Sphingosine 1-phosphate-metabolizing enzymes control influenza virus propagation and viral cytopathogenicity

Abstract

Sphingosine 1-phosphate (S1P)-metabolizing enzymes regulate the level of sphingolipids and have important biological functions. However, the effects of S1P-metabolizing enzymes on host defense against invading viruses remain unknown. In this study, we investigated the role of S1P-metabolizing enzymes in modulating cellular responses to influenza virus infection. Overexpression of S1P lyase (SPL), which induces the degradation of S1P, interfered with the amplification of infectious influenza virus. Accordingly, SPL-overexpressing cells were much more resistant than control cells to the cytopathic effects caused by influenza virus infection. SPL-mediated inhibition of virus-induced cell death was supported by impairment of the upregulation of the proapoptotic protein Bax, a critical factor for influenza virus cytopathogenicity. Importantly, influenza virus infection of SPL-overexpressing cells induced rapid activation of extracellular signal-regulated kinase (ERK) and STAT1 but not of p38 mitogen-activated protein kinase (MAPK), Akt, or c-Jun N-terminal kinase (JNK). Blockade of STAT1 expression or inhibition of Janus kinase (JAK) activity elevated the level of influenza virus replication in the cells, indicating that SPL protects cells from influenza virus via the activation of JAK/STAT signaling. In contrast to that of SPL, the overexpression of S1P-producing sphingosine kinase 1 heightened the cells' susceptibility to influenza virus infection, an effect that was reversed by the inhibition of its kinase activity, representing opposed enzymatic activity. These findings indicate that the modulation of S1P-metabolizing enzymes is crucial for controlling the host defense against infection with influenza virus. Thus, S1P-metabolizing enzymes are novel potential targets for the treatment of diseases caused by influenza virus infection.

FIG. 1.

Effect of SPL overexpression on influenza virus amplification. (A) HEK cells (filled squares) or SPL cells (open triangles) were infected with WSN virus at an MOI of 0.1. At 6, 12, 24, or 48 hpi (left) and at 3 or 4 dpi (right), viral titers were determined by a plaque assay. (B) HEK cells or SPL cells were either left uninfected (control [CTR]) or infected with WSN virus at an MOI of 1. At 2 dpi, influenza virus NP and actin were detected by Western blot analysis. (C) Cells were either left uninfected (CTR) or infected with WSN at an MOI of 0.1 and were analyzed for the expression of viral NP by flow cytometry at 3 dpi. The percentages of virus NP⁺ cells are given. SSC, side scatter. (D) SPL cells were either mock transfected (−) or transfected with control siRNA (si-CTR) or siRNA targeting SPL (si-SPL); then the cells were infected with WSN virus at an MOI of 1. Uninfected SPL cells are shown as CTR. At 2 dpi, Western blot analysis was performed to detect SPL, influenza virus NP, and actin (left); viral titers were quantified by a plaque assay (right).

FIG. 2.

Reduced CPEs in influenza virus-infected SPL cells. (A) HEK cells or SPL cells were either left uninfected (CTR) or infected with WSN virus at an MOI of 1. At 2, 3, or 4 dpi, cellular viability was monitored by using a trypan blue exclusion assay. The number of uninfected cells was set at 1.0, and the relative numbers of virus-infected groups were compared. Three separate wells per group were used. Values are means ± SEM. P values are included to show statistical significance. NS, no significant difference. (B) HEK cells or SPL cells were either left uninfected or infected with WSN virus at an MOI of 5. Cells were stained with annexin V at 1 dpi and were then analyzed by flow cytometry. Percentages of annexin V⁺ cells are shown. (C and D) HEK cells or SPL cells were infected with WSN virus at an MOI of 1. Cell lysates were used for Western blot analysis to detect Bax, Bcl-2, and actin (C) or uncleaved PARP, cleaved PARP, and actin (D) at 1 or 2 dpi.

FIG. 3.

