Flavivirus activates phosphatidylinositol 3-kinase signaling to block caspase-dependent apoptotic cell death at the early stage of virus infection.
Flaviviruses such as dengue virus (DEN) and Japanese encephalitis virus (JEV) are medically important in humans. The lipid kinase, phosphatidylinositol 3-kinase (PI3K) and its downstream target Akt have been implicated in the regulation of diverse cellular functions such as proliferation, and apoptosis. Since JEV and DEN appear to trigger apoptosis in cultured cells at a rather late stage of infection, we evaluated the possible roles of the PI3K/Akt signaling pathway in flavivirus-infected cells. We found that Akt phosphorylation was noticeable in the JEV- and DEN serotype 2 (DEN-2)-infected neuronal N18 cells in an early, transient, PI3K- and lipid raft-dependent manner. Blocking of PI3K activation by its specific inhibitor LY294002 or wortmannin greatly enhanced virus-induced cytopathic effects (CPEs), even at an early stage of infection, but had no effect on virus production. This severe CPE was characterized as apoptotic cell death as evidenced by TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) staining and cleavage of caspase-3 and poly(ADP-ribose) polymerase (PARP). Mechanically, the initiator and effector caspases involved are mainly caspase-9 and caspase-6, since only a pan-caspase inhibitor and the inhibitors preferentially target caspase-9 and -6, but not the ones antagonizing caspase-8, -3, or -7 alleviated the levels of PARP cleavage after virus infection and PI3K blockage. Furthermore, Bcl-2 appears to be a crucial mediator downstream of PI3K/Akt signaling, since overexpression of Bcl-2 reduced virus-induced apoptosis even when PI3K activation was repressed. Collectively, our results suggest an anti-apoptotic role for the PI3K/Akt pathway triggered by JEV and DEN-2 to protect infected cells from early apoptotic cell death.
Lee
Chyan-Jang
CJ
Graduate Institute of Life Sciences, Taipei, Taiwan, Republic of China.
Liao
Ching-Len
CL
Lin
Yi-Ling
YL
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D001483:Base Sequence; D061945:Caspase Inhibitors; D016923:Cell Death; D045744:Cell Line, Tumor; D017931:DNA Primers; D003716:Dengue Virus / Q000235:genetics / Q000502:physiology*; D004664:Encephalitis Virus, Japanese / Q000235:genetics / Q000502:physiology*; D004789:Enzyme Activation; D005416:Flavivirus / Q000502:physiology*; D007770:L-Lactate Dehydrogenase / Q000032:analysis; D051379:Mice; D009447:Neuroblastoma; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D014176:Protein Biosynthesis; D012367:RNA, Viral / Q000235:genetics; D015398:Signal Transduction; D014158:Transcription, Genetic
D061945:Caspase Inhibitors; D017931:DNA Primers; D011480:Protease Inhibitors; D012367:RNA, Viral; D007770:L-Lactate Dehydrogenase; D019869:Phosphatidylinositol 3-Kinases