expression level of HIF-1 protein in shrimp hemocytes. β-actin was used as a control. (D) Evaluation of the impact of HIF-1 silencing on the expression of TPI in shrimp. Shrimp were injected with HIF-1-siRNA. As a control, HIF-1-siRNA-scrambled was included in the injection. At 36 hr after injection, the expression level of TPI protein in the hemocytes of shrimp was analyzed by Western blot. β-actin was used as a control.

Figure 8

Mechanism of palmitic amide-triggered initiation of virus infection Schematic diagram of the underlying mechanism of palmitic amide packaged in virions during virus infection.