Coexpression of Bax and viral NP in influenza virus-infected HEK or SPL cells. (A) HEK cells or SPL cells were either left uninfected (dotted lines) or infected (solid lines) with WSN virus at an MOI of 1. (Left) At 2 dpi, the expression of viral NP was assessed by flow cytometry. Virus-treated cells (solid lines) were separated into NP-expressing (NP⁺) and nonexpressing (NP⁻) cells. (Right) The Bax expression of the NP⁺ cells (solid lines) and the NP⁻ cells (shaded areas) was compared with that of uninfected cells (dotted lines). (B) Cells were either left uninfected (CTR) or infected with WSN at an MOI of 1. They were fixed, permeabilized, and stained with DAPI, to detect nuclei, and with antibodies against viral NP and Bax at 2 dpi. Representative confocal images are shown (original magnification, ×200). Bar, 20 μm.

FIG. 4.

Analysis for the activation of JAK/STAT following influenza virus infection of SPL cells. HEK cells or SPL cells were either left uninfected or infected with WSN virus at an MOI of 1. (A) At 1 dpi, cell lysates were used for Western blot analysis to detect STAT1, pSTAT1, STAT2, pSTAT2, and actin. (B) At 0, 12, or 18 hpi, whole proteins were extracted and then used for Western blot analysis to detect STAT1, pSTAT1, and actin. (C) HEK or SPL cells were pretreated with fludarabine (1 μM), blocking STAT1 expression, and at 2 dpi, the expression of viral NP and actin was analyzed. (D) HEK cells (filled bars) and SPL cells (open bars) were either left uninfected (CTR) or infected with WSN virus at an MOI of 1 (left) or 10 (right). Reverse transcription followed by quantitative real-time PCR was performed to detect the relative mRNA level of IFN-β. Three reactions per sample were carried out for the right panel. Values are means ± SEM. (E) HEK or SPL cells were treated with inhibitors of JAK1 (J1 [10 μM for the left panel or 2 μM for the right panel]), JAK2 (J2 [2 μM]), or JAK3 (J3 [2 μM]) at 2 hpi. After 2 days, the levels of viral NP and actin were assessed by Western blot analysis.

FIG. 5.

Preferential activation of ERK, but not of p38, Akt, or JNK, in SPL cells. HEK cells or SPL cells were either left uninfected or infected with WSN virus at an MOI of 1. (A) At 1 or 2 dpi, whole proteins were used for Western blot analysis to record the expression of ERK, pERK, STAT1, pSTAT1, viral NP, and actin. (B) Cell lysates were obtained at 1 or 2 dpi and were analyzed by Western blotting to determine the levels of p38, p-p38, Akt, pAkt, JNK, pJNK, and actin.

FIG. 6.

Enhanced susceptibility of SK1 cells to influenza virus infection. (A) HEK cells (open triangles) or SK1 cells (filled squares) were infected with WSN virus at an MOI of 0.1. At 12, 18, or 48 hpi, virus titers were determined by a plaque assay. Three separate samples of virus-infected cells per group were used at each time point. Values are means ± SEM. (B) Western blot analysis was conducted at 2 dpi to detect viral NP and actin. (C) Cell lysates were used for Western blot analysis to detect STAT1, pSTAT1, and actin. (D) HEK cells were infected with WSN virus at an MOI of 1 and were treated with either a solvent control (CTR), FTY720 (1 μM), d-erythro-sphingosine (1 μM), or S1P (1 μM). At 2 dpi, total proteins were extracted for the detection of viral NP and actin by Western blot analysis. (E) SK1 cells were pretreated with DMS (0.1 μM) or its solvent control for 3 h and were then either left uninfected or infected with WSN virus at an MOI of 1. At 3 dpi, the expression of viral NP, Bax, Bcl-2, and actin was analyzed by Western blotting. (F) SK1 cells were pretreated with DMS (0.5 μM) or its solvent control and were then either left uninfected or infected with WSN virus at an MOI of 1. At 2 dpi, the expression of viral NP was detected by flow cytometry. Dotted line, non-virus-infected cells; solid line, virus-infected cells; shaded area, DMS-treated, virus-infected cells.