The transmembrane domain of hepatitis C virus E1 glycoprotein induces cell death.
The E1 protein of hepatitis C virus (HCV) shows the ability to induce cell lysis by the alteration of membrane permeability when expressed in Escherichia coli cells. This function seems to be an intrinsic property of a C-terminal hydrophobic region of E1 as permeability changes and cell lysis can be blocked by mutagenesis of specific amino acids in this domain. To establish whether the expression of E1 protein and its C-terminal domain was able to induce cell death also in eukaryotic cell, we cloned HCV sequences expressing the full-length E1 (E383), the C-terminal domain (SVP) and a mutant lacking the C-terminal region (E340) in the pRC/CMV expression vector. HepG2 cell line was co-transfected with empty vector or HCV expression plasmids and a reporter vector that expressed beta-galactosidase (beta-gal) to visualize co-transfected blue cells. At 60 h after transfection, the loss of blue cells, considered as a measure of cell death, was 31.5 and 64.3% for the E1 and SVP clones. On the contrary, the number of blue cells after transfection with E340 plasmid was similar to that observed with the control vector. The analysis by the terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) assay revealed an increased number of apoptotic cells at 48 h after transfection with E1 and SVP clones. Furthermore, cells transfected with SVP revealed a typical internucleosomal DNA fragmentation and the activation of caspase-3-like proteases as the specific inhibitor Ac-DEVD-CHO peptide partially blocked SVP apoptosis. These data indicate that the intracellular expression of HCV E1 protein and its C-terminal domain induces an apoptotic response in human hepatoma cell line.
Ciccaglione
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AR
Laboratory of Virology, Istituto Superiore di Sanità, Viale Regina Elena, 299-00161 Rome, Italy.
Marcantonio
C
C
Tritarelli
E
E
Equestre
M
M
Magurano
F
F
Costantino
A
A
Nicoletti
L
L
Rapicetta
M
M
D017209:Apoptosis; D002460:Cell Line; D002462:Cell Membrane; D002463:Cell Membrane Permeability / Q000187:drug effects* / Q000502:physiology; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000166:cytology / Q000187:drug effects; D016174:Hepacivirus / Q000472:pathogenicity; D006801:Humans; D014759:Viral Envelope Proteins / Q000494:pharmacology*
C087035:E1 protein, Hepatitis C virus; D014759:Viral Envelope Proteins