article: BCL2 family proteins including pro-survival proteins, BH3-only proteins and BAX/BAK proteins control mitochondria-mediated apoptosis to maintain cell homeostasis via the removal of damaged cells and pathogen-infected cells. In this study, we examined the roles of BCL2 proteins in the induction of apoptosis in cells upon infection with flaviviruses, such as Japanese encephalitis virus, Dengue virus and Zika virus. We showed that survival of the infected cells depends on BCLXL, a pro-survival BCL2 protein due to suppression of the expression of another pro-survival protein, MCL1. Treatment with BCLXL inhibitors, as well as deficient BCLXL gene expression, induced BAX/BAK-dependent apoptosis upon infection with flaviviruses. Flavivirus infection attenuates cellular protein synthesis, which confers reduction of short-half-life proteins like MCL1. Inhibition of BCLXL increased phagocytosis of virus-infected cells by macrophages, thereby suppressing viral dissemination and chemokine production. Furthermore, we examined the roles of BCLXL in the death of JEV-infected cells during in vivo infection. Haploinsufficiency of the BCLXL gene, as well as administration of BH3 mimetic compounds, increased survival rate after challenge of JEV infection and suppressed inflammation. These results suggest that BCLXL plays a crucial role in the survival of cells infected with flaviviruses, and that BCLXL may provide a novel antiviral target to suppress propagation of the family of Flaviviridae viruses.
  The genus Flavivirus belongs to the family Flaviviridae and includes the Japanese encephalitis virus (JEV), Dengue virus (DENV), West Nile virus (WNV) and Zika virus (ZIKV), all of which are mosquito-borne human pathogens [1,2]. The flaviviruses are internalized into dendritic cells, such as Langerhans cells [3], and keratinocytes in skin [4] through mosquito bites. Dendritic cells that are thus infected with flaviviruses are activated and migrate into the lymph nodes, facilitating viral spread into peripheral tissues [5] and the induction of visceral and/or central nervous system diseases. DENV infection may generate dengue haemorrhagic fever and dengue shock syndrome [6], while infection with either WNV or JEV can cause severe encephalitis [7,8]. Recently, an outbreak of ZIKV infection in Brazil was linked to birth defects, including microcephaly, and to Guillain-Barre syndrome in adults [9,10]. Therefore, the development of effective vaccines and antiviral drugs against the family of flaviviruses is an urgent need. Infection with flaviviruses induces apoptosis in cells by the production of reactive oxidative species, unfolded protein responses, and viral proteins [11], in addition to other types of cell death, such as necrosis and pyroptosis. Receptor-interacting serine/threonine-protein kinase-3 (RIPK3), a central player of necroptosis, has been shown to play an additional role during virus infection [12]. Infection with either DENV or hepatitis C virus (HCV) induces interleukin-1β production and pyroptosis [13]. The biological significance of pyroptosis in the pathogenicity of flavivirus infection remains largely unknown [14]. Apoptosis is a type of programmed cell death that removes excess cells during development, as well as during routine homeostasis [15–17]. B-cell lymphoma-2 (BCL2) family proteins play central roles in mitochondria-mediated apoptosis [18]. BCL2 family proteins have been divided into three groups: BH3-only proteins, BAX/BAK proteins and pro-survival proteins. Once an apoptotic stimulus such as DNA damage occurs, BH3-only proteins, such as BIM, BID, PUMA, BAD and NOXA, are activated and directly or indirectly activate BAX and BAK. Activated BAX/BAK forms homo-oligomers that permeabilize mitochondria and allow the release of pro-apoptotic factors such as cytochrome c, which promotes activation of caspases [19]. Pro-survival proteins, such as BCL2, BCLW, BCLXL, MCL1 and A1, sequester BH3-only proteins to prevent self-activation of BAX/BAK by interaction with the BH3 domains of BAX/BAK [20]. In a notable extension to cancer therapy, small molecule inhibitors have been developed, including ABT-737, ABT-263 (navitoclax, an orally available clinical derivative of ABT-737), ABT-199 (venetoclax) and A-1331852. ABT-199 and A-1331852 target BCL2 and BCLXL, respectively, while ABT-737 and ABT-263 target BCL2, BCLXL and BCLW. ABT-199 was approved by the United States Food and Drug Administration for the treatment of 17-p deleted chronic