ISKNV Triggers AMPK/mTOR-Mediated Autophagy Signaling through Oxidative Stress, Inducing Antioxidant Enzyme Expression and Enhancing Viral Replication in GF-1 Cells.

Infectious spleen and kidney necrosis virus (ISKNV) infections can induce the process of host cellular autophagy but have rarely been identified within the molecular autophagy signaling pathway. In the present study, we demonstrated that ISKNV induces ROS-mediated oxidative stress signals for the induction of 5'AMP-activated protein kinase/mechanistic target of rapamycin kinase (AMPK/mTOR)-mediated autophagy and upregulation of host antioxidant enzymes in fish GF-1 cells. We also examined ISKNV-induced oxidative stress, finding that reactive oxidative species (ROS) increased by 1.5-fold and 2.5-fold from day 2 to day 3, respectively, as assessed by the H2DCFDA assay for tracing hydrogen peroxide (H2O2), which was blocked by NAC treatment in fish GF-1 cells. Furthermore, ISKNV infection was shown to trigger oxidative stress/Nrf2 signaling from day 1 to day 3; this event was then correlated with the upregulation of antioxidant enzymes such as Cu/ZnSOD and MnSOD and was blocked by the antioxidant NAC. Using an MDC assay, TEM analysis and autophagy marker LC3-II/I ratio, we found that ROS stress can regulate autophagosome formation within the induction of autophagy, which was inhibited by NAC treatment in GF-1 cells. Through signal analysis, we found that AMPK/mTOR flux was modulated through inhibition of mTOR and activation of AMPK, indicating phosphorylation levels of mTOR Ser 2448 and AMPK Thr 172 from day 1 to day 3; however, this process was reversed by NAC treatment, which also caused a reduction in virus titer (TCID50%) of up to 1000 times by day 3 in GF-1 cells. Thus, ISKNV-induced oxidative stress signaling is blocked by antioxidant NAC, which can also either suppress mTOR/AMPK autophagic signals or reduce viral replication. These findings may provide the basis for the creation of DNA control and treatment strategies.

A Mitochondrion-Targeting Protein (B2) Primes ROS/Nrf2-Mediated Stress Signals, Triggering Apoptosis and Necroptosis in Lung Cancer.

The betanodavirus B2 protein targets mitochondria and triggers mitochondrion-mediated cell death signaling in lung cancer cells; however, its molecular mechanism remains unknown. In this study, we observed that B2 triggers hydrogen peroxide/Nrf2-involved stress signals in the dynamic regulation of non-small lung cancer cell (NSCLC)-programmed cell death. Here, the B2 protein works as a necrotic inducer that triggers lung cancer death via p53 upregulation and RIP3 expression, suggesting a new perspective on lung cancer therapy. We employed the B2 protein to target A549 lung cancer cells and solid tumors in NOD/SCID mice. Tumors were collected and processed for the hematoxylin and eosin staining of tissue and cell sections, and their sera were used for blood biochemistry analysis. We observed that B2 killed an A549 cell-induced solid tumor in NOD/SCID mice; however, the mutant ΔB2 did not. In NOD/SCID mice, B2 (but not ΔB2) induced both p53/Bax-mediated apoptosis and RIPK3-mediated necroptosis. Finally, immunochemistry analysis showed hydrogen peroxide /p38/Nrf2 stress strongly inhibited the production of tumor markers CD133, Thy1, and napsin, which correlate with migration and invasion in cancer cells. This B2-triggered, ROS/Nrf2-mediated stress signal triggered multiple signals via pathways that killed A549 lung cancer tumor cells in vivo. Our results provide novel insight into lung cancer management and drug therapy.

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.

