GSIV serine/threonine kinase can induce apoptotic cell death via p53 and pro-apoptotic gene Bax upregulation in fish cells.

Previous studies have shown that GSIV induces apoptotic cell death through upregulation of the pro-apoptotic genes Bax and Bak in Grouper fin cells (GF-1 cells). However, the role of viral genome-encoded protein(s) in this death process remains unknown. In this study, we demonstrated that the Giant seaperch iridovirus (GSIV) genome encoded a serine/threonine kinase (ST kinase) protein, and induced apoptotic cell death via a p53-mediated Bax upregulation approach and a downregulation of Bcl-2 in fish cells. The ST kinase expression profile was identified through Western blot analyses, which indicated that expression started at day 1 h post-infection (PI), increased up to day 3, and then decreased by day 5 PI. This profile indicated the role of ST kinase expression during the early and middle phases of viral replication. We then cloned the ST kinase gene and tested its function in fish cells. The ST kinase was transiently expressed and used to investigate possible novel protein functions. The transient expression of ST kinase in GF-1 cells resulted in apoptotic cell features, as revealed with Terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling (TUNEL) assays and Hoechst 33258 staining at 24 h (37 %) and 48 h post-transfection (PT) (49 %). Then, through studies on the mechanism of cell death, we found that ST kinase overexpression could upregulate the anti-stress gene p53 and the pro-apoptotic gene Bax at 48 h PT. Interestingly, this upregulation of p53 and Bax also correlated to alterations in the mitochondria function that induced loss of mitochondrial membrane potential (MMP) and activated the initiator caspase-9 and the effector caspase-3 in the downstream. Moreover, when the p53-dependent transcriptional downstream gene was blocked by a specific transcriptional inhibitor, it was found that pifithrin-α not only reduced Bax expression, but also averted cell death in GF-1 cells during the ST kinase overexpression. Taken altogether, these results suggested that aquatic GSIV ST kinase could induce apoptosis via upregulation of p53 and Bax expression, resulting in mitochondrial disruption, which activated a downstream caspases-mediated cell death pathway.

RGNNV induces mitochondria-mediated cell death via newly synthesized protein dependent pathway in fish cells.

The RNA nervous necrosis virus induces necrotic cell death in fish; however, the molecular mechanism remains unknown. In this study, we demonstrated that beta-nodavirus-induced mitochondria-mediated dependent cell death is through newly synthesized protein dependent pathway in replication cycle. We determined that newly synthesized protein dependent pathway is required for red-spotted grouper nervous necrosis virus (RGNNV)-induced cell death. UV irradiation of the virus effectively blocked viral replication and cell death. Next, RGNNV RNA-dependent RNA polymerase (RdRp or protein A) was cloned and its involvement in RNA genome replication and viral protein synthesis was analyzed. Protein A was initially expressed 48 h post-infection and localized to the cytoplasm. Knockdown of protein A expression completely blocked viral genomic replication and expression of viral protein expression RNA1 small hairpin RNA (shRNA) producing cell lines, which coincided with inhibition of phosphatidylserine exposure, mitochondria-mediated death signaling, and increased cell viability 72 h post-infection. Furthermore, RGNNV-induced mitochondria-mediated caspase-3-independent necrotic cell death is dependent on viral synthesized protein dependent pathway at the middle-late replication stage. Taken together, for instance these results suggested that RGNNV induces cell death may require newly synthesized protein for triggering host mitochondria-mediated cell death. These findings may provide new insights into RNA viral pathogenesis.

Zebrafish anti-apoptotic protein zfBcl-xL can block betanodavirus protein alpha-induced mitochondria-mediated secondary necrosis cell death.

Betanodavirus protein alpha induces cell apoptosis or secondary necrosis by a poorly understood process. In the present work, red spotted grouper nervous necrosis virus (RGNNV) RNA 2 was cloned and transfected into tissue culture cells (GF-1) which then underwent apoptosis or post-apoptotic necrosis. In the early apoptotic stage, progressive phosphatidylserine externalization was evident at 24h post-transfection (p.t.) by Annexin V-FLUOS staining. TUNEL assay revealed apoptotic cells at 24-72 h p.t, after which post-apoptotic necrotic cells were identified by acridine orange/ethidium bromide dual dye staining from 48 to 72 h p.t. Protein alpha induced progressive loss of mitochondrial membrane potential (MMP) which was detected in RNA2-transfected GF-1 cells at 24, 48, and 72 h p.t., which correlated with cytochrome c release, especially at 72 h p.t. To assess the effect of zfBcl-xL on cell death, RNA2-transfected cells were co-transfected with zfBcl-x(L). Co-transfection of GF-1 cells prevented loss of MMP at 24 h and 48 h p.t. and blocked initiator caspase-8 and effector caspase-3 activation at 48 h p.t. We conclude that RGNNV protein alpha induces apoptosis followed by secondary necrotic cell death through a mitochondria-mediated death pathway and activation of caspases-8 and -3.