lymphocytic leukaemia (CLL) [21], supporting the therapeutic use of induction of cell death by mitochondria-mediated apoptosis to eliminate unnecessary cells. In this study, we showed that cells infected with flaviviruses including JEV, DENV and ZIKV specifically induced apoptosis by inhibition of BCLXL through the suppression of MCL1 expression. Furthermore, we showed that host protein synthesis is impaired in cells upon infection with flaviviruses, leading to the decay of labile proteins such as MCL1. Treatment with inhibitors for BCLXL, as well as deficiency of BCLXL gene, induced BAX/BAK-dependent apoptosis in the infected cells. Finally, engulfment of virus-infected cells by macrophages was significantly enhanced by the inhibition of BCLXL without an increase of the production of pro-inflammatory cytokines, which led to the suppression of viral dissemination.
  Firstly, to examine the roles of BAX/BAK-dependent apoptosis during flavivirus-infected cells, we generated BAX and BAK double-knockout (BAX/BAKDKO) Huh7 cell lines (clones #29 and #47) and infected with Japanese encephalitis virus (JEV) (S1A Fig). Although Huh7 cells induced cell death at 4 days post-infection with JEV, BAX/BAKDKO cells exhibited a complete resistant to mitochondria mediated apoptosis [22], suggesting that cells infected with JEV induce mitochondria-mediated apoptosis at 4 days post-infection. However, BAX/BAKDKO cells also exhibited cell death at 5 and 6 days post-infection. The BAX/BAK-independent cell death might be induced by necrosis or pyroptosis rather than apoptosis as previously reported (Fig 1A) [12]. Recent clinical studies showed that survival of chronic lymphocytic leukaemia (CLL) is largely dependent on BCL2 [21], and that loss of a single Bclx allele attenuates MYC-induced lymphoma in vivo [23]. In addition, selective inhibition of MCL1 has been shown to induce apoptosis in acute myelogenous leukaemia cell lines without signs of toxicity to normal human haematopoietic progenitor cells [24]. It had been reported that cellular stress such as ER stress, DNA damage and innate immune response affects on the expression of BCL2 protein family [25–27]. Therefore, we speculated that virus infection alters the expression of BCL2 proteins through the induction of cellular stress. BH3-only proteins are known to exhibit specific interactions with pro-survival BCL2 proteins, including BCL2, BCLXL, BCLW and MCL1 [28]. BIM and PUMA bind to all pro-survival BCL2 proteins; BAD binds selectively to BCL2, BCLXL and BCLW; and NOXA binds specifically to MCL1 (S1B Fig). To investigate the roles of pro-survival BCL2 proteins in cells infected with flaviviruses, we generated Huh7 cell lines stably expressing a BIM-mutant possessing either a binding-defective BH3 domain (BIM-4EBH3), the BH3 domain from BAD (BIM-BADBH3), or the BH3 domain from NOXA (BIM-NOXABH3) (S1C and S1D Fig). Although lentivirus-induced expression of BIM-NOXABH3 or BIM-BADBH3 specifically killed Huh7 cells expressing BIM-BADBH3 or BIM-NOXABH3, respectively, parental and Huh7 cells expressing BIM-4EBH3 were resistant to killing through the expression of BIM-mutants (S1E Fig). Although no cell death was observed in parental Huh7 cells, or in cells expressing either BIM-4EBH3 or BIM-NOXABH3, upon infection with JEV and DENV, cells expressing BIM-BADBH3 showed a significant reduction in viability at 3 days-post infection (Fig 1B). To assess which pro-survival BCL2 proteins were responsible for the survival of cells infected with flaviviruses, the effects of BH3 mimetics on flavivirus-infected cells were determined. No cytotoxicity was observed in Huh7 cells solely treated with BH3 mimetics at 3days post treatment (S1F Fig). Cells infected with JEV, DENV and ZIKV, and treated with either ABT-737 [29] (targeting BCL2, BCLW and BCLXL) or A-1331852 [30] targeting BCLXL exhibited cell death at 3 days-post infection. In contrast, infected cells treated with ABT-199 [31] (targeting BCL2) showed no cell death (Fig 1C and S1B Fig). Treatment of ABT-737 (1 μM) significantly accelerated JEV, DENV and ZIKV induced cell death (Fig 1D). Next, to examine the roles of BCLXL inhibition on infectious virus production, we quantified viral titres by using a focus-forming assay. Treatment with ABT-737 (1 μM) exhibited no effect on the infectious particle production of JEV, DENV and ZIKV until 2-days post-infection, but significantly reduced the infectious titers at 3 and 4 days post-infection (Fig 1E), suggesting that induction of