Infectious spleen and kidney necrosis virus (ISKNV) infections can trigger host cell death and are correlated with viral replication; however, they have rarely been considered in terms of the host organelle involvement. In the present study, we demonstrated that ISKNV triggered an oxidative stress signal in the Nrf2-mediated oxidative stress response and induced stress signals for Bax/Bak-mediated host cell death in fish GF-1 cells. The results showed that after ISKNV infection, the levels of reactive oxidative species (ROS) increased by 60-80% from day 3 to day 5, as assessed by an H2DCFDA assay for tracing hydrogen peroxide (H2O2), which was correlated with up to a one-fold change in the fish GF-1 cells. Furthermore, we found that ISKNV infection induced Nrf2-mediated ROS stress signals from D1 to D5, which were correlated with the upregulation of antioxidant enzymes, such as catalase, SOD1, and SOD2; these effects were blocked by the antioxidants GSH and NAC. By analyzing Nrf2-mediated ROS stress signals for cell death regulation via an apoptotic assay, we found that treatment with antioxidants reduced annexin-V-positive signals by 10% (GSH) to 15% (NAC); moreover, necrotic-positive signals were reduced by 6% (GSH) and 32% (NAC) at day 5 (D5) in GF-1 cells, as indicated by PI staining. Furthermore, we found that Nrf2-mediated ROS stress regulated mitochondrion-mediated Bax/Bak death signals at D3 and D5; this was effectively blocked by antioxidant treatment in the GF-1 cells, as demonstrated by a JC1 assay (ΔΨm) and western blot analysis. In addition, we found that downstream signals for caspase-9 and -3 activation were apparently blocked by antioxidant treatment at D3 and D5. Finally, we found that treatment with GSH and NAC reduced major capsid protein (MCP) expression and virus titer (TCID50%) by up to 15-fold at D5 in GF-1 cells. Thus, our data suggest that ISKNV can induce ROS production, which triggers Nrf2-mediated stress signals. Then, these stress signals can regulate mitochondrion-mediated Bax/Bak apoptotic signaling, which is connected to downstream caspase-9 and -3 activation. If ISKNV-induced Nrf2-mediated stress signaling is blocked, then the antioxidants GSH and NAC can also suppress apoptotic signals or reduce viral replication. These findings may provide insights into the control and treatment of double-stranded DNA viruses.

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.

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 Proapoptotic Gene Bad Regulates Brain Development via p53-Mediated Stress Signals in Zebrafish.

Studies have shown that the BH3-only domain Bad regulates brain development via the control of programmed cell death (PCD), but very few studies have addressed its effect on the molecular signaling of brain development in the system. In this work, we examined the novel role of zebrafish Bad in initial programmed cell death for brain morphogenesis through the priming of p53-mediated stress signaling. In a biological function study on the knockdown of Bad by morpholino oligonucleotides, at 24 h post-fertilization (hpf) Bad defects induced abnormal hindbrain development, as determined in a tissue section by means of HE staining which traced the damaged hindbrain. Then, genome-wide approaches for monitoring either the upregulation of apoptotic-related genes (11.8%) or the downregulation of brain development-related genes (29%) at the 24 hpf stage were implemented. The p53/caspase-8-mediated apoptotic death pathway was strongly involved, with the pathway being strongly reversed in a p53 mutant (p53M214K) line during Bad knockdown. Furthermore, we propose the involvement of a p53-mediated stress signal which is correlated with regulating Bad loss-mediated brain defects. We found that some major genes in brain development, such as crybb1, pva1b5, irx4a, pax7a, and fabp7a, were dramatically restored in the p53M214K line, and brain development recovered to return movement behavior to normal. Our findings suggest that Bad is required for (PCD) control, exerting a p53 stress signal on caspase-8/tBid-mediated death signaling and brain development-related gene regulation.

Proapoptotic Bad Involved in Brain Development, When Severely Defected, Induces Dramatic Malformation in Zebrafish.

The BH3-only molecule Bad regulates cell death via its differential protein phosphorylation, but very few studies address its effect on early embryonic development in vertebrate systems. In this work, we examined the novel role of zebrafish Bad in the initial programmed cell death (PCD) for brain morphogenesis through reducing environmental stress and cell death signaling. Bad was considered to be a material factor that because of the knockdown of Bad by morpholino oligonucleotides, PCD was increased and the reactive oxygen species (ROS) level was enhanced, which correlated to trigger a p53/caspase-8 involving cell death signaling. This Bad knockdown-mediated environmental stress and enhanced cell dying can delay normal cell migration in the formation of the three germ layers, especially the ectoderm, for further brain development. Furthermore, Bad defects involved in three-germ-layers development at 8 hpf were identified by in situ hybridization approach on cyp26, rtla, and Sox17 pattern expression markers. Finally, the Bad knockdown-induced severely defected brain was examined by tissue section from 24 to 48 h postfertilization (hpf), which correlated to induce dramatic malformation in the hindbrain. Our data suggest that the BH3-only molecule Bad regulates brain development via controlling programmed cell death on overcoming environmental stress for reducing secondary cell death signaling, which suggests that correlates to brain developmental and neurological disorders in this model system.

