SARS-CoV Nucleocapsid Protein Induced Apoptosis of COS-1 Mediated by the Mitochondrial Pathway

Abstract

To investigate the apoptosis effect of SARS coronavirus nucleocapsid protein on cultured cell lines and to explore the possible pathway of apoptosis. pCDNA3.1(-)/his-myc vector containing the SARS coronavirus nucleocapsid gene (N), matric gene (M), spike gene (S) were transfected into COS-1, Huh-7 and HepG2 cells. Apoptosis induced by SARS coronavirus N protein under starvation of serum of COS-1 cells was monitored by Annexin V and electron microscopy assays. Intracellular reactive oxygen species (ROS) and mitochondrial membrane potential (DeltaPsim) were determined by flow cytometric assay. Cytochrome C, cleaved caspase (cysteine aspartic acid protease)-3, 9, and poly (ADP-ribose) polymerase (PARP) were detected by Western blot. After removal of serum in COS-1 cells, we observed the loss of DeltaPsim, the increase of ROS and cytochrome C release into cytosol and subsequent activation of caspase-3 and PARP cleavage. The pan-caspase inhibitor z-VAD-fmk can block the activation of caspase 3, 9 and PARP cleavage. In conclusion, SARS coronavirus N protein can induce apoptosis of COS-1 cells by activating mitochondrial pathway. SARS coronavirus M, S protein can not induce apoptosis in COS-1, HepG2 and Huh-7 and SARS coronavirus N protein can not induce apoptosis in HepG2 and Huh-7 by methods used in this study.

Hypoxia induces apoptosis and autophagic cell death in human periodontal ligament cells through HIF‐1α pathway

“…Previous studies have shown that overexpression of Bnip3 results in its integration into the outer mitochon-drial membrane with specific orientationits N-terminus in the cytoplasm and C-terminus in the membrane (41). After integration into the mitochondrial membrane, Bnip3 can interact with components of MPT pores to induce their rapid opening and dissipation of DΨm, followed by apoptotic cell death (42). We have also proven that CoCl 2 promoted activation of HIF-1a, upregulated expression of HIF-1a and Bnip3, and ultimately resulted in apoptosis of the cells; this could be attenuated by use of NAC, an inhibitor of HIF-1 a.…”

The SARS-CoV nucleocapsid protein: A protein with multifarious activities

“…However, this phenomenon was not observed in another cell line of epithelial lineage (huh7). Recently, Zhang et al (2007) have reported that serum starvation apoptosis of N expressing COS-1 cells involves activation of mitochondrial pathway. It remains to be studied whether this phenomenon is actually recapitulated in vivo.…”

Molecular Characterizations of Subcellular Localization Signals in the Nucleocapsid Protein of Porcine Epidemic Diarrhea Virus

“…Among the proteins, N, as the RNA-binding protein, play an important role in both virus RNA synthesis and modulating host cell processes, and phosphorylation may regulate these processes by exposing various functional motifs [4,5]. Several other functions have been postulated for the coronavirus N protein throughout the virus life cycle, including encapsidation, packaging, correct folding of the RNA molecule, the deregulation of the host cell cycle [6,7,8], inhibition of interferon production [9,10], up-regulation of COX2 production [11,12], up-regulation of AP1 activity [13], induction of apoptosis [14,15,16], association with host cell proteins [17], and RNA chaperone activity [18]. Therefore, it is clear that N is a multifunctional protein involved in biological processes related to the survival of PEDV.…”