cell death by the treatment with ABT-737 in viral infected cells reduces infectious virus production. We next determined whether ABT-737 treatment induces BAX/BAK-dependent cell death upon infection with flaviviruses. Although parental Huh7 cells exhibited cell death upon JEV infection and treatment with ABT-737, which activated caspase 3/7, BAX/BAKDKO cells were resistant to cell death under the same conditions, and no activation of caspase 3/7 was detected (Fig 1F and 1G). In addition, release of cytochrome c was observed in parental Huh7 cells infected with JEV and treated with ABT-737, but not in BAX/BAKDKO cells (S1G Fig). Although no increase of infectious particle production was observed in BAX/BAKDKO cells until 2-days post-infection, three to ten times of increase of infectious titer compared to parental Huh7 cells was observed in BAX/BAKDKO cells at 3 and 4 days post-infection (Fig 1H). Collectively, these results suggest that cell death is induced upon infection with JEV and treatment with ABT-737 through a BAX/BAK-dependent pathway. To confirm the BCLXL-dependent survival of cells upon infection with flaviviruses, we generated Huh7 cell lines that were deficient in either BCLXL or MCL1 (BCLXKO or MCL1KO) (Fig 2A). Expression of BIM-BADBH3 and BIM-NOXABH3 killed MCL1KO and BCLXKO cells, respectively, while expression of BIM-4EBH3 conferred resistance to both parental and KO cells (Fig 2B). Among the pro-survival proteins of BCL2 protein family, BCLX and MCL1 redundantly suppress activation of both of BAX and BAK. On the other hands, BCL2 and BCLW had been reported to suppress BAX but not BAK [32–36]. In addition, deficiency of BCLW in mice developed normally except spermatogenesis in male mice, suggesting that BCLW is important for apoptosis in testis [37]. To examine the redundancy of BCL2 proteins, we treated parental, MCL1KO and BCLXKO Huh7 cells with either A-1331852, BCLX-specific inhibitor or S63845, MCL1 selective inhibitor [24]. MCL1KO Huh7 cells treated with A-1331852 (1 μM) but not with S63845 (1 μM) and BCLXKO Huh7 cells treated with S63845 (1 μM) but not with A-1331852 (1 μM) induced cell death, while parental Huh7 cells are resistant to the treatment with either compounds but induced cell death by the treatment with both compounds (S2A Fig). These data suggest that BCLX and MCL1 redundantly suppress activation of BAX and BAK. The viabilities of BCLXKO cells upon infection with JEV, DENV or ZIKV were consistently lower than those of parental and MCL1KO Huh7 cells (Fig 2C), and we confirmed the induction of cell death upon infection with JEV in other independently established BCLXKO Huh7 cell lines (S2B and S2C Fig). Next, we determined the effect of BCLX inhibition on plaque formation. Furthermore, Huh7 cells did not form any plaque upon infection with JEV, DENV and ZIKV, however, BCLXKO Huh7 cells exhibited formation of clear plaques upon infection with these viruses (Fig 2D). Taken together, these results indicate that apoptosis is induced in BCLX-deficient cells upon infection with flaviviruses. BAX/BAK activation is suppressed by pro-survival BCL2 proteins [20]. To investigate how BCLXL participates in cell survival upon infection with flaviviruses, expression levels of BCLXL and MCL1 were determined in flavivirus-infected cells. Interestingly, expression of MCL1 decreased upon infection with JEV, DENV and ZIKV, in contrast to the continued stable expression of other BCL2 family proteins (Fig 3A). No MCL1 protein was detected in the insoluble fraction of cell lysates (S3A Fig), suggesting that the total amount of MCL1 protein is decreased by the infection with flaviviruses. MCL1 has been shown to be a short-lived protein that is degraded by the proteasome [32,38,39]. Northern blot analysis revealed stable expression of mRNA of MCL1 in JEV-infected cells (S3B Fig). Reduction of MCL1 protein by JEV infection was slightly restored by the treatment with a proteasome inhibitor (Ac-Leu-Leu-Nle-Aldehyde, ALLN; (20 μM), however, the expression level of MCL1 was not recoverable by the inhibition of proteasome (Fig 3B). Moreover, lysosome inhibitor treatment did not restore reduction of MCL1 protein (S3C Fig), suggesting that JEV infection suppresses host translation resulting in the reduction of MCL1 protein expression through proteasome-mediated degradation. A previous study showed that MULE (HUWE1) is a unique BH3-containing E3 ubiquitin ligase that is associated with suppression of MCL1 expression [40]. Suppression of MCL1 