Suppression of viral replication by stress-inducible GADD34 protein via the mammalian serine/threonine protein kinase mTOR pathway - PubMed (original) (raw)

Suppression of viral replication by stress-inducible GADD34 protein via the mammalian serine/threonine protein kinase mTOR pathway

Kahori Minami et al. J Virol. 2007 Oct.

Abstract

GADD34 is a protein that is induced by a variety of stressors, including DNA damage, heat shock, nutrient deprivation, energy depletion, and endoplasmic reticulum stress. Here, we demonstrated that GADD34 induced by vesicular stomatitis virus (VSV) infection suppressed viral replication in wild-type (WT) mouse embryo fibroblasts (MEFs), whereas replication was enhanced in GADD34-deficient (GADD34-KO) MEFs. Enhanced viral replication in GADD34-KO MEFs was reduced by retroviral gene rescue of GADD34. The level of VSV protein expression in GADD34-KO MEFs was significantly higher than that in WT MEFs. Neither phosphorylation of eIF2alpha nor cellular protein synthesis was correlated with viral replication in GADD34-KO MEFs. On the other hand, phosphorylation of S6 and 4EBP1, proteins downstream of mTOR, was suppressed by VSV infection in WT MEFs but not in GADD34-KO MEFs. GADD34 was able to associate with TSC1/2 and dephosphorylate TSC2 at Thr1462. VSV replication was higher in TSC2-null cells than in TSC2-expressing cells, and constitutively active Akt enhanced VSV replication. On the other hand, rapamycin, an mTOR inhibitor, significantly suppressed VSV replication in GADD34-KO MEFs. These findings demonstrate that GADD34 induced by VSV infection suppresses viral replication via mTOR pathway inhibition, indicating that cross talk between stress-inducible GADD34 and the mTOR signaling pathway plays a critical role in antiviral defense.

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Figures

FIG. 1.

FIG. 1.

Induction of GADD34 mRNA in WT and GADD34-KO MEFs. WT and GADD34-KO MEFs were infected with VSV. (A and B) Induction of GADD34 mRNA. WT and GADD34 MEFs were infected with VSV at MOIs of 0.3 (A) and 100 (B), and the induction of GADD34 mRNA was investigated by RT-PCR as described in Materials and Methods. Tunicamycin (Tn), an ER stress inducer, was used at a concentration of 2 μg/ml for 24 h as a positive control for the induction of GADD34 mRNA. GAPDH mRNA was monitored as an internal control. (C) Changes in cell morphology in WT and GADD34-KO MEFs infected with VSV at an MOI of 0.3. (D) Activation of caspases 3 and 12 in WT and GADD34-KO MEFs infected with VSV (MOI, 0.3). Immunoblot analyses were performed using antibodies to caspase 3, caspase 12, and α-tubulin as described in Materials and Methods.

FIG. 2.

FIG. 2.

Suppression of viral replication by GADD34. (A) Comparison of viral replication in WT (closed bars) and GADD34-KO (open bars) MEFs infected with VSV at MOIs of 0.3 (left) and 100 (right). The titers of infectious VSV at 24 h (MOI, 0.3) and 12 h (MOI, 100) after infection were assayed in Vero cells as described in Materials and Methods. Images of the plaque assay are also shown. Assays were performed in triplicate wells. Each bar indicates mean ± standard deviation. (B) Immunoblot analysis of exogenous GADD34 in GADD34-KO MEFs infected with retroviral vector containing the human GADD34 gene. α-Tubulin was used as an internal control. pCX and GADD34 indicate GADD34-KO MEFs infected with the vector virus, pCXbsr, or the GADD34-expressing virus, pCXbsr/GADD34, respectively. (C) Viral replication in GADD34-KO MEFs transduced with a control (pCX) (open bar) or GADD34-expressing (GADD34) (closed bar) vector. Infectious VSV titers at 24 h after infection (MOI, 0.3) were assayed in Vero cells. Images of the plaque assay are also shown. Assays were performed in triplicate wells. Each bar indicates mean ± standard deviation. (D) Induction of GADD34 mRNA by infection with MRSV or HSV-1. Cellular RNA was isolated from MEFs infected with MRSV (3 days after infection) or with HSV-1 (3 h after infection). (E) Replication of MRSV in WT (closed bar) and GADD34-KO (open bar) MEFs. The MRSV titer at 5 days after infection (MOI, 0.01) was evaluated by transformed-focus-forming ability in 3Y1 cells. Assays were performed in duplicate dishes. Each bar indicates mean ± standard deviation. (F) Replication of HSV-1 in WT (closed bar) and GADD34-KO (open bar) MEFs. Infectious HSV titers at 24 h after infection (MOI, 0.3) were assayed in Vero cells. Assays were performed in triplicate wells. Each bar indicates mean ± standard deviation.

FIG. 3.

FIG. 3.

