Article Figures & Data
Figures
- FIG. 1.
HCMV replication is inhibited by Torin1. Serum-starved confluent human fibroblasts were infected with HCMV at a multiplicity of 0.05 PFU/cell. Cell-free virus was quantified by a TCID50 assay, and error bars represent the standard errors of the means from two independent experiments, each performed in duplicate. (A) Torin1 inhibits HCMV replication to a greater extent than does rapamycin. Immediately following viral adsorption, cells were treated with vehicle alone (N) (black bars) (dimethyl sulfoxide [DMSO]), rapamycin (T) (gray bars) (20 nM), or Torin1 (T) (white bars) (250 nM). Supernatants were harvested every other day and replaced with fresh medium containing the appropriate treatment, and virus in the supernatant was assayed on the indicated days. (B) Inhibition of HCMV replication is dose dependent and does not result from cellular toxicity. Infected fibroblasts were treated with various doses of Torin1. Medium with drug was replaced every other day, and virus in the supernatant was assayed on day 8 postinfection (black bars). On day 8, a second set of cultures was washed twice, serum-free medium containing no drug was added to each well, and virus was assayed after an additional 8 days (16 days postinfection) (white bars). (C) Torin1 is not toxic to uninfected human fibroblasts. The viability of fibroblasts treated with Torin1 (250 nM) was monitored over a time course of 10 days by a trypan blue exclusion assay.
- FIG. 2.
Torin1 does not affect HCMV entry into fibroblasts. Serum-starved confluent fibroblasts were infected with HCMV at a multiplicity of 3 PFU/cell. (A) Torin1 does not block the entry of viral DNA. Serum-free confluent fibroblasts were pretreated with Torin1 (T) (250 nM) for 24 h prior to infection (Pre) or beginning immediately after adsorption at 1 hpi (Post). Control cultures received the vehicle in which Torin1 was dissolved (NT). At 2 hpi cells were harvested, and cell-associated viral DNA was quantified by real-time PCR analysis. Error bars represent the standard errors of the means from two independent experiments performed in duplicate. (B) Torin1 does not alter the accumulation of the HCMV IE1 protein. The level of IE1 was determined at 6 hpi by a Western blot assay using an IE1-specific monoclonal antibody. The image is representative of two independent experiments. (C) Torin1 does not alter the percentage of infected cells. The expression of a GFP marker gene present in the viral genome was monitored at 24 h after infection in the presence or absence of drug.
- FIG. 3.
Torin1 has little effect on the accumulation of an immediate-early protein and an early protein but inhibits the accumulation of HCMV DNA and a late protein. (A) Rapamycin-resistant mTOR activity is required for the accumulation of an some but not all HCMV proteins. Serum-starved confluent fibroblasts were infected with HCMV at a multiplicity of 3 PFU/cell and then incubated with vehicle (N) (DMSO), rapamycin (R) (20 nM), or Torin1 (T) (250 nM) immediately following adsorption. Cells were harvested at the indicated times, and the accumulation of the indicated proteins was analyzed by Western blotting. (B) Torin1 inhibits HCMV DNA accumulation. Serum-starved confluent human fibroblasts were infected with HCMV at a multiplicity of 0.05 PFU/cell and incubated with vehicle, rapamycin, or Torin1 as described above (A). At the indicated times DNA was isolated, and viral DNA was quantified by qPCR. Equivalent amounts of DNA were analyzed for each sample, and the results are normalized to the level of actin DNA per sample. (C) The levels of the viral late transcript UL99 are inhibited by Torin1 treatment. Fibroblasts were infected with HCMV at a multiplicity of 3 PFU/cell and treated with vehicle, rapamycin, or Torin1 as described above (A). At the indicated times the amount of UL99 RNA was determined by qPCR, and the results are normalized to the amount of actin RNA in each sample.
- FIG. 4.
Rapamycin-resistant mTOR activity is required for 4EBP1 phosphorylation and eIF4F complex integrity during HCMV infection. Serum-starved confluent human fibroblasts were infected with HCMV at a multiplicity of 3 PFU/cell. At 1 hpi, cultures were treated with the vehicle in which drugs were dissolved (N) (DMSO), rapamycin (R) (20 nM), or Torin1 (T) (250 nM). (A) At 48 hpi the phosphorylation status of mTORC1 targets was assessed by Western blot assay by using antibodies to phosphorylated targets (4EBP1-PT37/46 and rpS6-PS235/6) and total proteins. Tubulin was assayed as a loading control. (B) Same as above (A) except that cells were harvested at the indicated times. (C and D) After mock infection (M) or infection with HCMV (WT) at a multiplicity of 3 PFU/cell, cultures were harvested at the indicated times. Equivalent amounts of protein from each sample were incubated with m7GTP-Sepharose, and the isolated protein complexes were analyzed by Western blotting using the indicated antibodies to the eIF4F complex and 4EBP1. In all cases the results are representative of at least two independent experiments. lys, lysate.