expression in JEV-infected MULEKO cells was comparable to MCL1 in JEV-infected parental Huh7 cells (S3D and S3E Fig). Further, the Skp, Cullin and F-box containing complex (SCF complex) has also been shown to play a role in MCL1 degradation [41,42]. Although the overexpression of dominant negative Cullin-1 (DN-CUL1) led to an upregulation of MCL1 in non-infected cells, infection with JEV still suppressed MCL1 expression (S3F Fig). Additionally, overexpression of the BH3-only protein, NOXA, has been shown to induce suppression of MCL1 expression [32]. Suppression of MCL1 expression in JEV-infected NOXAKO cells was comparable with MCL1 in JEV-infected parental Huh7 cells (S3G and S3H Fig). Importantly, expression of MCL1K/R, which is an ubiquitination-defective mutant of MCL1, was still supressed upon JEV infection (S3I and S3J Fig). The ts20 cells are derived from Balb/c 3T3 cells and possesses a temperature-sensitive ubiquitin-activating E1 enzyme [43]. The ts20 cells were infected with JEV, then incubated for 6 h at 37°C (active E1) or 39°C (inactive E1), 2 days post-infection. Notably, the expression of p53, but not of MCL1, was restored by inhibition of E1 (S3K Fig), suggesting that ubiquitination is not involved in the suppression of MCL1 protein expression in cells infected with JEV. To examine the effects of JEV infection on protein synthesis, JEV-infected cells were labelled with [35S]-methionine and [35S]-cysteine. The total amount of protein synthesis was significantly reduced at both 2 and 3 days post-infection with JEV (Fig 3C, left panel). Additionally, protein synthesis of MCL1 was significantly reduced by infection with JEV (Fig 3C, right panel). In addition, expression of CCND1, another short half-life protein [44,45], was also suppressed by JEV infection (Fig 3D). We hypothesized that expression of short half-life proteins was non-specifically suppressed upon infection with flaviviruses because labile proteins were constitutively degraded under inhibition of newly protein synthesis. To clarify it, we generated Huh7 cell lines stably expressing either GFP or D2GFP, a destabilized form of GFP that contains a PEST sequence [46]. Expression of D2GFP and MCL1 was decreased upon infection with JEV in a dose-dependent manner at 2 days post infection, in contrast to the stable expression of GFP (Fig 3E). These results suggest that infection with flaviviruses induces inhibition of protein translation, leading to a reduction of short half-life proteins such as MCL1. Next, to examine the effect of viral proteins on MCL1 expression, we transfected the plasmids encoding JEV structural proteins (HA-Core and prM-E), non-structural proteins (HA-NS1, HA-NS2A, HA-NS2B, HA-NS3, HA-NS4A, HA-NS4B and HA-NS5) and control vector into Huh7 cells. Expression of viral proteins did not induce suppression of MCL1 expression (S3L Fig). These results suggest that viral propagtion is required for suppression of MCL1 expression. To examine the relationship between the suppression of MCL1 expression and a susceptibility to JEV propagation in other human cell lines, we selected cell lines with high and low susceptibility to JEV infection: IGR-OV1, H522 and SF-268 cells (high susceptibility) and OVCAR5 and HT29 cells (low susceptibility) (Fig 4A). Expression of MCL1 was reduced in the highly susceptible cell lines upon infection with JEV; in contrast, MCL1 was stably expressed in cell lines with low susceptibility (Fig 4B). Moreover, cell lines with high susceptibility were killed by the combination of infection with JEV and treatment with the BH3 mimetic ABT-737 (1 μM) (Fig 4C) or A-1331852 (1 μM) (S4A Fig); in contrast, cell lines with low susceptibility remained viable when receiving this treatment. In addition, the suppression of MCL1 expression was enhanced upon infection with JEV (Fig 4D), but suppressed in a dose-dependent manner by treatment with IFNα (Fig 4E). Further, the decrease in cell viability induced by the combination treatment was reversed upon treatment with IFNα (Fig 4F). These data suggest that viral replication is important for suppression of MCL1 expression and suppression of MCL1 expression is independent on IFN signalling pathway. U937 cells, a human lymphoma cell line, exhibited high, moderate and low susceptibility to ZIKV, JEV and DENV, respectively (Fig 4G). Suppression of MCL1 expression was consistent with the degree of susceptibility to each virus at 2 days post infection (Fig 4H). Treatment with the BH3 mimetic ABT-737 (1 μM) or