Overexpression of Bcl2 and Bcl2L1 Can Suppress Betanodavirus-Induced Type III Cell Death and Autophagy Induction in GF-1 Cells

: Betanodavirus infection induces viral nervous necrosis (VNN) in ﬁsh. However, the role of cell death and autophagy in the pathogenesis of VNN remains unknown. This study aimed to investigate the effect of red-spotted grouper nervous necrosis virus (RGNNV) infection on Bcl2 downregulation and overexpression on asymmetric interaction between cell death and autophagy. The mRFP-LC3 reporter system was used to identify autophagosome formation in GF-1 (Grouper ﬁn-1) ﬁsh cells. We found that the RGNNV could strongly induce autophagosome formation 36 h post-infection (hpi) after autophagy inhibitor 3-MA had downregulated anti-apoptotic genes such as Bcl2 and Bcl2L1 (Bcl-xL). We proposed that the overexpression of Bcl2 and Bcl2L1 can modulate both cell death and autophagy. Then, we found that it can also reduce either type III cell death or autophagy, which are mildly correlated with reduced viral replication. Our data suggest that RGNNV-induced Bcl2 downregulation correlates with the asymmetrical interaction between cell death induction and the autophagy process, which resembles viral replication.


Introduction
Betanodaviruses are the causative agents of viral nervous necrosis (VNN), an infectious neuropathological condition characterized by necrosis of the brain and retina in fish [1]. RGNNV infection causes massive mortality in larval and juvenile populations of several marine teleost species [2,3]. The infection correlates with the modulation of innate or acquired immunity [4,5], but very little is known about the molecular mechanisms underlying the pathogenesis of VNN.
The betanodavirus genome also includes two single-strand RNA molecules with positive polarity: RNA1 (3.1 kb) and RNA2 (1.4 kb), which lacks a 3 poly (A) extension [4]. RNA1 encodes a 110 kDa protein acting on an RNA-dependent RNA polymerase (protein A) for replication of the viral genome. RNA2 encodes a 42 kDa capsid protein [4,6] that can trigger post-apoptotic necrotic cell death through a cytochrome c release-mediated pathway in GF-1 cells [7]. In the middle-late replication stage, betanodaviruses were also shown to synthesize a sub-genomic RNA3 from the 3 terminus of RNA1 during genome replication, which encodes two non-structural proteins, B1 (absent in some strains) [8] and B2 (as a death gene) [1,9].
Recently, cell death has acquired multiple meanings. Apoptosis, type I cell death, has taken center stage as the principal mechanism of programmed cell death in mammalian tissues. Autophagy, type II cell death, is characterized by the massive accumulation of autophagic vacuoles in the cytoplasm of cells. The autophagic flow is activated as an adaptive response to a variety of extracellular and intracellular stresses, which include some factor changes such as nutrient deprivation, hormonal or therapeutic treatment, pathogenic infection, aggregated and misfolded proteins, and damaged organelles. Necroptosis, a programmed type III cell death, manifests with a distinctive morphology different from type I and type II [10,11].
