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. 2006 Aug 18;347(1):261-5.
doi: 10.1016/j.bbrc.2006.06.086. Epub 2006 Jun 22.

Mechanisms of establishment of persistent SARS-CoV-infected cells

Tetsuya Mizutani  1 , Shuetsu Fukushi, Koji Ishii, Yuko Sasaki, Tsuyoshi Kenri, Masayuki Saijo, Yumi Kanaji, Kinji Shirota, Ichiro Kurane, Shigeru Morikawa
Affiliations

Mechanisms of establishment of persistent SARS-CoV-infected cells

Tetsuya Mizutani et al. Biochem Biophys Res Commun. .

Abstract

Previously, we reported the establishment of cells with persistent SARS-CoV infection after apoptotic events and showed that both JNK and PI3K/Akt signaling pathways are important for persistence by treatment with inhibitors at the early stages of SARS-CoV infection. However, the mechanisms of establishment of persistent infection are still unclear. In this study, we investigated which signaling pathways play important roles in escape from apoptosis in cells infected with SARS-CoV. In persistently infected cells at 50h.p.i., PI3K/Akt, JNK, p38 MAPK and Bcl-2 were phosphorylated and the protein levels of Bcl-2 and Bcl-xL were increased. When surviving cells were treated with the JNK-specific inhibitor, SP600125, at 50h.p.i., all cells died, suggesting that the JNK signaling pathway is necessary for maintenance of persistently infected cells. Among the signaling pathways in persistently infected cells, Akt and JNK were phosphorylated in SARS-CoV-nucleocapsid (N) protein-expressing Vero E6 cells using vaccinia viral vector (DIs), strongly suggesting that N protein-induced phosphorylation of Akt and JNK are necessary to establish persistence. These results indicated that at least four proteins, Akt, JNK, Bcl-2 and Bcl-xL, are necessary for survival of persistently SARS-CoV-infected cells.

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Figures

Fig. 1
Fig. 1
Phosphorylation status of signaling pathways in persistently SARS-CoV-infected cells. Vero E6 cells were prepared at confluency in T-25 flasks with 2% fetal bovine serum (FBS) containing Dulbecco’s modified Eagle’s medium (DMEM), and infected with SARS-CoV at 5 m.o.i. At 50 h.p.i., surviving cells and controls were washed 5 times with 2% FCS containing DMEM (pipetting a total of 25 times). Mock-infected subconfluent cells, similar in number to surviving cells that had escaped from apoptosis by SARS-CoV infection, were also washed in the same manner. Western blotting analysis was performed using these protein samples.
Fig. 2
Fig. 2
Effects of JNK and PI3K/Akt inhibitors on cell viability of persistently SARS-CoV-infected cells. (A) Confluent Vero E6 cells in 24-well plates were infected with SARS-CoV for 50 h, and then LY294002 (10 μM) and SP600125 (20 μM) were added to the cells. All wells contained the same volume of DMSO. After incubation for 7 days, the cells were fixed with 10% formaldehyde and stained with 0.1% naphthol blue-black. (B) Stained cells were quantified by measuring the absorbance at OD660 with addition of NaOH. (C) Subconfluent cells were treated with inhibitors for 5 days, and then cells were counted using the WST-1 cell proliferation assay system .
Fig. 3
Fig. 3
Modulation of signaling pathways by N expression. Confluent Vero E6 cells in 24-well plates were infected with DIs-N and DIs-GFP at 5 m.o.i. Protein samples were obtained at 18 h.p.i. and Western blotting analysis was performed.
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