EPA and DHA can modulate cell death via inhibition of the Fas/tBid-mediated signaling pathway with ISKNV infection in grouper fin cell line (GF-1) cells.

Polyunsaturated fatty acids (PUFAs) play important roles in organisms, including the structure and liquidity of cell membranes, anti-oxidation and anti-inflammation. Very little has been done in terms of the effect of PUFAs on cell death, especially on DNA virus. In this study, we demonstrated that the infectious spleen and kidney necrosis virus (ISKNV) can induce host cell death via the apoptotic cell death pathway, which correlated to modulation by PUFAs in grouper fin cell line (GF-1) cells. We screened the PUFAs, including docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), for the ability of different dosages to prevent cell death in GF-1 cells with ISKNV infection. In the results, each 10 µM of DHA and EPA treatment enhanced host cell viability up to 80% at day 5 post-infection. Then, in Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay, DHA- and EPA-treated groups reduced TUNEL positive signals 50% in GF-1 cells with ISKNV infection. Then, through studies of the mechanism of cell death, we found that ISKNV can induce both the Bax/caspase-3 and Fas/caspase-8/tBid death signaling pathways in GF-1 cells, especially at day 5 post-infection. Furthermore, we found that DHA and EPA treatment can either prevent caspase-3 activation on 17-kDa form cleavage or Bid cleaved (15-kDa form) for activation by caspase-8, apparently. On the other hand, the anti-apoptotic gene Bcl-2 was upregulated 0.3-fold and 0.15-fold at day 3 and day 5, respectively, compared to ISKNV-infected and DHA-treated cells; that this did not happen in the EPA-treated group showed that different PUFAs trigger different signals. Finally, ISKNV-infected GF-1 cells treated with either DHA or EPA showed a 5-fold difference in viral titer at day 5. Taken together, these results suggest that optimal PUFA treatment can affect cell death signaling through both the intrinsic and extrinsic death pathways, reducing viral expression and viral titer in GF-1 cells. This finding may provide insight in DNA virus infection and control.

Nuclear targeting of the betanodavirus B1 protein via two arginine-rich domains induces G1/S cell cycle arrest mediated by upregulation of p53/p21.

The molecular functions of betanodavirus non-structural protein B and its role in host cell survival remain unclear. In the present study, we examined the roles of specific nuclear targeting domains in B1 localization as well as the effect of B1 nuclear localization on the cell cycle and host cell survival. The B1 protein of the Red spotted grouper nervous necrosis virus (RGNNV) was detected in GF-1 grouper cells as early as 24 hours post-infection (hpi). Using an EYFP-B1 fusion construct, we observed nuclear localization of the B1 protein (up to 99%) in GF-1 cells at 48 hpi. The nuclear localization of B1 was mediated by two arginine-rich nuclear targeting domains (B domain: 46RRSRR51; C domain: 63RDKRPRR70) and domain C was more important than domain B in this process. B1 nuclear localization correlated with upregulation of p53 and p21(wef1/cip1); downregulation of Cyclin D1, CDK4 and Mdm2; and G1/S cell cycle arrest in GF-1 cells. In conclusion, nuclear targeting of the RGNNV B1 protein via two targeting domains causes cell cycle arrest by up-regulating p53/p21 and down-regulating Mdm2, thereby regulating host cell survival.

Complete genome sequence and phylogenetic analysis of megalocytivirus RSIV-Ku: A natural recombination infectious spleen and kidney necrosis virus.

Megalocytiviruses are classified into three genotypes, infectious spleen and kidney necrosis virus (ISKNV), red seabream virus (RSIV), and turbo reddish body iridovirus (TRBIV), based on the major capsid protein and ATPase genes. However, only a few complete genome sequences have been obtained. This paper reports the complete genome sequence and phylogenetic analysis of an RSIV-Ku strain megalocytivirus. The genome sequence comprises 111,154 bp, has 132 putative open reading frames, and is homologous mostly to ISKNV, except for the sequence in the region 58981-66830, which is more closely related to that of the RSIV genotype. The results imply that RSIV-Ku is actually a natural recombinant virus.

