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Influenza virus differentially activates mTORC1 and mTORC2 signaling to maximize late stage replication.

ABSTRACT: Influenza A virus usurps host signaling factors to regulate its replication. One example is mTOR, a cellular regulator of protein synthesis, growth and motility. While the role of mTORC1 in viral infection has been studied, the mechanisms that induce mTORC1 activation and the substrates regulated by mTORC1 during influenza virus infection have not been established. In addition, the role of mTORC2 during influenza virus infection remains unknown. Here we show that mTORC2 and PDPK1 differentially phosphorylate AKT upon influenza virus infection. PDPK1-mediated phoshorylation of AKT at a distinct site is required for mTORC1 activation by influenza virus. On the other hand, the viral NS1 protein promotes phosphorylation of AKT at a different site via mTORC2, which is an activity dispensable for mTORC1 stimulation but known to regulate apoptosis. Influenza virus HA protein and down-regulation of the mTORC1 inhibitor REDD1 by the virus M2 protein promote mTORC1 activity. Systematic phosphoproteomics analysis performed in cells lacking the mTORC2 component Rictor in the absence or presence of Torin, an inhibitor of both mTORC1 and mTORC2, revealed mTORC1-dependent substrates regulated during infection. Members of pathways that regulate mTORC1 or are regulated by mTORC1 were identified, including constituents of the translation machinery that once activated can promote translation. mTORC1 activation supports viral protein expression and replication. As mTORC1 activation is optimal midway through the virus life cycle, the observed effects on viral protein expression likely support the late stages of influenza virus replication when infected cells undergo significant stress.

SUBMITTER: Kuss-Duerkop SK

PROVIDER: S-EPMC5617226 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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Influenza virus differentially activates mTORC1 and mTORC2 signaling to maximize late stage replication.

Kuss-Duerkop Sharon K SK Wang Juan J Mena Ignacio I White Kris K Metreveli Giorgi G Sakthivel Ramanavelan R Mata Miguel A MA Muñoz-Moreno Raquel R Chen Xiang X Krammer Florian F Diamond Michael S MS Chen Zhijian J ZJ García-Sastre Adolfo A Fontoura Beatriz M A BMA

PLoS pathogens 20170927 9

Influenza A virus usurps host signaling factors to regulate its replication. One example is mTOR, a cellular regulator of protein synthesis, growth and motility. While the role of mTORC1 in viral infection has been studied, the mechanisms that induce mTORC1 activation and the substrates regulated by mTORC1 during influenza virus infection have not been established. In addition, the role of mTORC2 during influenza virus infection remains unknown. Here we show that mTORC2 and PDPK1 differentially ...[more]

PMID: 28953980

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Project description:BACKGROUND:The mechanistic target of rapamycin (mTOR) pathway is a potential target to inhibit pathologic processes in choroidal neovascularization. However, the exact role of mTOR signaling in the development of CNV remains obscure. In this study, we assessed the role of mTORC1 and mTORC2 as well as the effect of rapamycin (sirolimus) on choroidal neovascularization (CNV) in a laser-induced mouse model. METHODS:In experiment A, we observed the natural course of CNV development and the dynamics of mTOR-related proteins during the 12?days after the laser injury. The expression of mTOR-related proteins was evaluated using Western blot (WB). Cryosections of CNV-induced mice were immunostained for the visualization of the vascular and extravascular components of the CNV. Experiment B was performed to confirm the critical period of mTOR signaling in the development of laser-induced CNV, we administered rapamycin before and/or during the active period of mTOR complexes. WB and immunofluorescence staining was performed to evaluate the mode of action and the effect of mTOR inhibition on CNV development. RESULTS:In experiment A, we detected high levels of p-mTOR S2448 and p-mTOR S2481 from the 5th to 12th day of laser injury. Immunofluorescence imaging of cryosections of mice sacrificed on day 7 revealed greater co-immunoreactivity of p-mTOR S2448 positive cells with CD11b and F4/80, while p-mTOR S2481 positive cells showed colocalization with CD31, ?-SMA, and cytokeratin. In experiment B, rapamycin injection during the active period of mTOR signaling demonstrated near-complete inhibition of CNV lesion as well as significant induction of autophagy. CONCLUSION:Our study suggests the mTOR as a critical player during CNV development in laser-induced mouse model through differentially acting with the mTORC1 and mTORC2. mTORC1 activity was high predominantly in inflammatory cells in CNV lesion, while mTORC2 activity was higher in vascular components and the RPE.

Project description:ObjectiveTetrameric α(2)-macroglobulin (α(2)M), a plasma panproteinase inhibitor, is activated upon interaction with a proteinase, and undergoes a major conformational change exposing a receptor recognition site in each of its subunits. Activated α(2)M (α(2)M*) binds to cancer cell surface GRP78 and triggers proliferative and antiapoptotic signaling. We have studied the role of α(2)M* in the regulation of mTORC1 and TORC2 signaling in the growth of human prostate cancer cells.MethodsEmploying immunoprecipitation techniques and Western blotting as well as kinase assays, activation of the mTORC1 and mTORC2 complexes, as well as down stream targets were studied. RNAi was also employed to silence expression of Raptor, Rictor, or GRP78 in parallel studies.ResultsStimulation of cells with α(2)M* promotes phosphorylation of mTOR, TSC2, S6-Kinase, 4EBP, Akt(T308), and Akt(S473) in a concentration and time-dependent manner. Rheb, Raptor, and Rictor also increased. α(2)M* treatment of cells elevated mTORC1 kinase activity as determined by kinase assays of mTOR or Raptor immunoprecipitates. mTORC1 activity was sensitive to LY294002 and rapamycin or transfection of cells with GRP78 dsRNA. Down regulation of Raptor expression by RNAi significantly reduced α(2)M*-induced S6-Kinase phosphorylation at T389 and kinase activity in Raptor immunoprecipitates. α(2)M*-treated cells demonstrate about a twofold increase in mTORC2 kinase activity as determined by kinase assay of Akt(S473) phosphorylation and levels of p-Akt(S473) in mTOR and Rictor immunoprecipitates. mTORC2 activity was sensitive to LY294002 and transfection of cells with GRP78 dsRNA, but insensitive to rapamycin. Down regulation of Rictor expression by RNAi significantly reduces α(2)M*-induced phosphorylation of Akt(S473) phosphorylation in Rictor immunoprecipitates.ConclusionBinding of α(2)M* to prostate cancer cell surface GRP78 upregulates mTORC1 and mTORC2 activation and promotes protein synthesis in the prostate cancer cells.

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Influenza virus differentially activates mTORC1 and mTORC2 signaling to maximize late stage replication.

Publications

Influenza virus differentially activates mTORC1 and mTORC2 signaling to maximize late stage replication.

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