The bovine viral diarrhea virus (BVDV) NS3 protein, when expressed alone in mammalian cells, induces apoptosis which correlates with caspase-8 and caspase-9 activation

Abstract

-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 NS3∆50 (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 NS3∆50-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 NS3∆50-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/NS3∆50 induces apoptosis initiated by caspase-8 activation and subsequent cytochrome c release-dependent caspase-9 activation. bovine viral diarrhea virus / NS3 protein / apoptosis / caspases

Expression of the NS3 protease of cytopathogenic bovine viral diarrhea virus results in the induction of apoptosis but does not block activation of the beta interferon promoter

“…Firstly, in the case of HCV, NS3 expressed in the absence of NS4A exhibits an aberrant subcellular localization (predominantly nuclear) and is rapidly degraded (half-life of 3 h), in comparison with NS3 expressed in the context of the NS3/4A complex (Wölk et al., 2000). Secondly, an N-terminal deletion of 50 residues had no effect on the ability of NS3 to induce apoptosis (St Louis et al, 2005), and, as previously demonstrated, such a deletion would be predicted to abolish protease activity . To clarify this situation, we used transient expression of wild-type and protease/helicase mutant derivatives of BVDV NS3, both alone and in the context of the NS2-3 fusion protein, which in all cases included NS4A.…”

“…not fused to NS2) is considered a marker of cytopathogenicity, it is not clear whether NS3 directly induces apoptosis. Previously, it was shown (St Louis et al, 2005) that BVDV NS3, expressed in the absence of NS4A from an adenovirus vector, and CSFV NS3 expressed as a green fluorescent protein fusion (Xu et al., 2007) were able to induce apoptosis. However, the physiological significance of these results is questionable for two reasons.…”

“…Furthermore, the dsRNA-activated proteins 29,59-oligoadenylate synthetase and protein kinase R may play a critical role in the induction of apoptosis following CP BVDV infection (Yamane et al, 2006). Transient expression of the NS3 proteins from BVDV and CSFV (without the presence of NS4A, the NS3 co-factor; Xu et al, 1997) has been shown to induce apoptosis (St Louis et al, 2005;Xu et al, 2007). However, the mechanism by which CP BVDV and NS3/4A induce these effects has yet to be elucidated.…”

Bovine Viral Diarrhea Virus Infections

“…84 The viral protein NS3 induces apoptosis via activation of caspase 8 and the cytochrome c release-dependent caspase 9, thus inducing both extrinsic and intrinsic pathways. 140 Induction of moderate amounts of caspase 8 is associated with apoptosis of T-lymphocyte-rich interfollicular areas and marked apoptosis of B lymphocytes in lymphoid follicles during infection of ileal Peyer patches. 116 This finding suggests a role of the extrinsic pathway of apoptosis in gut-associated lymphoid tissue (GALT).…”

Apoptosis in Bovine Viral Diarrhea Virus (BVDV)–Induced Mucosal Disease Lesions

“…3 Thus, the majority of the in vitro studies point to an involvement of the intrinsic rather than the extrinsic apoptosis pathway in cell death caused by cp BVDV infection. 14,37,48 However, it is still unclear if this mechanism also participates in the development of lesions present in animals dying of MD and whether it is correlated with BVDV antigen expression in vivo. The aim of this retrospective study was, therefore, to investigate the role of the various apoptosis pathways in the pathogenesis of lesions in animals suffering from MD.…”