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Blocking the PI3K/AKT pathway enhances mammalian reovirus replication by repressing IFN-stimulated genes.

Tian J, Zhang X, Wu H, Liu C, Li Z, Hu X, Su S, Wang LF, Qu L - Front Microbiol (2015)

Bottom Line: Virus infection induced phosphorylation of focal adhesion kinase (FAK) but not Gab1, and blockage of FAK phosphorylation suppressed Akt phosphorylation.We also found that reovirus infection activated the IFN-stimulated response element (ISRE) in an interferon-independent manner and up-regulated IFN-stimulated genes (ISGs) via the PI3K/Akt/EMSY pathway.Suppression of PI3K/Akt activation impaired the induction of ISRE and down-regulated the expression of ISGs.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences , Harbin, China.

ABSTRACT
Many host cellular signaling pathways were activated and exploited by virus infection for more efficient replication. The PI3K/Akt pathway has recently attracted considerable interest due to its role in regulating virus replication. This study demonstrated for the first time that the mammalian reovirus strains Masked Palm Civet/China/2004 (MPC/04) and Bat/China/2003 (B/03) can induce transient activation of the PI3K/Akt pathway early in infection in vitro. When UV-treated, both viruses activated PI3K/Akt signaling, indicating that the virus/receptor interaction was sufficient to activate PI3K/Akt. Reovirus virions can use both clathrin- and caveolae-mediated endocytosis, but only chlorpromazine, a specific inhibitor of clathrin-mediated endocytosis, or siRNA targeting clathrin suppressed Akt phosphorylation. We also identified the upstream molecules of the PI3K pathway. Virus infection induced phosphorylation of focal adhesion kinase (FAK) but not Gab1, and blockage of FAK phosphorylation suppressed Akt phosphorylation. Blockage of PI3K/Akt activation increased virus RNA synthesis and viral yield. We also found that reovirus infection activated the IFN-stimulated response element (ISRE) in an interferon-independent manner and up-regulated IFN-stimulated genes (ISGs) via the PI3K/Akt/EMSY pathway. Suppression of PI3K/Akt activation impaired the induction of ISRE and down-regulated the expression of ISGs. Overexpression of ISG15 and Viperin inhibited virus replication, and knockdown of either enhanced virus replication. Collectively, these results demonstrate that PI3K/Akt activated by mammalian reovirus serves as a pathway for sensing and then inhibiting virus replication/infection.

No MeSH data available.


Related in: MedlinePlus

Role of clathrin-mediated endocytosis in the activation of PI3K/Akt early in infection. Serum-starved A549 cells were pretreated with various concentrations of chlorpromazine (Chl; 5–50 μM) and genistein (Gen; 50–300 μM) for 30 min, and the cells were then infected with MPC/04 (A,C) and B/03 (B,D) at a MOI of 5. A mock-infected lane was included in each panel as a negative control. After 30 min p.i., the cell lysates were collected and separated by SDS-PAGE. The levels of p-Akt (Ser473), total Akt and GAPDH were determined by Western blot analysis as detailed in Methods. (E,G) A549 cells were transfected with 15 pmol/well of negative control (Negative siRNA) or clathrin siRNA. 24 h after transfection, cells were infected with MPC/04 (E) or B/03 (G) at a MOI of 5. After 30 min, the cell lysates were collected and separated by SDS-PAGE. The levels of p-Akt (Ser473), total Akt, clathrin and GAPDH were determined by Western blot analysis. (F,H) Quantification of relative p-Akt band intensities to Akt in (E) and (G). The results were confirmed in three independent experiments. (I) A549 cells pretreated with 50 μM Chl or 200 μM Gen for 1 h or transfected with 15 pmol/well of negative control (Negative siRNA) or clathrin siRNA for 12 h were infected with MPC/04 or B/03 at a MOI of 5. Viral RNA levels were determined at 24 h post infection. The data shown in I represent the results of three independent experiments. *p < 0.05. Error bars indicate SD.
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Figure 4: Role of clathrin-mediated endocytosis in the activation of PI3K/Akt early in infection. Serum-starved A549 cells were pretreated with various concentrations of chlorpromazine (Chl; 5–50 μM) and genistein (Gen; 50–300 μM) for 30 min, and the cells were then infected with MPC/04 (A,C) and B/03 (B,D) at a MOI of 5. A mock-infected lane was included in each panel as a negative control. After 30 min p.i., the cell lysates were collected and separated by SDS-PAGE. The levels of p-Akt (Ser473), total Akt and GAPDH were determined by Western blot analysis as detailed in Methods. (E,G) A549 cells were transfected with 15 pmol/well of negative control (Negative siRNA) or clathrin siRNA. 24 h after transfection, cells were infected with MPC/04 (E) or B/03 (G) at a MOI of 5. After 30 min, the cell lysates were collected and separated by SDS-PAGE. The levels of p-Akt (Ser473), total Akt, clathrin and GAPDH were determined by Western blot analysis. (F,H) Quantification of relative p-Akt band intensities to Akt in (E) and (G). The results were confirmed in three independent experiments. (I) A549 cells pretreated with 50 μM Chl or 200 μM Gen for 1 h or transfected with 15 pmol/well of negative control (Negative siRNA) or clathrin siRNA for 12 h were infected with MPC/04 or B/03 at a MOI of 5. Viral RNA levels were determined at 24 h post infection. The data shown in I represent the results of three independent experiments. *p < 0.05. Error bars indicate SD.

