Dissection of IPNV and VP3 induce cell death in CHSE-214 cells

• Main Authors: Yen-Ting Liu, 劉嫣婷
• Other Authors: Jiann-Ruey Hong
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/33272732780797993530

碩士 === 國立成功大學 === 生物科技研究所碩博士班 === 96 === Infectious Pancreatic Necrosis Virus (IPNV) is one of the widespread fish pathogen and infects many economically important finfish and shellfish. IPNV-infected chinook salmon embryo cells (CHSE-214) could induce cell death through apoptosis. Further, we have found that the submajor capsid protein VP3 can induce apoptosis in ZLE cell line, but its cell death mechanism is still unknown. In this thesis, we further want to know VP3 death mechanism during IPNV infection. First, we have found that VP3 could target into mitochondria at early-middle replication stage with the immunofluorescence staining assay and Western blot from whole mitochondria. On the other hand, IPNV-induced CHSE-214 cell death through mitochondrial membrane potential loss could be blocked by overexpression of zfBcl-xL. Then, with using of antisense RNA approach for knockdown of VP3 expression during IPNV infection, in the results, (1) can enhance the cell viability, (2) inhibit either PS exposure or mitochondrial membrane potential (MMP) loss, (3) knock down the Bad expression, and (4) reduce the virus titer. Furthermore, VP3 and a series truncated forms were constructed for expression in ZLE cells. We found that the VP3 death motif may be located between 80-158 amino residues. Then, we found the VP3 may have a tyrosine kinase activity by immunoprecipitation assay between 3 hour and 12 hour post-infection and VP3 may reserve as a substrate for either tyrosine or serine/threonine kinases, but their functions are still further examined. Taken our results suggest that VP3 may play a crucial role on pro-apoptotic function, which may up-regulate the pro-apoptotic gene Bad and induces the loss of MMP in CHSE-214 cells with IPNV infection. This finding may thus provide an important insight into the understanding of IPNV pathogenesis and disease control.