A-1331852 (1 μM) enhanced cell death in U937 cells that were also infected with ZIKV (Fig 4I), but not in cells that were also infected with JEV at 3 days post infection (S4B Fig). Taken together, these data suggest that suppression of MCL1 is required for an efficient viral propagation. Primary targets of JEV, DENV and ZIKV include haematopoietic and skin cells; therefore, we next examined the effects of BCLX inhibition during flavivirus infection by using host cells closer to human infection. Monocyte-derived dendritic cells (MDDCs) have been shown to be highly susceptible to DENV infection [47]. Treatment with ABT737 (1 μM) significantly increased cell death in MDDCs that were also infected with DENV (S4C Fig). In addition, treatment with ABT-737 (1 μM) or A-1331852 (1 μM) significantly increased cell death in human melanoma cell lines, UACC257 and UACC62, upon infection with JEV (S4D Fig), and in mouse embryonic fibroblasts (MEFs) infected with JEV, DENV or ZIKV at 2 days post infection (S4E Fig). These results suggest that suppression of MCL1 expression and induction of cell death by the treatment with an inhibitor for BCLX are conserved in a variety of human and mouse cells upon infection with flaviviruses. An arthropod-borne flavivirus primarily infects dendritic cells, such as Langerhans cells, and keratinocytes in skin [3]. These infected cells are stimulated and then migrate into the lymph nodes [48], leading to propagation within the lymphatic system. We hypothesized that the induction of cell death by BCLXL inhibition in flavivirus-infected cells would accelerate exposure of ‘eat-me’ signal on the surface of the cells and engulfment of infected cells by phagocytes, thus inhibiting dissemination of virus infection. To examine the involvement of phagocytosis in JEV-infected cells treated with the BH3 mimetic ABT-737, we performed an in vitro phagocytosis assay using peritoneal exudate cells (PECs) as model phagocytes (Fig 5A). Both of BCLXKO and ABT-737-treated Huh7 cells infected with JEV exhibited a significant increase of engulfment by PECs at 2 and 3 days post-infection, even though no cell death was observed at 2 days post-infection (Fig 5B). In contrast, no phagocytosis was observed in JEV-infected Huh7 cells (Fig 5B). To examine whether PECs produce chemokines or cytokines upon co-culture with JEV infected cells, BCLXKO and ABT-737-treated Huh7 cells infected with JEV were overlaid onto PECs at 1,2 and 3 days post-infection. Production of chemokines and cytokines in the supernatants was determined after 24 h of incubation. Significant amounts of chemokines and cytokines were observed in the supernatants of co-cultured PECs with JEV infected but not with mock-infected Huh7 cells (Fig 5C). Inhibition of BCLXL in JEV-infected Huh7 cells suppressed the production of pro-inflammatory cytokines by PECs, including CCL5, CCL2 and CXCL10, while no significant difference in the expression of TNF-α and IL-6 was observed (Fig 5C). To examine whether infectious particles are released from the co-culture system, the supernatants were also collected and infectious titers were determined after 24 h of co-culture. Infectious titers were significantly reduced in the supernatants of PECs that were co-cultured with either BCLXKO or ABT-737-treated Huh7 cells, compared with PECs that were co-cultured with parental Huh7 cells at 2 and 3 days post-infection with JEV (Fig 5D). Additionally, we confirmed that JEV could propagate in PECs (S5 Fig). These results suggest that the inhibition of BCLXL in viral-infected cells accelerates efficient engulfment by phagocytes, thereby suppressing the production of chemokines and infectious particles. To further examine the biological significance of BCLXL inhibition in virus-infected cells, mice that were heterozygotic for Bclx were generated using a CRISPR/Cas9 system (S6A Fig), because homozygous mice died around E13 due to massive cell death of immature hemopoietic cells and neurons [49]. We obtained F1 mice and generated Bclx+/- mice (F2) by a cross with wild-type mice. We established three Bclx+/- lines from separate lines of F1 mice (No. 32, No. 40 and No. 44, S6A Fig). Bclx+/- mice expressed less BCLX protein in the spleen compared to wild type mice (S6B Fig). We injected lethal amounts of JEV into the footpad of each mouse (subcutaneous challenge). Bclx+/- mice exhibited higher survival rates against footpad challenge with JEV, compared with wild-type mice (Fig 6A). Expression of viral RNA, as well as