Enhanced viability of a nervous necrosis virus-infected stable cell line over-expressing a fusion product of the zfBcl-xL and green fluorescent protein genes.
Nervous necrosis virus (NNV) infection induces host cell apoptosis by an ill-understood process. We utilized a fusion between enhanced green fluorescent protein (EGFP) and the zfBcl-x(L) gene in GL-av cells to select for zfBcl-x(L) stable cell lines and to assess the effectiveness of the anti-apoptotic protein Bcl-x(L) in circumventing NNV-induced cell death. Stable EGFP and EGFP-Bcl-x(L)-expressing clones were obtained at high purity within 2.5-3 months. In the latter, the EGFP-Bcl-x(L) fusion protein (approximately 58.2 kDa, as ascertained by Western blot) was predominantly targeted to mitochondria. We assayed for apoptosis in red-spotted grouper NNV Tainan no. 1 (RGNNV TN1)-infected cells with terminal deoxynucleotidyl transferase (TdT)-mediated end labelling (TUNEL) of DNA at different virus doses. NNV infection of NNV Bcl-x(L) GL-av cell line revealed a protective effect, with a decrease in TUNEL-positive cells of 7%, 8% and 31.8% at 24, 48 and 72 h, respectively. In addition, RGNNV infection of the Bcl-x(L) GL-av cell line revealed a protective effect, with an enhanced viability of 3%, 40% and 73% at 24, 48, and 72 h, respectively. We conclude that NNV-induced apoptotic cell death can be lessened in transgenic grouper fish cells.
Chen
S-P
SP
Laboratory of Molecular Virology and Biotechnology, National Cheng-Kung University, Tainan, Taiwan.
Yang
H-L
HL
Lin
H-Y
HY
Chen
M-C
MC
Wu
J-L
JL
Hong
J-R
JR
D000818:Animals; D017209:Apoptosis / Q000502:physiology; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D005393:Fish Diseases / Q000821:virology*; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics*; D020287:In Situ Nick-End Labeling / Q000662:veterinary; D008099:Liver / Q000166:cytology; D030381:Nodaviridae / Q000472:pathogenicity*; D010473:Perciformes / Q000821:virology*; D012327:RNA Virus Infections / Q000662:veterinary* / Q000821:virology; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis; D051020:bcl-X Protein / Q000096:biosynthesis / Q000235:genetics*
D011994:Recombinant Proteins; D051020:bcl-X Protein; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins