Infectious Spleen and Kidney Necrosis Virus (ISKNV) Triggers Mitochondria-Mediated Dynamic Interaction Signals via an Imbalance of Bax/Bak over Bcl-2/Bcl-xL in Fish Cells

Abstract

The molecular pathogenesis of infectious spleen and kidney necrosis virus (ISKNV) infections is important but has rarely been studied in connection to host organelle behavior. In the present study, we demonstrated that ISKNV can induce host cell death via a pro-apoptotic Bcl-2 and anti-apoptotic Bcl-2 family member imbalance in mitochondrial membrane potential (MMP or ΔΨm) regulation in GF-1 cells. The results of our study on ISKNV infection showed that it can induce host cell death by up to 80% at day 5 post-infection. Subsequently, in an apoptotic assay, ISKNV infection was seen to induce an increase in Annexin-V-positive signals by 20% and in propidium iodide (PI) staining-positive signals by up to 30% at day 5 (D5) in GF-1 cells. Then, through our studies on the mechanism of cell death in mitochondria function, we found that ISKNV can induce MMP loss by up to 58% and 78% at days 4 and 5 with a JC1 dye staining assay. Furthermore, we found that pro-apoptotic members Bax and Bak were upregulated from the early replication stage (day one) to the late stage (day 5), but the expression profiles were very dynamically different. On the other hand, by Western blotted analysis, the anti-apoptotic members Bcl-2 and Bcl-xL were upregulated very quickly at the same time from day one (two-fold) and continued to maintain this level at day five. Finally, we found that pro-apoptotic death signals strongly activated the downstream signals of caspase-9 and -3. Taken together, these results suggest that ISKNV infection can induce Bax/Bak-mediated cell death signaling downstream of caspase-9 and -3 activation. During the viral replication cycle with the cell death induction process, the anti-apoptotic members Bcl-2/Bcl-xL interacted with the pro-apoptotic members Bax/Bak to maintain the mitochondrial function in the dynamic interaction so as to maintain the MMP in GF-1 cells. These findings may provide insights into DNA-virus control and treatment.

The role of BCL-2 family proteins in regulating apoptosis and cancer therapy

“…Therefore, BAK plays an important role in apoptosis and is an important regulatory factor in the apoptosis process ( 56 ). BAK protein encoded by BAK gene can promote apoptosis directly or indirectly: 1) BAK alone can promote apoptosis by inhibiting the apoptosis activity of BCL-2 and BCL-XL ( 57 ), or BAK neutralizes the apoptosis inhibitory protein of virus, promoting apoptosis ( 58 ). 2) BAK either directly activates the apoptosis pathway ( 59 ) or is activated as part of the cell death process to promote apoptosis.…”

Infectious spleen and kidney necrosis virus induces the reactive oxidative species/Nrf2-mediated oxidative stress response for the regulation of mitochondrion-mediated Bax/Bak cell death signals in GF-1 cells

“…Complex I and complex III of the electron transport chain are the major sites of ROS production (Turrens and Boveris, 1980;Sugioka et al, 1988). The inhibition of complex I inhibition by rotenone has been shown to increase ROS generation in submitochondrial particles (Chen et al, 2022). ns, no significant different.…”

“…Naturally infected Pagrus major (red seabream) were collected in 2016 from the Kaohsiung Prefecture; these fish were the source of RSIV-KU (accession number: Kt781098) that was used to infect the GF-1 cells used in this study. RSIV-KU is an ISKNV-like strain that has had its complete viral genome sequenced (Shiu et al, 2018;10.3389/fmicb.2022.958476 Chen et al, 2022). The virus was purified and stored at −80 • C until use.…”

“…At different time points after infection, the cells were washed, fixed, and permeabilized with PBS containing 0.2% Triton X-100-PBS for 5 min on ice. Then, they were incubated with Mito-ID JC1 dye for 30 min at 28 • C, washed with PBS, suspended in LB medium, and analyzed using fluorescence microscopy (Chen et al, 2007(Chen et al, , 2022. The evaluation was performed using fluorescence microscopy with an excitation wavelength of 488 nm and a 515 nm long-pass filter for detection.…”

“…The culture media were aspirated at the end of each time point, after which the cells were washed with PBS and lysed in 0.3 ml of lysis buffer (10 mM Tris base, 20% glycerol, 10 mM sodium dodecyl sulfate, and 2% ß-mercaptoethanol; pH 6.8). The proteins were separated via sodium dodecyl sulfate polyacrylamide gel electrophoresis, electroblotted, and then subjected to immunodetection, as previously described (Hong et al, 1999;Chen et al, 2022). The membranes were incubated with a dilution of anti-mouse Bax MAb (1:1,000 (GeneTex, GTX 116093), or anti-mouse ß-actin MAb (1:2,500; Chemicon, Temecula, CA, USA) along with a dilution of the appropriate secondary antibodies (1:7,500 to 1:10,000), which included peroxidase-labeled goat antimouse (Amersham, Piscataway, NJ, USA) and goat anti-rabbit (Amersham) antibodies.…”