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. 2007 Aug 1;81(20):11106–11115. doi: 10.1128/JVI.01063-07

Suppression of Viral Replication by Stress-Inducible GADD34 Protein via the Mammalian Serine/Threonine Protein Kinase mTOR Pathway▿

Kahori Minami 1, Yukihiro Tambe 2, Ryosuke Watanabe 1, Takahiro Isono 3, Masataka Haneda 4, Ken-ichi Isobe 4, Toshiyuki Kobayashi 5, Okio Hino 5, Hidetoshi Okabe 1, Tokuhiro Chano 1,6, Hirokazu Inoue 2,*
PMCID: PMC2045534  PMID: 17670836

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 eIF2α 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.

GADD34 (growth arrest and DNA damage protein 34) belongs to a family of proteins whose expression is increased by growth arrest and DNA damage (12, 43). It is also a major regulator of translation during conditions of cell stress such as heat shock (16), nutrient deprivation (26), energy depletion (41), and exposure to agents that cause the improper folding of proteins in the endoplasmic reticulum (ER) (27). Cells respond to stress by turning off protein synthesis through the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) at serine 51 (14). Phosphorylated eIF2α induces the expression of GADD34, which forms a functional complex with protein phosphatase 1 (PP1) to dephosphorylate eIF2α, which in turn leads to the restoration of protein synthesis (27). A study of GADD34-deficient mice has demonstrated that GADD34 is required for eIF2α dephosphorylation and recovery from a shutoff of protein synthesis in response to ER stress (22). These processes appear to be critically involved in translational control during conditions of cell stress. Although viral infection has been shown to induce GADD34 through the phosphorylation of eIF2α by PKR and PERK (7), the role played by GADD34 in viral infection remains to be elucidated. Another regulator of the translational machinery in response to cellular stress is target of rapamycin (TOR), which is an evolutionarily conserved serine/threonine protein kinase. The mammalian homologue of TOR, mTOR, stimulates protein synthesis by phosphorylating the major mTOR targets, ribosomal protein S6 kinase (S6K) and eukaryotic initiation factor 4E binding protein (4EBP1). mTOR plays a pivotal role in controlling cell growth via its regulation of translation by the phosphorylation of S6K and 4EBP1 (11, 19, 35, 42). Phosphorylation of S6K and 4EBP1 transduces nutrient and growth factor signals by modifying the rate of protein synthesis. The tuberous sclerosis complex 1 (TSC1) and TSC2 genes encode the tumor suppressors hamartin and tuberin, respectively, which are potent inhibitors of the mTOR pathway (2, 11, 19, 35, 42). Various types of cellular stress, such as glucose (18) or amino acid starvation (13) and hypoxia (5), activate TSC1/TSC2 function and inhibit mTOR function, thereby leading to the suppression of protein synthesis (30).

Although mTOR- and GADD34-mediated signaling pathways have been thought to be independently regulated, we have previously shown that GADD34 induced by energy depletion forms a stable complex with TSC1/2 and inhibits mTOR signaling (41). This finding suggested that cross talk between GADD34 and mTOR signaling pathways regulates the protein synthetic machinery in response to environmental stress. In the present study, we investigated the role of GADD34 in antiviral protection using GADD34-deficient (GADD34-KO) mouse embryo fibroblasts (MEFs) infected with vesicular stomatitis virus (VSV) and revealed that GADD34 suppresses viral replication via inhibition of the mTOR pathway.

MATERIALS AND METHODS

Materials.

Anti-human GADD34 (H-193), anti-TSC2 (C-20), anti-eIF2α (FL-315), and anti-Myc (9E10) antibodies were purchased from Santa Cruz Biotechnology. Antihemagglutinin (anti-HA) (3F9) rat high-affinity antibody was obtained from Roche. Anti-α-tubulin (DM1A) and anti-β-actin antibodies were purchased from Sigma. Anti-phospho-eIF2α (Ser51) antibody was purchased from Biosource International. Anti-phospho-S6 (Ser240/244), anti-phospho-4EBP1 (Thr37/46), anti-TSC1, anti-Akt, anti-phospho-Akt (Ser473) anti-caspase 3, and anti-caspase 12 antibodies were purchased from Cell Signaling Technology. Anti-VSV antibody was provided by Bin Gotoh (Shiga University of Medical Science). Rapamycin was purchased from the LC Laboratory. A full-length human GADD34 (GenBank accession no. BC003067) was purchased from Open Biosystems and subcloned into Myc-tagged pcDNA3 vector or pCXbsr retroviral vector (1).