A number of studies have investigated the molecular mechanisms by which the redspotted grouper nervous necrosis virus (RGNNV) induces apoptosis or necrosis in host cells [12,13]. RGNNV infection induces cell death that correlates with the loss of mitochondrial membrane potential (MMP) and the release of cytochrome c at the mid-replication stage. This is regulated by the zebrafish anti-apoptotic protein Bcl-2 [13], a member of the BCL-2 family [14], and blocked by bongkrekic acid, an inhibitor of the mitochondrial permeability transition pore [12,14]. Furthermore, betanodavirus-induced mitochondriamediated cell death can be blocked by the protein synthesis inhibitor cycloheximide [14], indicating that newly synthesized proteins from the viral genome are necessary for cell necrosis. Recent studies of the viral genome revealed the role of two viral death factors: protein α, which can induce the caspase-3-and caspase-9-dependent signaling pathway [7,15]; and protein B2, a non-structural protein that can induce BAX (BCL-2 associated X, apoptosis regulator)-mediated cell death [9] and cause ATP-depletion-mediated necrosis [16], which is dependent on the expression of the zebrafish anti-apoptotic gene Bcl2L1 (BCL-2 like 1) [9,14]. A recent study showed that B2 can induce DNM1L/Drp1 (dynamin 1 like)-mediated mitochondrial fragmentation [17]. However, little is known as to whether betanodaviruses induce necrosis or necroptosis.
Autophagy is a universal cell defense mechanism against intracellular microbes, which are delivered to lysosomes for degradation [18]. Autophagy contributes to the activation of antiviral innate immunity [19,20] and the adaptive immune response by delivering virusderived peptides to lymphocytes for presentation to major histocompatibility complex molecules [21][22][23]. Autophagy and autophagy genes likely play both anti-viral and proviral roles in the life cycle and pathogenesis of many different virus families. With respect to anti-viral roles, autophagy proteins function by targeting viral components or virions for lysosomal degradation in a process termed xenophagy. They also play a role in the initiation of innate and adaptive immune system responses to viral infections. Consistent with this anti-viral role of host autophagy, some viruses encode virulence factors that interact with the host autophagy machinery to block its execution. On the other hand, other viruses appear to use components of the autophagic machinery to foster their own intracellular growth or non-lytic cellular egress. As the details of the role(s) of autophagy in viral pathogenesis become clearer, new anti-viral therapies could be developed to inhibit the beneficial and enhance the destructive aspects of autophagy on the viral life cycle [20].
The mechanistic target of rapamycin (MTOR) is a protein kinase that regulates autophagy by stimulating protein synthesis and inhibiting induction of autophagy [24].
The class III phosphatidylinositol 3-kinase (PtdIns3K) plays an important role in many biological processes, including the activation of MTOR. The chemical 3-methyladenine (3-MA) has been reported to inhibit autophagy by blocking autophagosome formation via the inhibition of class III Ptdlns3K [25]. However, 3-MA plays a dual role in autophagy. Prolonged treatment with 3-MA promotes autophagy under nutrient-rich conditions, whereas 3-MA inhibits starvation-induced autophagy [25]. Recently, regarding the molecular pathogenesis of RGNNV infection, the symmetrical or asymmetrical interaction between cell death induction and autophagy during viral replication has become a topic of interest.
Numerous viruses have evolved molecular mechanisms to allow them to escape or inhibit autophagy, thereby increasing their infectivity [20], but little has been discovered about how an RNNV virus induces autophagy. In the present study, we investigated the role of Bcl2 family crosstalk between cell death and autophagy. Our findings in the molecular pathogenesis of RGNNV infection may help to identify potential targets that could be used for the prevention or treatment of RNA viruses.