- FIG. 5.
MCMV replication is inhibited by Torin1. (A) Torin1 but not rapamycin inhibits the production of MCMV progeny. MEFs were infected with MCMV at a multiplicity of 0.05 PFU/cell and treated with vehicle (black bars) (DMSO), rapamycin (gray bars) (20 nM), or Torin1 (white bars) (250 nM). Fresh serum-free medium containing drugs was added every other day. At the indicated times, cell-free supernatants were harvested, and the amount of virus in the supernatant was quantified by the TCID50 method. Error bars represent the standard errors of the means form two independent experiments performed in duplicate. (B) MEFs were infected with MCMV at a multiplicity of 3 PFU/cell and treated with vehicle (N), rapamycin (R), or Torin1 (T) as described above (A) or were treated with LY294002 (LY) (20 μM). At 48 hpi the phosphorylation state of the indicated mTORC1 targets was analyzed by a Western blot assay by using antibodies to phosphorylated targets (4EBP1-PT37/46 and rpS6-PS235/6) and total proteins. The results are representative of three independent experiments.
- FIG. 6.
mTORC2 and its target Akt are not the source of rapamycin-resistant mTOR activity. (A) MCMV growth is inhibited by Torin1 in Rictor-null MEFs. Confluent serum-starved cells were infected with MCMV at a multiplicity of 0.05 PFU/cell, and vehicle (black bars) (DMSO), rapamycin (gray bars) (20 nM), or Torin1 (white bars) (250 nM) was added at 1 hpi. At 6 days postinfection the amount of MCMV in cell-free supernatants was determined by the TCID50 method. (B) Torin1 blocks 4EBP1 phosphorylation in Rictor-null MEFs. MEFs were mock infected (M) or infected with MCMV (WT) at a multiplicity of 3 PFU/cell and treated with vehicle (N), rapamycin (R), or Torin1 (T) as described above (A). At 48 hpi, the phosphorylation state of mTORC1 targets was assessed by Western blotting using antibodies specific for the indicated proteins. (C) Confirmation of the genotype of Rictor-null MEFs. Total DNA was isolated from wild-type and Rictor-null MEFs, and the genotype was confirmed by use of PCR. (D) Same as above (A) except that Akt1- and Akt2-null MEFs were used. (E) Same as above (B) except that Akt1- and Akt2-null MEFs were used. For B and E, the error bars represent the standard errors of the means from at least two independent experiments, each performed in duplicate. For C and E, tubulin was assayed as a loading control. (F) Akt is not expressed in Akt1- and Akt2-null MEFs. Protein from wild-type or mutant MEFs was analyzed by Western blotting by use of an antibody specific for Akt.
- FIG. 7.
Deletion of the mTORC1 target 4EBP1 rescues replication of MCMV in the presence of Torin1. (A) MCMV growth is not inhibited by Torin1 in 4EBP1-null MEFs. Confluent serum-starved cells were infected with MCMV at a multiplicity of 0.05 PFU/cell, and vehicle (black bars) (DMSO), rapamycin (gray bars) (20 nM), or Torin1 (white bars) (250 nM) was added at 1 hpi. At 6 days postinfection the amount of MCMV in cell-free supernatants was determined by the TCID50 method. The error bars represent the standard errors of the means from three independent experiments, each performed in duplicate. (B) Torin1 does not exclude eIF4G or eIF4A from the cap-binding complex in 4EBP1-null MEFs. Cells were infected with MCMV at a multiplicity of 3 PFU/cell and treated with vehicle (N), rapamycin (R), or Torin1 (T) as described above (A). At 48 hpi equal amounts of protein from cell lysates were incubated with m7G-Sepharose. The presence of eIF4F complex components bound by the cap analog was determined by Western blotting. The results are representative of two independent experiments.
- FIG. 8.
Rapamycin-resistant mTOR activity is required for lytic replication by representative alpha- and gammaherpesviruses. (A) Confluent serum-starved MEFs were infected at a multiplicity of 0.05 PFU/cell with HSV-1 or γHV68. The amount of virus in cell-free supernatants was determined by the TCID50 method at 72 hpi for HSV-1 (left) and at 6 days postinfection for γHV68 (right). Black bars represent vehicle-treated samples (N) (DMSO), gray bars represent rapamycin-treated samples (R) (20 nM), and white bars represent Torin1-treated samples (T) (250 nM). The error bars represent the standard errors of the means from at least two independent experiments. (B) Confluent MEF monolayers were infected with HSV-1 at a multiplicity of 3 PFU/cell. Infected cell lysates were harvested at 8 hpi, and equal amounts of protein were analyzed by Western blotting. (C) WT or 4EBP1-null MEFs were infected with HSV-1 at a multiplicity of 0.05 PFU/cell. The amount of cell-free virus present in the supernatant at 72 hpi was quantified by the TCID50 method. The error bars represent the standard errors of the means from two independent experiments.