Betanodavirus B2 protein triggers apoptosis and necroptosis in lung cancer cells that suppresses autophagy.

The betanodavirus B2 protein targets the mitochondria and acts as a "death factor", but its effect on lung cancer cells is unknown. We examined the effect of the B2 protein on triggering apoptosis or necroptosis via P53-dependent and P53-independent pathways and increased in suppression of autophagy. The B2 protein targets the mitochondria of A549 (P53+/+) and H1299 (P53-/-) lung cancer cells due to a specific signal sequence (41RTFVISAHAA50). This triggers generation of reactive oxygen species within the mitochondria, and a minor stress response in A549 cells, but a strong stress response in H1299 cells. We examined the molecular mechanism of this cell death pathway, and found that B2 protein induces the P53/Bax-mediated apoptotic pathway in A549 cells, and that a P53 specific inhibitor (pifithrin-α) switches this response to RIP3-mediated necroptosis. On the other hand, B2 induces RIP3-mediated necroptosis pathway in H1299 cells, and a necroptosis inhibitor (necrostatin-1) switches this response to the apoptotic pathway. Both types of cell death signals inhibited autophagy via a tightly increased balance of beclin-1 and Bcl-2. Thus, B2 protein triggers P53-dependent apoptosis in A549 cells and ROS/RIP3-mediated necroptosis in H1299 cells, and crosstalk of these pathways limits initiation of autophagy. These findings provide new insights into the possible control and treatment of lung cancer.

Anti-apoptotic genes Bcl-2 and Bcl-xL overexpression can block iridovirus serine/threonine kinase-induced Bax/mitochondria-mediated cell death in GF-1 cells.

Although serine/threonine (ST) kinase is known to induce host cell death in GF-1 cells, it remains unclear how ST kinase induces mitochondrial function loss. In the present study, we addressed the issue of mitochondrial function loss by determining whether the Bcl-2 family members Bcl-2 and Bcl-xL can prevent ST kinase-induced cell death activity via interacting with the pro-apoptotic gene Bax. Grouper fin cells (GF-1) carrying EGFP-Bal-xL and EGFP-Bcl-2 fused genes were selected, established in cell culture, and used to examine the involvement of Bcl-2 and Bcl-xL overexpression in protection of GF-1 cells from the effects of the giant sea perch iridovirus (GSIV) ST kinase gene. Using the TUNEL assay, we found that EGFP-Bcl-2 and EGFP-Bcl-xL reduced GSIV ST kinase-induced apoptosis to 20% all at 24 h and 48 h post-transfection (pt). Also, Bcl-2 and Bcl-xL substantially reduced the percentage of cells with GSIV ST kinase-induced loss of mitochondrial membrane potential (Δψps) at 24 and 48 hpt, respectively, and this reduction correlated with a 30% and 50% enhancement of host cell viability at 24 and 48 hpt as compared with vector control. Moreover, analysis of the effect of Bcl-2 and Bcl-xL interaction with Bax targeted to mitochondria during ST kinase expression at 48 hpt found that Bcl-2 and Bcl-xL also interacted with Bax to block cytochrome c release. Finally, Bcl-2 and Bcl-xL overexpression caused blockage of ST kinase function at 48 hpt, which was correlated with preventing caspase-9 and -3 cleavage and activation, thereby blocking downstream death signaling events. Taken together, our results suggest that the ST kinase-induced Bax/mitochondria-mediated cell death pathway can be blocked by the interaction of Bcl-2 and Bcl-xL with Bax to inhibit cytochrome c release during MMP loss. This rescue activity also correlated with inhibition of caspase-9 and -3 activation, thereby enhancing cell viability.

Complete Genome Sequence of a Giant Sea Perch Iridovirus in Kaohsiung, Taiwan.