Cells and viruses.

GADD34-KO (22) and wild-type (WT) MEFs were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). TSC2-null (TSC2−) cells were derived from Eker rat renal carcinoma (28). Stable TSC2-expressing (TSC2+) cells were generated from TSC2− cells by the introduction of the plasmid expressing human TSC2 cDNA (GenBank accession no. NM000548) (28). TSC2− and TSC2+ cells were cultured in RPMI medium supplemented with 10% FBS. F2408 is a rat fibroblast cell line. F2408 cells containing the expression plasmid for constitutively active (myristylated) Akt (pcDNA3-Myr-Akt) and the vector plasmid (pcDNA3) were cultured in DMEM supplemented with 5% FBS. 293T, Vero, and 3Y1 cells were cultured in DMEM supplemented with 10% FBS. VSV (New Jersey strain), herpes simplex virus type 1 (HSV-1) (KOS strain), and murine Rous sarcoma virus (MRSV), a murine recombinant retrovirus containing the v-src oncogene which contains murine leukemia virus as a helper (29), were used in this study. The recombinant retrovirus expressing GADD34 (pCXbsr/GADD34) was constructed by insertion of the human GADD34 cDNA (GenBank accession no. BC003067) into a retroviral vector, pCXbsr (1). GADD34-KO MEFs were transduced with pCXbsr/GADD34 or pCXbsr according to a previously described method (38). Virus-infected cells were selected in the presence of blasticidin (5 μg/ml), and blasticidin-resistant cells were collected, expanded, and used in subsequent experiments.

Plaque assay for VSV and HSV-1.

Inoculation of 5 × 105 WT or GADD34-KO MEF cells was carried out using 60-mm dishes containing DMEM supplemented with 10% FBS. After 24 h of incubation, the virus was added to the culture at a multiplicity of infection (MOI) of 0.3 or 100 PFU per cell. After incubation for 1 h at 37°C, the cells were washed once with phosphate-buffered saline (PBS), and 5 ml of DMEM containing 2% FBS was added to the cultures. Virus-infected cultures were then further incubated for 24 h (MOI, 0.3) or 14 h (MOI, 100). The cells, together with the culture medium, were harvested, and the virus stock was prepared by freeze-thawing and centrifuging the lysate at 2,000 rpm for 20 min to eliminate the cell debris. The titer of the virus stock was assayed for infectivity by the plaque method using Vero cells. Monolayered Vero cells were infected with several dilutions of the viral stock and were incubated for 1 h at 37°C. Unadsorbed viruses were then removed by washing, and an overlay of DMEM supplemented with 2% FBS and 0.4% methylcellulose was added to the cultures. The cultures were incubated until plaques were readily visible (∼24 h) and were then fixed in formaldehyde and stained with 1% crystal violet in 20% ethanol.

Focus assay for MRSV.

Inoculation of 2 × 105 of MEF cells was carried out in 60-mm dishes containing DMEM supplemented with 10% FBS. After 16 h of incubation, the cells were treated with Polybrene (2 μg/ml) for 30 min and infected with MRSV (29). Three days after infection, the culture medium was recovered from the MRSV-infected MEFs and was filtrated; the titer was then assayed in 3Y1 cells. Ten days after infection, the number of transformed foci was counted.

35S metabolic labeling.

Virus- and mock-infected cell cultures were incubated for 13 h in DMEM supplemented with dialyzed 2% FBS, and newly synthesized proteins were labeled with Tran35S-label (100 μCi/dish) (MP Biomedical, Inc.) in methionine- and cysteine-free DMEM supplemented with dialyzed 2% FBS for 30 min. The labeled cells were lysed in radioimmunoprecipitation assay (RIPA) buffer containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), and 1% protease inhibitor cocktail (Nacalai Tesque) and were centrifuged for 20 min at 10,000 × g at 4°C. Twenty microliters of the supernatant was precipitated with 1 ml of trichloroacetic acid, and the radioactivity of the labeled proteins was measured using a liquid scintillation counter. The protein contents of the lysates were also assayed using a Bio-Rad protein assay kit. 35S incorporation was indicated as counts per minute per protein (μg).

Immunoprecipitation.

