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.
Mannová
Petra
P
Public Health Sciences Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. North, M5-A864, Box 19024, Seattle, WA 98109-1024, USA.
Beretta
Laura
L
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