Activation of the N-Ras-PI3K-Akt-mTOR pathway by hepatitis C virus: control of cell survival and viral replication.
The hepatitis C virus (HCV) replication complex is localized within detergent-resistant membranes or lipid rafts. We analyzed the protein contents of detergent-resistant fractions isolated from Huh7 cells expressing a self-replicating full-length HCV-1b genome. Using two-dimensional gel electrophoresis followed by mass spectrometry, we identified N-Ras as one of the proteins in which expression was increased in the detergent-resistant fractions from HCV genomic replicon clones compared to control cells. N-Ras is an activator of the phosphatidylinositol-3-kinase (PI3K)-Akt pathway. We found that the activities of PI3K and Akt, as well as the activity of their downstream target, mTOR, in the HCV-replicating cells were increased. Both PI3K-Akt- and mTOR-dependent pathways have been shown to promote cell survival. In agreement with this, HCV replicon cells were resistant to serum starvation-induced apoptosis. We also characterized the role of this pathway in HCV replication. Reduction of N-Ras expression by transfection of N-Ras small interfering RNA (siRNA) resulted in increased replication of HCV. We observed a similar increase in HCV replication in cells treated with the PI3K inhibitor LY294002 and in cells transfected with mTOR siRNA. Taken together, these data suggest that increased N-Ras levels in subcellular sites of HCV replication and stimulation of the prosurvival PI3K-Akt pathway and mTOR by HCV not only protect cells against apoptosis but also contribute to the maintenance of steady-state levels of HCV replication. These effects may contribute to the establishment of persistent infection by HCV.
Public Health Sciences Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. North, M5-A864, Box 19024, Seattle, WA 98109-1024, USA.
Petra
P
Mannová
Laura
L
Beretta
D017209:Apoptosis; D045744:Cell Line, Tumor; D016174:Hepacivirus / Q000502:physiology*; D006801:Humans; D021962:Membrane Microdomains / Q000821:virology; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology*; D011494:Protein Kinases / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D011518:Proto-Oncogene Proteins / Q000502:physiology*; D051057:Proto-Oncogene Proteins c-akt; D015398:Signal Transduction; D058570:TOR Serine-Threonine Kinases; D014779:Virus Replication; D018631:ras Proteins / Q000502:physiology*
D019869:Phosphatidylinositol 3-Kinases; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases; D018631:ras Proteins; C546842:MTOR protein, human; C494918:AKT1 protein, human