The cells were lysed in RIPA buffer containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1% protease inhibitor cocktail (Nacalai Tesque), and 1% protein phosphatase inhibitor cocktail (Sigma) and were centrifuged for 30 min at 10,000 × g at 4°C. The supernatant was incubated with primary antibody at 4°C for 1 h. The immunocomplexes were bound to protein G-Sepharose for 1 h at 4°C and washed three times with RIPA buffer. The proteins bound to the protein G-Sepharose were eluted by adding Laemmli SDS sample buffer containing 62.5 mM Tris-HCl (pH 6.8), 10% glycerol, 5% 2-mercaptoethanol, 2% SDS, and 0.01% bromophenol blue and boiling for 3 min. After centrifugation at 10,000 × g for 2 min, the supernatant was analyzed by immunoblotting.

Immunoblot analysis.

The cells were lysed in Laemmli SDS sample buffer. A sample of cell lysate was subjected to SDS-polyacrylamide gel electrophoresis, and the separated proteins were electrotransferred to membrane filters (Immobilon-P; Millipore). After blocking of the filters with TBS-T (10 mM Tris-HCl [pH 7.6], 150 mM sodium chloride, 0.1% Tween 20) containing 5% bovine serum albumin (BSA), the filters were incubated overnight with the indicated primary antibodies in TBS-T containing 2% BSA at 4°C. The filters were then washed in TBS-T and were incubated for 1 h in horseradish peroxidase-conjugated anti-mouse, anti-rabbit, or anti-rat immunoglobulin G (IgG) (GE Healthcare Bio-Science Corp.) diluted 1:20,000 in TBS-T containing 2% BSA. After several washes with TBS-T, the immunoreactivity was detected using the ECL system (GE Healthcare Bio-Science Corp.) according to the procedures recommended by the manufacturer.

Immunofluorescence assay.

Cells were cultured overnight on Biocoat poly-l-lysine cellware chamber slides (four well; Becton Dickinson) to 70% confluence and then were infected with VSV at an MOI of 100. After incubation, the cells were fixed with 1% paraformaldehyde for 10 min, permeabilized with 0.1% Triton X-100 for 5 min, and incubated overnight with rabbit anti-VSV serum at a dilution of 1:100. The coverslips were then washed with PBS and were then incubated with Alexa 488 anti-rabbit IgG antibody-conjugated secondary antibody (Molecular Probes) and propidium iodide (PI). After the coverslips were washed with PBS to remove excess fluorescent dye, they were mounted with Prolong Antifade (Molecular Probes). The specimens were observed and photographed under identical conditions using a fluorescence microscope fitted with a charge-coupled device camera (Leica Microsystems).

Reverse transcription-PCR (RT-PCR).

Total RNA was isolated from the cells using TRIzol (Invitrogen), and the first-strand cDNA was synthesized using Superscript III reverse transcriptase and oligo(dT)12-18 primer (Invitrogen). The following gene primer sequences were used: murine GADD34, 5′-CTGCAAGGGGCTGATAAGAG (forward) and 5′-AGGGGTCAGCCTTGTTTTCT (reverse); murine GAPDH (glyceraldehyde-3-phosphate dehydrogenase), 5′-CATGACAACTTTGGCATTGTG (forward) and 5′-GTTGAAGTCGCAGGAGACAAC (reverse). The PCR consisted of 26 cycles for GADD34 and 21 cycles for GAPDH according to the following protocol: 20 s of denaturation at 95°C, 20 s of annealing at 55°C, and 30 s of extension at 72°C.

RESULTS

Enhancement of viral replication in GADD34-KO MEFs infected with VSV.

