St-Louis Marie-Claude, Massie Bernard, Archambault Denis
Department of Biological Sciences, University of Québec at Montréal, PO Box 8888, Succursale Centre-Ville, Montréal, Québec, H3C 3P8, Canada.
Vet Res. 2005 Mar-Apr;36(2):213-27. doi: 10.1051/vetres:2004059.
The bovine viral diarrhea virus (BVDV) strains exist as two biotypes, cytopathic (cp) and noncytopathic (ncp), according to their effects on tissue culture cells. It has been previously reported that cell death associated to cp BVDV in vitro is mediated by apoptosis. Here, experiments were conducted to determine the involvement of the NS3 protein in the induction of apoptosis. The NS3- and NS3Delta50 (deleted from the NH2-terminal 50 amino acids)-cDNA encoding sequences of BVDV NADL cp reference strain were cloned into adenoviral vectors (AdV) from which the BVDV gene of interest could be expressed from a tetracycline-responsive promoter. A549tTA cells infected in vitro with NS3 or NS3Delta50-expressing AdV showed cytopathic changes characterized by cell rounding and detachment, and nucleus chromatin condensation. DNA fragmentation assays, cytochrome c release, and activation of cellular caspases performed on these infected cells clearly correlated with the observed cytopathic changes with apoptosis. The BVDV NS3Delta50-induced apoptotic process was inhibited by caspase-8- and -9-specific peptide inhibitors (Z-IETD-FMK and Z-LEHD-FMK). Furthermore, apoptosis was inhibited in cells expressing the R1 subunit of herpes simplex virus type 2 ribonucleotide reductase (HSV2-R1) or hsp70, two proteins which are known to inhibit apoptosis associated with caspase-8 activation and cytochrome c release-dependent caspase-9 activation, respectively. Given that HSV2-R1, a specific inhibitor of the caspase-8 activation pathway, efficiently suppressed apoptosis and also prevented caspase-9 activation, the overall results indicate that the BVDV NS3/NS3Delta50 induces apoptosis initiated by caspase-8 activation and subsequent cytochrome c release-dependent caspase-9 activation.
根据牛病毒性腹泻病毒（BVDV）毒株对组织培养细胞的影响，其存在细胞病变型（cp）和非细胞病变型（ncp）两种生物型。此前已有报道称，体外与cp BVDV相关的细胞死亡是由凋亡介导的。在此，进行了实验以确定NS3蛋白在凋亡诱导中的作用。将BVDV NADL cp参考毒株的NS3和NS3Delta50（从氨基末端50个氨基酸处缺失）编码序列克隆到腺病毒载体（AdV）中，感兴趣的BVDV基因可从四环素反应性启动子表达。体外感染表达NS3或NS3Delta50的AdV的A549tTA细胞出现细胞病变变化，特征为细胞变圆、脱落以及细胞核染色质浓缩。对这些感染细胞进行的DNA片段化分析、细胞色素c释放以及细胞半胱天冬酶激活实验与观察到的伴有凋亡的细胞病变变化明显相关。BVDV NS3Delta50诱导的凋亡过程被半胱天冬酶-8和-9特异性肽抑制剂（Z-IETD-FMK和Z-LEHD-FMK）抑制。此外，在表达2型单纯疱疹病毒核糖核苷酸还原酶R1亚基（HSV2-R1）或热休克蛋白70（hsp70）的细胞中凋亡受到抑制，已知这两种蛋白分别抑制与半胱天冬酶-8激活相关的凋亡以及细胞色素c释放依赖性半胱天冬酶-9激活相关的凋亡。鉴于HSV2-R1是半胱天冬酶-8激活途径的特异性抑制剂，能有效抑制凋亡并防止半胱天冬酶-9激活，总体结果表明BVDV NS3/NS3Delta50诱导由半胱天冬酶-8激活引发且随后依赖细胞色素c释放的半胱天冬酶-9激活的凋亡。