Ccl5 and Cxcl10, but not Tnfα, Il6 Ccl2, Ifna and Ifnb, was significantly decreased in the footpads of Bclx+/- mice at 5 days post-infection (Fig 6B and 6C and S6C Fig). In contrast, the expression of Ccl5, Cxcl10, Tnfα and Il6 was comparable in wild-type and Bclx+/- mice upon stimulation with Poly (I: C) (Fig 6D and S6D Fig). In addition, oral administration of a clinically relevant dose of ABT-263 (50mg kg-1), an analogue of the BH3 mimetic ABT-737, conferred prolonged survival against subcutaneous challenge of JEV (Fig 6E). In contrast, Bclx+/- mice exhibited similar survival rates and viral RNA production upon intracerebral challenge of JEV with wild-type mice (Fig 6F and 6G), suggesting that the suppression of virus growth in peripheral tissues participates in the delay of death and higher percentage of survival in Bclx+/- mice against JEV challenge. These results suggest that viral propagation within flavivirus-infected cells can be inhibited by the suppression of BCLXL and subsequent induction of apoptosis.
  In this study, we showed that flavivirus infection suppresses expression of labile proteins such as MCL1 without observable cytotoxicity. Clear cell death, through activation of caspase 3/7, occurred only when BCLXL was inhibited. Moreover, we showed that treatment with BCLXL inhibitor (A-1331852) or deficiency of the BCLXL gene induces cell death upon infection with flaviviruses in several cancer cell lines and primary MEFs. Previous report showed that BCLXL and MCL1 predominantly inhibit activation of BAK [50], suggesting that a mechanism to inhibit BCLXL may induce BAK-dependent apoptosis in flavivirus-infected cells, therefore inhibition of BCLX alone could enhanced cell death in virus-infected cells. In addition, viral infection decreased MCL1 expression in a variety of human and murine cell lines. Although the magnitude of suppression of MCL1 expression varied depending on cell line, our data suggest that suppression of MCL1 expression upon propagation of flaviviruses is a common feature. Legionella infection also induces suppression of MCL1 expression [51]. Although the molecular mechanisms of MCL1 protein expression in cells infected with Legionella remain unclear, the suppression of MCL1 through shut off of host translation might be part of the cellular innate response against infection with intracellular pathogens. MCL1 expression is strictly controlled by transcriptional and post-transcriptional mechanisms [52]. In this study, we showed that flavivirus infection suppresses protein synthesis, thereby reducing the quantity of proteins with short half-lives, such as MCL1. In addition, recent reports have shown that infection with flaviviruses represses translation of host proteins, but does not repress viral proteins, which are independent of PKR and eIF2α [53]. Further, infection with mammalian orthoreovirus has been shown to inhibit host translation by compartmentalization of translation machinery within the viral replication complex [54]. Protein translation is regulated by ribosomal proteins [55]. DENV NS1 protein specifically binds to the ribosomal protein RPL18 to sequester in perinuclear region and to regulate viral translation and replication [56]. RPLP1 and RPLP2 identified by genome-wide RNAi screening were host factors to contribute DENV and yellow fever virus (YFV) infection [57]. We previously reported that heterogeneous nuclear ribonucleoprotein A2/B1 (HNRNPA2B1) interact with JEV core protein and facilitate JEV replication through the translocation of core protein from nucleus to cytoplasm [58]. In addition, infection with HCV has been shown to induce accumulation of adenosine 5′-triphosphate in the viral replication complex [59]. Because expression of JEV proteins exhibited no effect on host translation (S3L Fig), viral propagation might induce shut-off of host translation by unknown mechanism rather than PKR and eIF2α. On the other hand, viruses counteract host responses through the rearrangement of membrane structures to restrict the components of translation machinery into the viral replication complex. Our data indicate that inhibition of BCLXL in viral-infected cells activates phagocytosis by macrophages, leading to the suppression of infectious particle production and suggest that inhibition of BCLXL function as an anti-viral effect. Although the survival of mice that were intracerebrally inoculated with JEV was not significantly different between wild-type and Bclx+/- mice, Bclx+/- mice showed higher