Cell and Virus Lines
Grouper fin-1 (GF-1), cells derived from the fin tissue of a grouper (Epinephelus coioides), were grown at 28 • C in Leibovitz's L-15 medium (Thermo Fisher Scientic Inc., 11415-114, Waltham, MA, USA) and supplemented with 5% fetal bovine serum and 25 µg/mL gentamycin. Naturally infected red grouper larvae, collected in 2002 from the Tainan Prefecture, were the source of the RGNNV Tainan No. 1 (RGNNV TN1 strain) used to infect GF-1 cells. The virus was purified as described by Mori et al. [26] and stored at −80 • C until use. The viral titer was determined using the TCID 50 (50% tissue culture infective dose) assay, as reported by Dobos et al. [27].

Selection of mRFP-LC3-Producing GF-1 Cells
Vector-producing (pmRFP-C1 as a negative control) (Addgene, 21075; deposited by Tamotsu Yoshimori Lab) and pmRFP-LC3-producing cells were obtained by transfection and selection with G418 (800 µg/mL; Invivogen, ant-gn-1). Transcription of the inserted sequences in these vectors was driven by the immediate-early promoter of human cytomegalovirus. The selection time varied from 2 to 2.5 months according to cell properties [14].

Autophagosome Formation Assay
The mRFP and mRFP-LC3-producing cells (10 5 cells/mL) were cultured to monolayer confluence in 60 mm diameter Petri dishes for 20 h, rinsed twice with PBS (pH 7.4; Gibco, 10010023, Waltham, MA, USA), and serum-starved for 48 h as a positive control. Cells were then pre-treated with autophagy inhibitor (2 mM 3-MA; Acros, 379791000, Geel, Belgium) for 2 h, and infected with RGNNV (multiplicity of infection (MOI) = 1) for 0, 24, 36 and 48 h at 28 • C. At the end of each incubation time, the slides were observed under fluorescence microscopy (Olympus IX70). The red fluorescent images were captured at 300× to document the formation of RFP-LC3 puncta or dots indicative of autophagosomes or autolysosomes. The formation of RFP-LC3 puncta or dots was counted to 10 as a positive cell. The number of mRFP dots was determined as previously described [28]. Fluorescent puncta were counted manually in at least 3 independent experiments, and at least 200 cells were examined in each experiment.

Regulation of Autophagy by Overexpression of Bcl2 and Bcl2L1 in GF-1 Cells
Flag-Bcl2L1 and Flag-Bcl2-producing cells were obtained by transfection of GF-1 cells were transfected with pFlag (p3XFLAG-Myc-CMV), pFlag-Bcl2, and pFlag-Bcl2L1, respectively as previously described [31] by using Lipofectamine-Plus (Invitrogen, 11668500, Waltham, MA, USA), according to the manufacturer's instructions. Positive clones were selected with G418 (800 mg/mL). Transcription of the Bcl2 and Bcl2L1 genes in these vectors is driven by the immediate-early promoter of human cytomegalovirus. The selection time was about 2.5-3 months from a single colony, depending on cell properties [32].

Statistical Analysis
The fraction of cells positive for ANXA5 and PI in each sample was determined from counts of 200 cells. Results are expressed as means ± SEMs. Data were analyzed using the paired or unpaired Student's t-test or ANOVA with multiple comparisons, as appropriate. p < 0.05 was considered statistically significant.

Betanodavirus Induces Autophagy Process in GF-1 Cells
The mRFP-LC3 reporter system was used to identify autophagosome formation in GF-1 fish cells; cells with serum starvation were used as a positive control. Autophagosome formations were found at 36 h in serum starving cells ( Figure 1A) and RGNNV-infected cells ( Figure 1A). In RGNNV-infected cells, autophagosome formation was significantly increased in a time-dependent manner, with a~20% increase at 24 h,~40% increase at 36 h, and~60% increase at 48 h ( Figure 1B) compared to control cells.

3-Methyladenine (3-MA) Treatment Reduced RGNNV-Induced Autophagy and Blocked Bcl2 Family Downregulation in GF-1 Cells
There was a reduction of autophagosome formation in RGNNV-infected cells treated with 3-MA compared to untreated control by using the mRFP-LC3 reporter system ( Figure 2A). The autophagy inhibitor 3-MA underwent titration and received 2 mM as an optimal condition, which did not induce cell damage, and was then further tested. The 3-MA treatment significantly eliminated the autophagosome formation by~20% at 24 h, 40% at 36 h, and~55% at 48 h (p < 0.05) ( Figure 2B).

3-Methyladenine (3-MA) Treatment Reduced RGNNV-Induced Autophagy and Blocked Bcl2 Family Downregulation in GF-1 Cells
There was a reduction of autophagosome formation in RGNNV-infected cells treated with 3-MA compared to untreated control by using the mRFP-LC3 reporter system ( Figure 2A). The autophagy inhibitor 3-MA underwent titration and received 2 mM as an optimal condition, which did not induce cell damage, and was then further tested. The 3-MA treatment significantly eliminated the autophagosome formation by ~20% at 24 h, ~40% at 36 h, and ~55% at 48 h (p < 0.05) ( Figure 2B). Furthermore, treatment with 3-MA recovered RGNNV-induced BCL2 and BCL2L1 downregulation compared to untreated RGNNV-infected cells ( Figure 2C). The 3-MA treatment significantly inhibited BCL2 downregulation ~0.6-fold and BCL2L 0.45-fold at 36 h (p < 0.05) ( Figure 2D). Whether the downregulation of proteins in the BCL2 family correlated with the RGNNV-induced autophagy and cell death was then examined. We designed and produced Bcl2 and Bcl2L1-producing GF-1 cells for these experiments and confirmed that the expression of Bcl2 ( Figure 3A) and Bcl2L1 ( Figure 3B) were stable in GF-1 cells. Then, the effect of Bcl2 and Bcl2L1 overexpression on the regulation of autophagy-mediated cell death was examined. The results from the LC3-II:LC3-1 ratio at 36 and 48 h showed that the upregulation of Bcl2 and Bcl2L1 had eliminated the RGNNV-induced increase in LC3-II:LC3-1 ratio, indicating that overexpression of Bcl2 and Bcl2L1 may inhibit RGNNV-induced autophagy ( Figure 3C,D).