To clarify the role played by GADD34 in viral infection, WT and GADD34-KO MEF cells were infected with VSV, and the subsequent induction of GADD34 mRNA was examined by RT-PCR. Tunicamycin, an ER stress inducer, was used as a positive control for the induction of GADD34 mRNA in WT MEFs (Fig. 1A and B). As shown in Fig. 1, the expression of GADD34 mRNA was induced from 9 and 3 h after VSV infection at MOIs of 0.3 (Fig. 1A) and 100 (Fig. 1B), respectively, in WT MEFs, but no induction was observed in GADD34-KO MEFs. In GADD34-KO MEFs infected with VSV (MOI, 0.3), cell death was observed at 18 h after infection, whereas only negligible cell death was seen in WT MEFs (Fig. 1C). As shown in Fig. 1D, marked activation of caspases 3 and 12 was detected starting 14 h after infection in GADD34-KO MEFs, whereas both caspases were weakly activated starting 14 h after infection in WT MEFs, indicating that the cell death in VSV-infected MEFs was caspase-mediated apoptosis. To evaluate viral replication in WT and GADD34-KO MEFs, the titers of infectious VSV produced in virus-infected MEFs were examined in Vero cells using a plaque assay. At 24 h (MOI, 0.3) or 14 h (MOI, 100) after infection, the production of infectious VSV in GADD34-KO MEFs was markedly elevated at MOIs of 0.3 (42-fold) and 100 (20-fold), compared with that in WT MEFs (Fig. 2A), suggesting that GADD34 suppresses VSV replication. To verify the suppressive function of GADD34 with respect to viral replication, we introduced GADD34 into GADD34-KO MEFs using a retroviral vector (Fig. 2B) and examined VSV replication in these cells. As shown in Fig. 2C, GADD34 rescue into GADD34-KO MEFs significantly suppressed the replication of VSV. Taken together, these results indicate that the induction of GADD34 by VSV is responsible for the suppression of viral replication.

FIG. 1.

FIG. 1.

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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.

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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.

To assess whether or not GADD34 is also able to suppress the replication of other viruses, WT and GADD34-KO MEFs were infected with MRSV, a murine recombinant retrovirus containing the v-src oncogene (29), or HSV-1. As shown in Fig. 2D, induction of GADD34 mRNA was observed only in WT MEFs infected with MRSV and HSV-1. In the case of MRSV, viral replication was estimated based on viral ability to form transformed foci in 3Y1 cells. MRSV replication was markedly elevated in GADD34-KO MEFs compared with that in WT MEFs (Fig. 2E). On the other hand, the replication of HSV-1, which was examined by plaque assay in Vero cells, was similar in WT and GADD34-KO MEFs (Fig. 2F). These results indicate that the effect exerted by GADD34 on viral replication is not limited to VSV, although this effect is not common among viruses.

To exclude the possibility that the observed differences in viral replication between WT and GADD34-KO MEFs resulted from different efficiencies of viral infection, the expression of VSV proteins was evaluated by immunofluorescence assay with anti-VSV serum. As shown in Fig. 3A, the percentage of the cells expressing viral proteins (green fluorescence) was similar in WT (96%) and GADD34-KO (100%) MEFs, but the immunofluorescence intensity in each cell was stronger in GADD34-KO MEFs than in WT MEFs at 6 h after VSV infection. Immunoblot analyses revealed markedly higher levels of expression of VSV proteins in GADD34-KO MEFs than in WT MEFs at 14 h after VSV infection (Fig. 3B). These results indicate that although VSV similarly infected WT and GADD34-KO MEFs, the production of viral proteins was low in WT MEFs and elevated in GADD34-KO MEFs, suggesting that the suppression of VSV replication in WT MEFs was due to the reduced production of VSV proteins.

FIG. 3.

FIG. 3.

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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.

Phosphorylation of eIF2α and cellular protein synthesis in WT and GADD34-KO MEFs infected with VSV.

During viral infection, eIF2α is phosphorylated at Ser51 by PKR and PERK to turn off cellular protein synthesis. To clarify the mechanism of suppression of viral replication by GADD34, we initially evaluated the changes in phosphorylation of eIF2α at Ser51 in WT and GADD34-KO MEFs infected with VSV (MOI, 100). As shown in Fig. 3C, phosphorylation of eIF2α at Ser51 was increased at 6 h but decreased at 14 h after infection in WT MEFs, suggesting that VSV infection increases the phosphorylation of eIF2α during the early phase of infection (6 h) but that GADD34 induced by phosphorylated eIF2α associates with PP1 to dephosphorylate eIF2α in the late phase of infection (14 h). On the other hand, the baseline level (0 h) of phosphorylated eIF2α in GADD34-KO MEFs was higher than that in WT MEFs (0 h) and increased further at 6 h after VSV infection. This latter, increased level of phosphorylation was retained even during the late phase of infection (14 h). These results indicate that increased phosphorylation of eIF2α is not always correlated with a suppression of viral replication. To clarify whether or not enhanced VSV replication in GADD34-KO cells is due to a dysregulation of cellular protein synthesis, we assessed the effects of VSV infection on cellular protein synthesis in WT and GADD34-KO MEFs. VSV- or mock-infected cells were labeled with [35S]methionine for 30 min at 13 h after infection (MOI, 100), and the levels of incorporated radioactivity were compared. As shown in Fig. 3D, the ongoing protein synthesis in virus-infected cells had significantly decreased in both WT and GADD34-KO MEFs, indicating that translational repression by eIF2α phosphorylation does not lead to the suppression of VSV replication in GADD34-KO MEFs.

