Human cytomegalovirus infection maintains mTOR activity and its perinuclear localization during amino acid deprivation - PubMed (original) (raw)

Human cytomegalovirus infection maintains mTOR activity and its perinuclear localization during amino acid deprivation

Amy J Clippinger et al. J Virol. 2011 Sep.

Abstract

The mammalian target of rapamycin (mTOR) kinase is present in 2 functionally distinct complexes, mTOR complex 1 (mTORC1) and complex 2 (mTORC2). Active mTORC1 mediates phosphorylation of eIF4E-binding protein (4E-BP) and p70 S6 kinase (S6K), which is important for maintaining translation. During human cytomegalovirus (HCMV) infection, cellular stress responses are activated that normally inhibit mTORC1; however, previous data show that HCMV infection circumvents stress responses and maintains mTOR kinase activity. Amino acid deprivation is a stress response that normally inhibits mTORC1 activity. Amino acids can signal to mTORC1 through the Rag proteins, which promote the colocalization of mTORC1 with its activator Rheb-GTP in a perinuclear region, thereby inducing 4E-BP and S6K phosphorylation. As expected, our results show that amino acid depletion in mock-infected cells caused loss of mTORC1 activity and loss of the perinuclear localization; however, there was no loss of activity or perinuclear localization in HCMV-infected cells where the perinuclear localization of Rheb-GTP and mTOR coincided with the perinuclear assembly compartment (AC). This suggested that HCMV infection bypasses normal Rag-dependent amino acid signaling. This was demonstrated by short hairpin RNA (shRNA) depletion of Rag proteins, which had little effect on mTORC1 activity in infected cells but inhibited activity in mock-infected cells. Our data show that HCMV maintains mTORC1 activity in an amino acid- and Rag-independent manner through the colocalization of mTOR and Rheb-GTP, which occurs in association with the formation of the AC, thus bypassing inhibition that may result from lowered amino acid levels.

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Figures

Fig. 1.

Fig. 1.

The PI3K-Akt-TSC-mTOR pathway. The general PI3K-Akt-TSC-mTOR pathway is outlined for the activation of mTOR kinase in mTORC1 and subsequent phosphorylation (P) of 4E-BP and S6K. The HCMV UL38 protein protects HCMV-infected cells from many types of cellular stress that signal through the TSC. Amino acid deprivation inhibits mTOR activity through a mechanism that does not depend on the TSC. Amino acid availability can be signaled to mTORC1 through the Rag proteins by regulating the localization of mTORC1 to a perinuclear compartment that contains its activator, Rheb-GTP (35). PI3K, phosphatidylinositol 3-kinase; Akt, protein kinase B; TSC, tuberous sclerosis complex; Rheb, Ras homology enriched in brain; mTORC1, mammalian target of rapamycin complex 1; Rag, Ras-related GTP-binding proteins; 4E-BP, eIF4E binding protein; S6K, p70 S6 kinase.

Fig. 2.

Fig. 2.

mTORC1 activity is maintained in the absence of amino acid signaling. Western blot analysis of mock- or HCMV-infected HFs (A) or U373s (B) following incubation in complete medium (lanes 1 and 6) or in medium lacking amino acids (lanes 2 and 7) for 50 min. Following 50 min of amino acid starvation, amino acids were restored for 3, 10, or 30 min before extraction (lanes 3 to 5 and 8 to 10). All samples were harvested at 48 hpi and analyzed for expression of total or phospho-S6K, total or phospho-4E-BP1, and actin. N/A, not applicable.

Fig. 3.

Fig. 3.

mTOR kinase remains active following depletion of the Rag proteins in HCMV-infected cells. Western analysis of mock- or HCMV-infected cells treated with shRNA specific for luciferase (Luc) or Rags A and B (Rag). Cells were harvested 48 h after mock or HCMV infection (48 hpi), and expression of Rags A and B, phospho-S6K, and total- and phospho-4E-BP1 and expression of the viral early protein UL44 and actin were examined.

Fig. 4.

Fig. 4.

HCMV growth is moderately inhibited following knockdown of the Rag proteins. An HCMV infection time course (0, 72, and 96 hpi) analysis was performed with U373 cells treated with luciferase- (Luc) or Rag A and B (Rag)-specific shRNA. (A) Viral titer was determined at 72 and 96 hpi for cells treated with luciferase-specific (black bars) or Rag-specific (gray bars) shRNAs. (B) Western blot analysis of corresponding protein samples shows effective knockdown of the Rag proteins at these time points; mock is equivalent to that at the zero time point of analysis described for panel A.

Fig. 5.

Fig. 5.

HCMV infection fails to activate mTOR kinase in Rheb-depleted cells. (A) Western analysis of samples from a viral infection time course analysis show that levels of Rheb are increased during HCMV infection of U373 cells. (B) Western analysis of mock- or HCMV-infected cells treated with shRNA specific for luciferase (L) or Rheb (R). U373 cells were harvested at 48 hpi, and expression of Rheb, phosphorylated S6K, and 4E-BP1 and expression of the viral early and late proteins UL44 and pp28 were examined. (C) An HCMV infection time course analysis was performed with U373 cells treated with luciferase (L)- or Rheb (R)-specific shRNA. Viral titer was determined at 72 and 96 hpi for cells treated with a luciferase-specific (black bars) or Rheb-specific (gray bars) shRNA.

Fig. 6.

Fig. 6.

Amino acid (AA) regulation of mTOR localization in normal U373 cells. U373 cells were maintained in complete medium (+AA), medium lacking amino acids for 50 min (−AA), or in medium lacking amino acids for 50 min and subjected to the restoration of amino acids for 30 min (−AA +AA). Cells were stained for mTOR (green), and the nuclei were visualized with DAPI (blue). Under complete-medium conditions, 70% of the cells showed definable mTOR-containing perinuclear regions; this dropped to less than 10% when the amino acids were removed and returned to 70 to 80% when the amino acids were restored.

Fig. 7.

Fig. 7.

mTOR and Rheb localize to the perinuclear viral assembly compartment during HCMV infection. (A) At 72 hpi, HCMV-infected U373 cells were maintained in complete medium (+AA), medium lacking amino acids for 50 min (−AA), or in medium lacking amino acids for 50 min and subjected to the restoration of amino acids for 30 min (−AA +AA). Cells were stained for mTOR (green), HCMV gB (red), and DAPI (blue). Colocalized staining of mTOR and gB was observed in 80% of the cell. (B) Colocalization of mTOR and Rheb in the viral assembly compartment. At 72 hpi, HCMV-infected U373 cells were stained for mTOR (red), Rheb (green), and DAPI (blue).

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