Overexpression of Zebrafish Bcl2 (Bcl2) and Zebrafish Bcl2 Like 1 (Bcl2L1) Reduced Autophagy and Protected Against Type III Cell Death
Whether the downregulation of proteins in the BCL2 family correlated with the RGNNV-induced autophagy and cell death was then examined. We designed and produced Bcl2 and Bcl2L1-producing GF-1 cells for these experiments and confirmed that the expression of Bcl2 ( Figure 3A) and Bcl2L1 ( Figure 3B) were stable in GF-1 cells. Then, the effect of Bcl2 and Bcl2L1 overexpression on the regulation of autophagy-mediated cell death was examined. The results from the LC3-II:LC3-1 ratio at 36 and 48 h showed that the upregulation of Bcl2 and Bcl2L1 had eliminated the RGNNV-induced increase in LC3-II:LC3-1 ratio, indicating that overexpression of Bcl2 and Bcl2L1 may inhibit RGNNVinduced autophagy ( Figure 3C,D).
Upregulation of RIPK3 during necroptosis was suppressed more efficiently by Bcl2L1 overexpressed cells than by Bcl2 at 36 h and 48 h, compared with untreated RGNNV-infected cells ( Figure 3E). Upregulation of RIPK3 during necroptosis was suppressed more efficiently by Bcl2L1 overexpressed cells than by Bcl2 at 36 h and 48 h, compared with untreated RGNNVinfected cells ( Figure 3E).

Blockage of Cell Death by Bcl2 and Bcl2L1 Overexpression and Reduced Viral Titers
Results from the ANXA5/annexin A5 and PI staining analysis indicated that Bcl2 and Bcl2L1 overexpression also significantly abolished RGNNV-induced cell death ( Figure 4). Our data showed that RGNNV-induced autophagy regulated host cell death by increasing signals that promote cell death and decreasing signals that prevent cell death. In addition, Bcl2 and Bcl2L1 overexpression in cells significantly inhibited RGNNV-induced the expression of viral death factors (B2 and protein α) by about 25% ( Figure 3C  Results are shown as means ± SEMs. * p < 0.05; ** p < 0.01.

Blockage of Cell Death by Bcl2 and Bcl2L1 Overexpression and Reduced Viral Titers
Results from the ANXA5/annexin A5 and PI staining analysis indicated that Bcl2 and Bcl2L1 overexpression also significantly abolished RGNNV-induced cell death ( Figure 4). Our data showed that RGNNV-induced autophagy regulated host cell death by increasing signals that promote cell death and decreasing signals that prevent cell death. In addition, Bcl2 and Bcl2L1 overexpression in cells significantly inhibited RGNNVinduced the expression of viral death factors (B2 and protein α) by about 25% (Figure 3C Our study showed that RGNNV induced autophagy of GF-1 cells during the early stage of viral replication and led to a dramatic increase in viral protein expression. Viral infection was correlated with the increased necroptosis in host cells and the downregulation of the anti-apoptotic BCL2 and BCL2L1 proteins. Our finding suggested that overexpression of the Bcl2 family can regulate host cell death and the viral-controlling autophagosome, which correlated to reduced viral expression.

Discussion
DNA and RNA viruses have evolved molecular mechanisms to allow them to escape or inhibit autophagy, thereby, increasing their infectivity [20], but RNNV virus-induced autophagy is still rare. As a result, how the autophagy process interacts with necroptosis is relatively unknown. In the present study, we investigated the role of Bcl2 family asymmetric crosstalk between cell death type III and autophagy. Our results in the molecular pathogenesis of RGNNV infection may help to identify potential targets that could be used for the prevention or treatment of RGNNV.