We report here the complete genome sequence of a megalocytivirus strain, GSIV-K1, isolated from a farmed giant sea perch (Lates calcarifer) in Kaohsiung, Taiwan. GSIV-K1 causes mortality in farmed marine fish, including giant sea perch and groupers. The genome sequence is nearly identical to the genome of the orange-spotted grouper iridovirus.

Knockdown of zebrafish YY1a can downregulate the phosphatidylserine (PS) receptor expression, leading to induce the abnormal brain and heart development.

BACKGROUND: Yin Yang 1 (YY1) is a ubiquitously expressed GLI-Kruppel zinc finger-containing transcriptional regulator. YY1 plays a fundamental role in normal biologic processes such as embryogenesis, differentiation, and cellular proliferation. YY1 effects on the genes involved in these processes are mediated via initiation, activation, or repression of transcription depending upon the context in which it binds. The role of the multifunctional transcription factor Yin Yang 1 (YY1) in tissue development is poorly understood. In the present, we investigated YY1a role in developing zebrafish on PSR-mediated apoptotic cell engulfment during organic morphogenesis. RESULTS: YY1a is first expressed 0.5 h post-fertilization (hpf), in the whole embryo 12 hpf, and in brain, eyes, and heart 72 hpf by in situ hybridization assay. The nucleotide sequence of zebrafish YY1a transcription factor (clone zfYY1a; HQ 166834) was found to be similar to that of zebrafish YY1a (99 % sequence identity; NM 212617). With the loss-of-function assay, YY1a knockdown by a morpholino oligonucleotide led to downregulation of the phosphatidylserine engulfing receptor zfPSR during embryonic segmentation and to the accumulation of a large number of dead apoptotic cells throughout the entire early embryo, especially in the posterior area. Up to 24 hpf, these cells interfered with embryonic cell migration and cell-cell interactions that normally occur in the brain, heart, eye, and notochord. Finally, with gain-of-function assay, defective morphants could be rescued by injecting both YY1a mRNA and PSR mRNA and trigger resumption of normal development. CONCLUSIONS: Taken together, our results suggest that YY1a regulates PS receptor expression that linked to function of PSR-phagocyte mediated apoptotic cell engulfment during development, especially the development of organs such as the brain and heart. YY1a/PSR-mediated engulfing system may involve in diseases.

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.

Aquatic viruses induce host cell death pathways and its application.

Virus infections of mammalian and animal cells consist of a series of events. As intracellular parasites, viruses rely on the use of host cellular machinery. Through the use of cell culture and molecular approaches over the past decade, our knowledge of the biology of aquatic viruses has grown exponentially. The increase in aquaculture operations worldwide has provided new approaches for the transmission of aquatic viruses that include RNA and DNA viruses. Therefore, the struggle between the virus and the host for control of the cell's death machinery is crucial for survival. Viruses are obligatory intracellular parasites and, as such, must modulate apoptotic pathways to control the lifespan of their host to complete their replication cycle. This paper updates the discussion on the detailed mechanisms of action that various aquatic viruses use to induce cell death pathways in the host, such as Bad-mediated, mitochondria-mediated, ROS-mediated and Fas-mediated cell death circuits. Understanding how viruses exploit the apoptotic pathways of their hosts may provide great opportunities for the development of future potential therapeutic strategies and pathogenic insights into different aquatic viral diseases.

Giant seaperch iridovirus (GSIV) induces mitochondria-mediated cell death that is suppressed by bongkrekic acid and cycloheximide in a fish cell line.

Giant seaperch iridovirus (GSIV) induces cell death by an unknown mechanism. We postulated that this mechanism involves mitochondria-mediated cell death. Cell viability assays revealed a steady increase in dead grouper fin cells (GF-1) after GSIV infection, from 11% at 2 days post-infection (dpi) to 67% at 5 dpi. Annexin V/PI staining revealed GSIV infection induced apoptosis in a steadily increasing fraction of cells, from 4% at 1 dpi to 29% at 5 dpi. Furthermore, post-apoptotic necrosis was apparent at 4 and 5 dpi in the late replication stage. In the early replication stage, JC-1 dye revealed mitochondrial membrane potential (ΔΨm) loss in 42% of infected cells at 1 dpi, increasing to 98% at 3 dpi. Phosphatidylserine (PS) exposure and loss of ΔΨm from apoptosis/necrosis was attenuated by treatment with the adenine nucleotide translocase inhibitor bongkrekic acid (BKA) and the protein synthesis inhibitor cyclohexamide (CHX). These data suggest GSIV induces GF-1 apoptotic/necrotic cell death through pathways that require newly synthesized protein and involve the mitochondrial function.