Suppression of mTOR pathway by GADD34 in MEFs infected with VSV.

Another important regulatory mechanism of protein synthesis is the mTOR signaling pathway, which is known to be suppressed by stressors such as energy depletion, nutrient deprivation, and hypoxia, via the activation of TSC1/2 (11, 19, 30, 35, 42). Recently, we reported that GADD34 formed a stable complex with TSC1/2, dephosphorylated TSC2, and inhibited mTOR signaling during conditions of glucose starvation (41). To assess the involvement of the mTOR pathway in the suppression of viral replication by GADD34, we investigated changes in the phosphorylation of S6 and 4EBP1, both proteins downstream of mTOR, in WT and GADD34-KO MEFs infected with VSV (MOI, 100). As shown in Fig. 4A, the phosphorylation of S6 (Ser240/244) and 4EBP1 (Thr37/46) was suppressed at 14 h after infection in WT MEFs but not in GADD34-KO MEFs. These results indicate that the GADD34 induced by VSV infection serves to suppress the mTOR pathway. To evaluate the ability of GADD34 to negatively regulate the mTOR pathway via TSC1/2, 293T cells were cotransfected with Myc-tagged GADD34 and HA-tagged TSC2/Myc-tagged TSC1; the association of GADD34 with TSC1/2 and the phosphorylation of TSC2 at Thr1462, which is known to be phosphorylated by Akt to activate the mTOR pathway (17), were then investigated. As shown in Fig. 4B, TSC2 immunoprecipitated with anti-HA antibody interacted with Myc-tagged GADD34 and TSC1, which indicated that GADD34 is able to form a complex with TSC1/2. In the same cotransfection experiment, we also evaluated the phosphorylation of TSC2 at Thr1462. As shown in Fig. 4C, TSC2 was significantly dephosphorylated by GADD34. Phosphorylation of endogenous Akt at Ser473 was not affected by the expression of GADD34, indicating that the decreased phosphorylation of TSC2 at Thr1462 is not mediated by Akt kinase (Fig. 4C). To confirm the interaction of GADD34 and the mTOR pathway, the effect of GADD34 on the phosphorylation of TSC2, S6, and 4EBP1 was examined in GADD34-KO MEFs. As shown in Fig. 4D, ectopic expression of GADD34 by retroviral vector markedly suppressed phosphorylation of TSC2 (Thr1462), S6 (Ser240/244), and 4EBP1 (Thr37/46) in GADD34-KO MEFs. These results suggest that GADD34 functions as a negative regulator of the mTOR pathway by binding to and dephosphorylating TSC2 during viral infection.

FIG. 4.

FIG. 4.

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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.

Involvement of TSC2 and mTOR in the suppression of viral replication.

TSC2 is a potent negative regulator of mTOR. If the regulation of TSC2 by GADD34 indeed plays a critical role in the suppression of VSV replication, then enhanced viral replication would be expected in TSC2-null cells compared to that in TSC2-expressing cells. To assess the involvement of TSC2 in the suppression of VSV replication, we compared viral replication in TSC2− and TSC2+ cells following VSV infection. Immunoblot analyses verified that TSC2 protein was expressed only in TSC2+ cells and not in TSC2− cells (Fig. 5A). TSC1 protein was expressed in both types of cells, although the level of expression in TSC2− cells was lower than that in TSC2+ cells (Fig. 5A). Similar levels of GADD34 mRNA were induced in both types of cells after VSV infection (Fig. 5B). In TSC2− cells, however, viral replication was elevated (3.5-fold) compared with that in TSC2+ cells (Fig. 5C), thus indicating that TSC2 is involved in the suppression of viral replication by GADD34. To additionally evaluate whether the mTOR pathway plays a critical role in VSV replication, the effect of constitutively active (myristylated) Akt, which activates the mTOR pathway via phosphorylation of TSC2, was investigated in a rat fibroblast cell line, F2408. As shown in Fig. 5D, the ectopic expression of myristylated Akt enhanced phosphorylation of S6, a downstream protein of mTOR, in F2408 cells. VSV replication was also elevated (7.4-fold) in the cells expressing myristylated Akt compared with control cells (Fig. 5E). Furthermore, to confirm the involvement of mTOR in the suppression of viral replication by GADD34, we examined the effect of rapamycin, a specific inhibitor of mTOR kinase, on viral replication in GADD34-KO MEFs. As shown in Fig. 5F, treatment with rapamycin suppressed viral replication in a dose-dependent manner in GADD34-KO MEFs, confirming that the mTOR pathway is involved in the suppression of viral replication. Taken together, these results indicate that GADD34 induced by VSV infection inhibits the mTOR pathway by activating TSC1/2 and that the TSC-mTOR pathway plays a critical role in the suppression of viral replication (Fig. 6).