Mentions: Because Akt phosphorylation occurs within 5–15 min p.i., it is possible that cellular endocytosis play key roles in the activation of PI3K/Akt. Thus, we examined whether PI3K/Akt activation depends on cellular endocytosis. Chlorpromazine (Chl), an inhibitor of clathrin-mediated endocytosis, completely suppressed Akt phosphorylation at a concentration of 10–50 μM (Figures 4A,B) and inhibited reovirus RNA production (Figure 4I). Genistein (Gen), an inhibitor of caveolar endocytosis, did not decrease the levels of p-Akt (Figures 4C,D) at a concentration of 300 μM, but it did inhibit the caveolar pathway and reovirus replication in A549 cells at 200 μM (Figure 4I). To confirm that clathrin-mediated endocytosis is required for the induction of PI3K/Akt, cells were transfected with siRNA targeting clathrin. Knockdown of clathrin reduced the levels of p-Akt (Figures 4E–H) and reduced reovirus RNA production (Figure 4I).


Blocking the PI3K/AKT pathway enhances mammalian reovirus replication by repressing IFN-stimulated genes.

Tian J, Zhang X, Wu H, Liu C, Li Z, Hu X, Su S, Wang LF, Qu L - Front Microbiol (2015)

Role of clathrin-mediated endocytosis in the activation of PI3K/Akt early in infection. Serum-starved A549 cells were pretreated with various concentrations of chlorpromazine (Chl; 5–50 μM) and genistein (Gen; 50–300 μM) for 30 min, and the cells were then infected with MPC/04 (A,C) and B/03 (B,D) at a MOI of 5. A mock-infected lane was included in each panel as a negative control. After 30 min p.i., the cell lysates were collected and separated by SDS-PAGE. The levels of p-Akt (Ser473), total Akt and GAPDH were determined by Western blot analysis as detailed in Methods. (E,G) A549 cells were transfected with 15 pmol/well of negative control (Negative siRNA) or clathrin siRNA. 24 h after transfection, cells were infected with MPC/04 (E) or B/03 (G) at a MOI of 5. After 30 min, the cell lysates were collected and separated by SDS-PAGE. The levels of p-Akt (Ser473), total Akt, clathrin and GAPDH were determined by Western blot analysis. (F,H) Quantification of relative p-Akt band intensities to Akt in (E) and (G). The results were confirmed in three independent experiments. (I) A549 cells pretreated with 50 μM Chl or 200 μM Gen for 1 h or transfected with 15 pmol/well of negative control (Negative siRNA) or clathrin siRNA for 12 h were infected with MPC/04 or B/03 at a MOI of 5. Viral RNA levels were determined at 24 h post infection. The data shown in I represent the results of three independent experiments. *p < 0.05. Error bars indicate SD.
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Figure 4: Role of clathrin-mediated endocytosis in the activation of PI3K/Akt early in infection. Serum-starved A549 cells were pretreated with various concentrations of chlorpromazine (Chl; 5–50 μM) and genistein (Gen; 50–300 μM) for 30 min, and the cells were then infected with MPC/04 (A,C) and B/03 (B,D) at a MOI of 5. A mock-infected lane was included in each panel as a negative control. After 30 min p.i., the cell lysates were collected and separated by SDS-PAGE. The levels of p-Akt (Ser473), total Akt and GAPDH were determined by Western blot analysis as detailed in Methods. (E,G) A549 cells were transfected with 15 pmol/well of negative control (Negative siRNA) or clathrin siRNA. 24 h after transfection, cells were infected with MPC/04 (E) or B/03 (G) at a MOI of 5. After 30 min, the cell lysates were collected and separated by SDS-PAGE. The levels of p-Akt (Ser473), total Akt, clathrin and GAPDH were determined by Western blot analysis. (F,H) Quantification of relative p-Akt band intensities to Akt in (E) and (G). The results were confirmed in three independent experiments. (I) A549 cells pretreated with 50 μM Chl or 200 μM Gen for 1 h or transfected with 15 pmol/well of negative control (Negative siRNA) or clathrin siRNA for 12 h were infected with MPC/04 or B/03 at a MOI of 5. Viral RNA levels were determined at 24 h post infection. The data shown in I represent the results of three independent experiments. *p < 0.05. Error bars indicate SD.
