Individual expression of poliovirus 2Apro and 3Cpro induces activation of caspase-3 and PARP cleavage in HeLa cells.
The expression of individual viral genes enables the study of their effects on cellular functions. Our group previously generated stable HeLa cell lines that efficiently express poliovirus proteases 2A (clone 2A7d) and 3C (clone 3C7) under the control of tetracycline [Virology 266 (2000a) 352; J. Virol. 74 (2000b) 2383]. Upon induction of these proteases, the cells undergo drastic morphological alterations and eventually die. The present paper characterizes, in detail, the cellular and molecular events that lead to cell death in these lines. Several signs of apoptosis were observed in both 2A7d- and 3C7-induced cells, such as nuclear fragmentation, DNA breakdown (as determined by TUNEL), and phosphatidylserine translocation. Protease 2A induces the cleavage of poly-ADP-ribose-polymerase (PARP). This is blocked by the caspase-3 inhibitor DEVD in both 2A7d-On and 3C7-On cells suggesting that this enzyme might account for PARP cleavage in both cell lines. The results indicate that both poliovirus proteases induce apoptosis by mechanisms involving caspase activation, although the kinetics of apoptosis differs.
Calandria
Carlos
C
Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain. ccalandria@cbm.uam.es
Irurzun
Alicia
A
Barco
Angel
A
Carrasco
Luis
L
D000086042:3C Viral Proteases; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D017955:Poliovirus / Q000201:enzymology* / Q000235:genetics; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics / Q000378:metabolism*; D013752:Tetracycline / Q000494:pharmacology; D014764:Viral Proteins / Q000378:metabolism*
D061945:Caspase Inhibitors; D014764:Viral Proteins; D011065:Poly(ADP-ribose) Polymerases; D020169:Caspases; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases; C057400:picornain 2A, Picornavirus; D013752:Tetracycline