FIG. 5.

FIG. 5.

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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.

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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.

DISCUSSION

During viral infection, double-stranded RNA-dependent protein kinase (PKR) is activated and phosphorylates eIF2α at Ser51 (21). Phosphorylated eIF2α leads to the suppression of cellular protein synthesis. Another eIF2α kinase, the PKR-like ER-resident protein kinase PERK, also contributes to cellular resistance to viral infections (3, 33). These processes have been thought to play critical roles in translational control and antiviral host defenses. Although GADD34 has been shown to be induced by eIF2α, which is phosphorylated by PKR and PERK (7), the physiological significance with respect to viral infection has not been fully elucidated. In the present study, WT and GADD34-KO MEF cells were used to demonstrate that GADD34 is induced by VSV infection and that it plays a critical role in the suppression of viral replication. This suppression of viral replication by GADD34 was mediated by inhibition of the mTOR signaling pathway. GADD34 was able to bind to TSC1/2 and dephosphorylate TSC2 at Thr1462. Activation of the mTOR pathway by the loss of TSC2 or the expression of constitutively active Akt enhanced VSV replication. Furthermore, viral replication in GADD34-KO cells was significantly inhibited by treatment with rapamycin. These findings suggest that an interaction between stress-inducible GADD34 and the mTOR pathway contributes to the antiviral host defense (Fig. 6).

Although GADD34-KO MEFs were highly sensitive to viral replication of VSV and MRSV, no difference in the replication of HSV-1 was observed between WT and GADD34-KO MEFs. Unlike other viruses, HSV-1 encodes the γ1 34.5 protein, which is homologous to and antagonizes the C-terminal region of GADD34 (8). This protein may help viruses evade cellular responses against viral infection in WT MEFs. This finding also supports the protective role of GADD34 in viral infection. To elucidate the mechanism of GADD34 suppression of viral replication, we initially focused on the phosphorylation status of eIF2α in VSV-infected cells, because increased phosphorylation of eIF2α has been thought to lead to the complete suppression of cellular protein synthesis, resulting in the suppression of viral replication (21, 36, 37). However, the global suppression of cellular protein synthesis via enhanced phosphorylation of eIF2α was not correlated with an inhibition of viral replication by GADD34. The production of VSV proteins and viral replication were markedly enhanced in GADD34-KO MEFs, irrespective of increased phosphorylation of eIF2α and the suppression of cellular protein synthesis. These findings mean that cellular translational control by eIF2α does not always affect viral translation and replication. Another cellular mechanism is required to account for why GADD34-KO cells are highly sensitive to VSV replication.

We then turned our attention to the mTOR pathway, which is also known to regulate translational machinery in response to cellular stress. The mTOR signaling pathway contributes to the protein synthetic machinery response to environmental stressors such as energy depletion, nutrient deprivation, and hypoxia (5, 13, 18, 30). Unlike eIF2α, mTOR is involved in the translational regulation of more limited types of mRNAs, which contain a 5′-TOP (tract of pyrimidines) sequence or a 5′-cap structure, via its downstream effectors, S6K and 4EBP1 (24, 31). The mTOR activity monitored by the phosphorylation of S6 (a downstream target of S6K), and 4EBP1 was inhibited in VSV-infected WT MEFs but not in GADD34-KO MEFs, which suggested the contribution of mTOR-mediated translational control to the cellular protection against viral infection. This idea was further supported by the following experiments. First, TSC2-null cells showed high sensitivity to VSV replication. Second, activation of mTOR by constitutively active Akt also enhanced VSV replication. Third, rapamycin, a specific inhibitor of mTOR, inhibited VSV replication in GADD34-KO MEFs. The discrepancy between reduction of cellular protein synthesis and enhancement of viral protein synthesis in GADD34 KO MEFs may be explained by the differences in the kinds of mRNAs regulated by eIF2α and mTOR. Although viral replication is frequently accompanied by a “shutoff” of cellular protein synthesis to selectively enhance viral protein synthesis (15, 34), the mechanism of action is not fully understood. Elucidation of the regulation of viral protein synthesis by mTOR signaling may provide novel insights into this problem; further investigation of the molecular mechanism will be necessary.