Giant seaperch iridovirus infection upregulates Bas and Bak expression, leading to apoptotic death of fish cells.

The giant seaperch iridovirus (GSIV) induces host cell apoptosis by a poorly-understood process. In this study, GSIV is shown to upregulate the pro-apoptotic death genes Bax and Bak at the middle replication stage, and factors in the grouper fin cell line (GF-1) are shown to modulate this process. Studying the mechanism of cell death, we found that upregulated, de novo-synthesized Bax and Bak proteins formed heterodimers. This up-regulation process correlated with mitochondrial membrane potential (MMP) loss, increased caspase-3 activity, and increased apoptotic cell death. All effects were diminished by treatment of infected GF-1 cells with the protein synthesis inhibitor cycloheximide. Interestingly, overexpression of the anti-apoptotic gene Bcl-xL also diminished GSIV-induced mitochondria-mediated cell death, increasing host cell viability and decreasing MMP loss at the early replication stage. Our data suggest that GSIV induces GF-1 apoptotic cell death through up-regulation of the pro-apoptotic genes Bax and Bak, which are regulated by Bcl-xL overexpression on mitochondria in GF-1 cells.

Interferon regulatory factor-1 (IRF-1) is involved in the induction of phosphatidylserine receptor (PSR) in response to dsRNA virus infection and contributes to apoptotic cell clearance in CHSE-214 cell.

The phosphatidylserine receptor (PSR) recognizes a surface marker on apoptotic cells and initiates engulfment. This receptor is important for effective apoptotic cell clearance and maintains normal tissue homeostasis and regulation of the immune response. However, the regulation of PSR expression remains poorly understood. In this study, we determined that interferon regulatory factor-1 (IRF-1) was dramatically upregulated upon viral infection in the fish cell. We observed apoptosis in virus-infected cells and found that both PSR and IRF-1 increased simultaneously. Based on a bioinformatics promoter assay, IRF-1 binding sites were identified in the PSR promoter. Compared to normal viral infection, we found that PSR expression was delayed, viral replication was increased and virus-induced apoptosis was inhibited following IRF-1 suppression with morpholino oligonucleotides. A luciferase assay to analyze promoter activity revealed a decreasing trend after the deletion of the IRF-1 binding site on PSR promoter. The results of this study indicated that infectious pancreatic necrosis virus (IPNV) infection induced both the apoptotic and interferon (IFN) pathways, and IRF-1 was involved in regulating PSR expression to induce anti-viral effects. Therefore, this work suggests that PSR expression in salmonid cells during IPNV infection is activated when IRF-1 binds the PSR promoter. This is the first report to show the potential role of IRF-1 in triggering the induction of apoptotic cell clearance-related genes during viral infection and demonstrates the extensive crosstalk between the apoptotic and innate immune response pathways.

RNA Viruses: ROS-Mediated Cell Death.

Reactive oxygen species (ROS) are well known for being both beneficial and deleterious. The main thrust of this review is to investigate the role of ROS in ribonucleic acid (RNA) virus pathogenesis. Much evidences has accumulated over the past decade, suggesting that patients infected with RNA viruses are under chronic oxidative stress. Changes to the body's antioxidant defense system, in relation to SOD, ascorbic acid, selenium, carotenoids, and glutathione, have been reported in various tissues of RNA-virus infected patients. This review focuses on RNA viruses and retroviruses, giving particular attention to the human influenza virus, Hepatitis c virus (HCV), human immunodeficiency virus (HIV), and the aquatic Betanodavirus. Oxidative stress via RNA virus infections can contribute to several aspects of viral disease pathogenesis including apoptosis, loss of immune function, viral replication, inflammatory response, and loss of body weight. We focus on how ROS production is correlated with host cell death. Moreover, ROS may play an important role as a signal molecule in the regulation of viral replication and organelle function, potentially providing new insights in the prevention and treatment of RNA viruses and retrovirus infections.