Mentions: Because Akt phosphorylation occurs within 5–15 min p.i., it is possible that cellular endocytosis play key roles in the activation of PI3K/Akt. Thus, we examined whether PI3K/Akt activation depends on cellular endocytosis. Chlorpromazine (Chl), an inhibitor of clathrin-mediated endocytosis, completely suppressed Akt phosphorylation at a concentration of 10–50 μM (Figures 4A,B) and inhibited reovirus RNA production (Figure 4I). Genistein (Gen), an inhibitor of caveolar endocytosis, did not decrease the levels of p-Akt (Figures 4C,D) at a concentration of 300 μM, but it did inhibit the caveolar pathway and reovirus replication in A549 cells at 200 μM (Figure 4I). To confirm that clathrin-mediated endocytosis is required for the induction of PI3K/Akt, cells were transfected with siRNA targeting clathrin. Knockdown of clathrin reduced the levels of p-Akt (Figures 4E–H) and reduced reovirus RNA production (Figure 4I).

Bottom Line: Virus infection induced phosphorylation of focal adhesion kinase (FAK) but not Gab1, and blockage of FAK phosphorylation suppressed Akt phosphorylation.We also found that reovirus infection activated the IFN-stimulated response element (ISRE) in an interferon-independent manner and up-regulated IFN-stimulated genes (ISGs) via the PI3K/Akt/EMSY pathway.Suppression of PI3K/Akt activation impaired the induction of ISRE and down-regulated the expression of ISGs.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences , Harbin, China.

ABSTRACT
Many host cellular signaling pathways were activated and exploited by virus infection for more efficient replication. The PI3K/Akt pathway has recently attracted considerable interest due to its role in regulating virus replication. This study demonstrated for the first time that the mammalian reovirus strains Masked Palm Civet/China/2004 (MPC/04) and Bat/China/2003 (B/03) can induce transient activation of the PI3K/Akt pathway early in infection in vitro. When UV-treated, both viruses activated PI3K/Akt signaling, indicating that the virus/receptor interaction was sufficient to activate PI3K/Akt. Reovirus virions can use both clathrin- and caveolae-mediated endocytosis, but only chlorpromazine, a specific inhibitor of clathrin-mediated endocytosis, or siRNA targeting clathrin suppressed Akt phosphorylation. We also identified the upstream molecules of the PI3K pathway. Virus infection induced phosphorylation of focal adhesion kinase (FAK) but not Gab1, and blockage of FAK phosphorylation suppressed Akt phosphorylation. Blockage of PI3K/Akt activation increased virus RNA synthesis and viral yield. We also found that reovirus infection activated the IFN-stimulated response element (ISRE) in an interferon-independent manner and up-regulated IFN-stimulated genes (ISGs) via the PI3K/Akt/EMSY pathway. Suppression of PI3K/Akt activation impaired the induction of ISRE and down-regulated the expression of ISGs. Overexpression of ISG15 and Viperin inhibited virus replication, and knockdown of either enhanced virus replication. Collectively, these results demonstrate that PI3K/Akt activated by mammalian reovirus serves as a pathway for sensing and then inhibiting virus replication/infection.

No MeSH data available.


Related in: MedlinePlus