TSC1 and TSC2 are known to be potent inhibitors of the mTOR pathway. Various signals from growth factors or cellular stresses affect TSC1/TSC2 function to regulate mTOR activity, in turn leading to the control of protein synthesis. Growth factor signals such as insulin activate Akt, which phosphorylates and inactivates TSC2, resulting in the activation of mTOR (17). On the other hand, energy depletion activates AMP-activated protein kinase, which phosphorylates and activates TSC2, leading to the inhibition of mTOR (18). Thus, TSC2 is a key molecule in the regulation of mTOR signaling. Our previous report indicated that GADD34 interacts with TSC2 to negatively regulate mTOR upon glucose depletion (41). In the present study, we demonstrated that expression of GADD34 enabled binding to TSC1/2 and the subsequent dephosphorylation of TSC2 at Thr1462, a site phosphorylated by Akt, in 293T and GADD34-KO MEF cells. Although GADD34 does not function as a phosphatase by itself, GADD34 has been shown to bind to the serine/threonine phosphatase PP1 at its C-terminal KVRF domain, recruits PP1 to various molecules, and modulates their phosphorylated status and the respective enzymatic activities (6). PP1 is most likely associated with GADD34 function in the dephosphorylation of TSC2 to inhibit the mTOR pathway. Previously, we have shown that a C-terminal mutant lacking the PP1 binding motif exerted no inhibitory effects on S6K phosphorylation, although the WT form and the C-terminal domain of GADD34 were able to inhibit the phosphorylation of S6K (41), suggesting the involvement of PP1 in dephosphorylation of TSC2 by GADD34.

Since a variety of cellular stresses induce GADD34 expression (4, 16, 26, 27, 39, 41), the inhibition of mTOR function by GADD34 might be fundamental to the cellular response to various stresses. This study in particular provided novel insights for gaining a better understanding of the functional relationship between mTOR-mediated translational control and the cellular response to viral infection. Recently, mTOR has been shown to suppress autophagy, which is also involved in the defense against viral infections (10). The mTOR-mediated regulation of autophagy may also contribute to the suppression of viral replication by GADD34. Altered signaling by mTOR has been shown to be linked to several diseases, including cancer, diabetes, neurodegenerative disorders, and obesity (9, 20, 23, 25, 40). A specific inhibitor of mTOR, rapamycin, is known to be an anticancer drug and is successfully used in clinical applications. Rapamycin has also been shown to reverse Huntington's disease in Drosophila and mouse models (32). In the search for novel treatments for infectious diseases, mTOR signaling modulation by GADD34 and/or rapamycin remains of considerable interest.

Acknowledgments

We thank Hiroko Kita (Department of Microbiology, Shiga University of Medical Science) for technical assistance. We thank K. L. Guan (University of Michigan Medical School) for the pcDNA3-Myc-TSC1 and pcDNA3-HA-TSC2 plasmids. We also thank Masahiro Aoki (Kyoto University) for the Myr-Akt expression plasmid. VSV (New Jersey strain) was provided by the National Institute of Animal Health, Japan. Anti-VSV serum was a generous gift from Bin Gotoh (Department of Microbiology, Shiga University of Medical Science).

This project was supported by grants-in-aid for PRESTO, JST (to T. Chano), a Grant for Scientific Research on Priority Areas (no. 17013038 and 18012022) (to T. Chano), and a Grant-in Aid for Scientific Research (C) (no. 17590341) (to H. Inoue) from the Ministry of Education, Science, Sports, and Culture of Japan.

Footnotes

▿

Published ahead of print on 1 August 2007.

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