Viral and cellular protein synthesis and phosphorylation of eIF2α in WT and GADD34-KO MEFs infected with VSV. (A) Immunofluorescence assay of the expression of viral proteins in WT and GADD34-KO MEFs infected with VSV (MOI, 100). The assay was performed at 6 h after infection, using a diluted rabbit anti-VSV serum and an Alexa 488-labeled anti-rabbit IgG according to the procedure described in Materials and Methods. PI staining was also performed. Green fluorescence indicates the expression of VSV proteins. Red fluorescence indicates that the nucleus was stained by PI. Fluorescence was observed under a fluorescence microscope. The percentage of green fluorescence-positive cells among red fluorescence-positive cells is indicated at the right. (B) Immunoblot analysis of viral proteins in WT and GADD34-KO MEFs infected with VSV (MOI, 100; 14 h after infection). The blot was probed with a diluted rabbit anti-VSV serum. The expression of β-actin was used as an internal control. (C) Phosphorylation of eIF2α. Immunoblot analyses were performed using anti-phospho-eIF2α (Ser51) and anti-eIF2α antibodies as described in Materials and Methods. The intensity of each band was quantified, and the ratio of phosphorylated eIF2α (p-eIF2α/eIF2α) is also presented. The expression of β-actin was used as an internal control. (D) Effects of viral infection on cellular protein synthesis in WT and GADD34-KO MEFs. Cells were infected with VSV (MOI, 100), incubated for 13 h, and labeled with Tran35S-label for 30 min. The labeled cells were lysed in RIPA buffer, and 35S incorporation was measured as described in Materials and Methods. Assays were performed in duplicate dishes. Each bar indicates mean ± standard deviation.

FIG. 4.

FIG. 4.

Suppression of the mTOR pathway by GADD34. (A) Changes in the phosphorylation of S6 and 4EBP1 in WT and GADD34-KO MEFs infected with VSV (MOI, 100). Immunoblot analyses were performed using the indicated antibodies as described in Materials and Methods. The intensity of each band was quantified, and the ratio of phosphorylation (p-S6/S6, p-4EBP1/4EBP1) is also presented. The expression of β-actin was used as an internal control. (B) Interaction between GADD34 and TSC1/2. HA-tagged TSC2/Myc-tagged TSC1 was cotransfected with Myc-tagged GADD34 in 293T cells. The protein complex immunoprecipitated by anti-HA antibody was analyzed using anti-Myc and anti-HA antibodies. IP, immunoprecipitation; IB, immunoblotting. (C) Suppression of the phosphorylation of TSC2 at Thr1462 by GADD34 in 293T cells. HA-tagged TSC2/Myc-tagged TSC1 was cotransfected with Myc-tagged GADD34 in 293T cells. The phosphorylation of TSC2 at Thr1462 and of Akt at Ser473 and the expression of TSC2, TSC1 (Myc), and GADD34 (Myc) were analyzed by immunoblotting with the indicated antibodies. (D) Suppression of the phosphorylation of TSC2, S6, and 4EBP1 by ectopic expression of GADD34 in GADD34-KO MEFs. GADD34 was introduced into GADD34-KO MEFs with retroviral vector as shown in Fig. 2B. The phosphorylation and the expression of TSC2, S6, and 4EBP1 were analyzed by immunoblotting with the indicated antibodies.

FIG. 5.

FIG. 5.

Involvement of the mTOR pathway in the suppression of viral replication. (A) Immunoblot analysis of TSC1/2 in TSC2− and TSC2+ cells. (B) Induction of GADD34 mRNA in TSC2− and TSC2+ cells infected with VSV (MOI, 100, 5 h after infection). Isolation of RNA and RT-PCR were performed as described in Materials and Methods. GAPDH mRNA was monitored as an internal control. (C) Viral replication in TSC2− and TSC2+ cells after VSV infection. Titers of VSV at 5 h after infection were assayed in Vero cells. Assays were performed in triplicate wells. Each bar indicates mean ± standard deviation. (D) Immunoblot analysis of phosphorylation of exogenous myristylated Akt and endogenous S6. (E) Viral replication in F2408 cells expressing myristylated Akt (Myr-Akt) and control cells (Vec.). Both cells were infected with VSV (MOI, 0.3), and the titers of VSV at 18 h after infection were determined in Vero cells. Assays were performed in duplicate wells. Each bar indicates mean ± standard deviation. (F) Effects of rapamycin on viral replication in GADD34-KO MEFs. WT and GADD34-KO MEFs were infected with VSV (MOI, 0.3) and cultured with rapamycin (50 and 100 nM). As the rapamycin stock was resolved in dimethyl sulfoxide, 1% dimethyl sulfoxide was added to all cultures. The production of infectious VSV at 12 h after infection was determined in Vero cells. Assays were performed in triplicate wells. Each value indicates mean ± standard deviation.

FIG. 6.

FIG. 6.

Schematic model of the role played by GADD34 and the mTOR pathway in the suppression of viral replication. GADD34 is induced by VSV infection and plays a critical role in the suppression of viral replication. Induced GADD34 interacts with TSC2 to inhibit the mTOR pathway, leading to a suppression of viral protein synthesis and viral replication.

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