Betanodavirus-Induced Autophagy
Recently, autophagy has emerged as an important mechanism for controlling intracellular pathogens. A variety of bacteria, including Mycobacteria, Salmonella, Shigella, and Listeria, are recognized by autophagy, but the specific signals that mediate recognition Our study showed that RGNNV induced autophagy of GF-1 cells during the early stage of viral replication and led to a dramatic increase in viral protein expression. Viral infection was correlated with the increased necroptosis in host cells and the downregulation of the anti-apoptotic BCL2 and BCL2L1 proteins. Our finding suggested that overexpression of the Bcl2 family can regulate host cell death and the viral-controlling autophagosome, which correlated to reduced viral expression.

Discussion
DNA and RNA viruses have evolved molecular mechanisms to allow them to escape or inhibit autophagy, thereby, increasing their infectivity [20], but RNNV virus-induced autophagy is still rare. As a result, how the autophagy process interacts with necroptosis is relatively unknown. In the present study, we investigated the role of Bcl2 family asymmetric crosstalk between cell death type III and autophagy. Our results in the molecular pathogenesis of RGNNV infection may help to identify potential targets that could be used for the prevention or treatment of RGNNV.

Betanodavirus-Induced Autophagy
Recently, autophagy has emerged as an important mechanism for controlling intracellular pathogens. A variety of bacteria, including Mycobacteria, Salmonella, Shigella, and Listeria, are recognized by autophagy, but the specific signals that mediate recognition of intracellular pathogens by the autophagy machinery have not yet been well defined. Up to now the ubiquitin associated with intracellular pathogens has promoted targeted autophagosome formation and bacteria pathogen destruction. On the other hand, p62 and NDP52 mediate different pathways of selective autophagy for Shigella and Listeria, which provide novel insight into the mechanisms by which adaptor proteins target bacteria to autophagy [31]. In a viral pathogen infection, such as with DNA virus Herpes simplex type 1 (HSV-1), infection can provoke autophagy under conditions that inhibit viral gene expression. The induction of autophagy occurs very early after infection with HSV-1, and de novo protein synthesis is not required for the response [32][33][34].
Although certain RNA viruses have been previously shown to induce autophagy (e.g., the human immunodeficiency virus (HIV), hepatitis C virus (HCV), and influenza) [35][36][37], there are few reports that examine the effect of betanodaviruses on autophagy. We showed that RGNNV infection induced the formation of autophagosomes (Figure 1) in GF-1 cells by mRFP-LC3 at 36 hpf. In our analysis, 3-MA inhibited phosphatidylinositol 3-kinase (PI3K), which plays an important role in many biological processes, including controlling the activation of mTOR, a key regulator of autophagy. Apparently 3-MA blocked the formation of autophagosomes, which was consistent with previous studies where RGNNV-induced autophagy was inhibited by 3-MA [25]. On the other hand, several lysosomal inhibitors, such as bafilomycin A 1 (BafA 1 ), protease inhibitors and chloroquine (CQ), have been used interchangeably to block autophagy in in vitro experiments, assuming that they all primarily block lysosomal degradation. Among them, only CQ and its derivate hydroxychloroquine (HCQ) are FDA-approved drugs and are, thus, currently the principal compounds used in clinical trials aimed to treat tumors through autophagy inhibition. However, the precise mechanism of how CQ blocks autophagy remains to be firmly demonstrated; this will hopefully be uncovered in future testing [38].

Crosstalk between Autophagy and Necroptosis Cell Death Signals
The MTOR and BECN1 proteins play major roles in the regulation of autophagy [24,39,40]. BECN1 is part of a complex that regulates class III phosphatidylinositol 3-phosphate (Ptdlns3P) and hVps34 (human vacuolar protein sorting 34), whose activity is essential during autophagosome formation. Activators such as UVRAG (UV radiation resistance associated), Bif-1 (Bax-interacting factor) and Ambra-1 (the activating molecule in Beclin-1regulated autophagy-1), bind with the BECN1 complex to increase phosphatidylinositol 3-monophosphate production. In contrast, proteins in the BCL2 family of anti-apoptotic proteins, such as BCL2 and BCL2L1, bind to BECN1 and act as inhibitors [24,40,41].
The antiapoptotic protein Bcl2 functions either as an antiapoptotic protein or as an anti-autophagy protein via its inhibitory interaction with Beclin 1 [42], which recently functioned in the lysosomal degradation pathway of autophagy and induced autophagic cell death in cancer cells. Furthermore, Bcl2/Bcl2L1 can bind Beclin 1 and inhibit Beclin 1-dependent autophagic cell death in cancer cells. Then, the BH3 domain on Beclin binds to the BH3 binding groove of Bcl2L1 to block Bcl2L1 function.
In summary (Figure 6), we found that RGNNV infection could induce host cell death and autophagy, which also correlated with anti-Bcl2 member downregulation, such as Bcl2 and Bcl2L1, at the early-middle viral replication stage (12-24 hpi). Then, blockage of autophagy initiation by 3-MA apparently reduced autophagy and Bcl2 downregulation at 36 hpi. On the other hand, overexpression of the Bcl2 family members Bcl2 and Bcl2L1 either dramatically reduced type III cell death or suppressed autophagy initiation. We hypothesized that RGNNV induced cell death and autophagy by asymmetrical interaction, not by direct crosstalk. There seemed to be an upstream or downstream correlation in this system, but this requires further research. at 36 hpi. On the other hand, overexpression of the Bcl2 family members Bcl2 and Bcl2L1 either dramatically reduced type III cell death or suppressed autophagy initiation. We hypothesized that RGNNV induced cell death and autophagy by asymmetrical interaction, not by direct crosstalk. There seemed to be an upstream or downstream correlation in this system, but this requires further research. Then, those stress signals can regulate multiple signaling pathways either to induce cell death or trigger an autophagy flow in the middle replication stage that correlates with anti-apoptotic member Bcl2 and Bcl2L1 downregulation. In autophagy inhibitor treatment, 3-MA can suppress autophagy initiation. Furthermore, if overexpression of Bcl2 and Bcl2L1 genes can also either block cell death and reduce the viral titer or suppress the autophagy process. The correlations between cell death and autophagy, appear to be asymmetrical upstream or downstream correlation interactions.

Conclusions
RGNNV infection in GF-1 cells can induce autophagosome formation and Bcl-2 downregulation at different post-infection times; however, Bcl-2 downregulation is blocked by the autophagy inhibitor 3-MA. Then, extracellular Bcl-2 and Bcl2L1 overexpression can suppress host cell death type III and autophagy caused by RGNNV infection. Our results suggest that RGNNV-induced cell death and autophagy by asymmetrical interaction was regulated by the anti-apoptotic members Bcl2 and Bcl2L1 and autophagy inhibitor 3-MA via the inhibition of class III Ptdlns3K.  Then, those stress signals can regulate multiple signaling pathways either to induce cell death or trigger an autophagy flow in the middle replication stage that correlates with anti-apoptotic member Bcl2 and Bcl2L1 downregulation. In autophagy inhibitor treatment, 3-MA can suppress autophagy initiation. Furthermore, if overexpression of Bcl2 and Bcl2L1 genes can also either block cell death and reduce the viral titer or suppress the autophagy process. The correlations between cell death and autophagy, appear to be asymmetrical upstream or downstream correlation interactions.

Conclusions
RGNNV infection in GF-1 cells can induce autophagosome formation and Bcl-2 downregulation at different post-infection times; however, Bcl-2 downregulation is blocked by the autophagy inhibitor 3-MA. Then, extracellular Bcl-2 and Bcl2L1 overexpression can suppress host cell death type III and autophagy caused by RGNNV infection. Our results suggest that RGNNV-induced cell death and autophagy by asymmetrical interaction was regulated by the anti-apoptotic members Bcl2 and Bcl2L1 and autophagy inhibitor 3-MA via the inhibition of class III Ptdlns3K.