survival rate against subcutaneous challenge by JEV, compared with their wild-type counterparts. Our data suggest that phagocytosis plays an important role in removing apoptotic. JEV-infected cells after BCLX inhibition. Microglial cells, a resident subset of macrophages in the brain, and hematogenous macrophages play a role in phagocytosis in the brain and peripheral tissues, respectively. These two-types of macrophage have phagocytotic activity but have some different functions [60]. The functional difference of the microglia and hematogenous macrophages might be involved in the phagocytosis-mediated removals of virus-infected cells by BCLX inhibition in intracerebral or subcutaneous infection. Collectively, these data suggest that apoptosis induced by inhibition of BCLXL attenuates viral virulence in peripheral tissues. The production of cytokines and chemokines during viral infection is strongly associated with viral pathogenicity [61], especially, deficiency of several chemokine receptors such as CCR5 and CXCR3 promotes severity of pathogenicity against WNV infection [62]. We showed that the inhibition of BCLXL specifically induces apoptosis and impaired production of CCL5 and CXCL10, which are ligands for CCR5 and CXCR3, respectively, during in vitro and in vivo viral infections. Impairment of these chemokines may contribute attenuation of pathogenicity by inhibition of BCLXL. In addition, administration of the BH3 mimetic, ABT-263, into wild-type mice increased survival rate after subcutaneous JEV challenge. We also showed that production of viral RNA and chemokines was impaired in Bclx+/- mice (Fig 6B and 6C). In addition, treatment of wild type and Bclx+/- mice with polyIC exhibited no effect on chemokine induction (Fig 6D). These data suggest that enhanced apoptosis in Bclx+/- and ABT-263-treated mice upon infection with JEV promotes engulfment by phagocytes, which leads to reduces viral production and the number of leukocytes migrating to the infection site due to lower production of chemokines. Treatment with inhibitors of BCLX such as ABT-263 was demonstrated to cause thrombocytopenia due to their neutralization of BCLXL in circulating platelets without bone marrow toxicity [30,63,64]. It was also demonstrated that thrombocytopenia induced by ABT-263 is dose-dependent and reversible effect [64], therefore it is possibility to control thrombocytopenia by appropriate terms of treatment or amounts of BCLX inhibitors [65,66]. It is also another possibility to develop the drug delivery system to virus-infected cells to avoid the unexpected side effects. Further studies are needed to use inhibitors of BCLX for antiviral therapy. Based on these results, we propose the model as shown in Fig 7 to explain the regulation of BCL2 family proteins upon infection with flaviviruses. In this study, we showed that infection with flaviviruses reduces expression of MCL1 but remaining BCLXL delayed apoptosis in infected cells. This delay of apoptosis is crucial for viral dissemination to neighbouring cells, leading to high pathogenicity. On the other hand, impairment of BCLXL accelerates apoptosis in cells infected with flavivirus and dissemination is reduced by the removal of infected cells through phagocytosis, leading to local infection and low pathogenicity in vivo. In conclusion, we have demonstrated that BCLXL is a critical cell survival factor during infection with flaviviruses, and that inhibition of BCLXL by treatment with BH3 mimetics restricts the production of infectious particles in vitro and in vivo. We showed that expression of pro-survival MCL1 is reduced upon infection with flaviviruses. Although further studies are needed to clarify the molecular mechanisms, the translational suppression induced by viral infection is suggested to participate in the instability of unstable proteins including MCL1. Bclx+/- mice exhibited impairment of pathogenicity to JEV infection, together with the enhanced viral clearance by phagocytosis of infected cells through the stimulation of apoptosis by BCLXL inhibition. In addition, Bclx+/- mice, and wild-type mice that received ABT-263, exhibited prolonged survival rates after JEV challenge compared with untreated wild-type mice. These data suggest that BCLXL may serve as a novel antiviral target that facilitates suppression of the propagation of a broad range of the family Flaviviridae viruses.
  Huh7,293T, U937, and Vero cells were obtained from the National Institute of Infectious Diseases and cultured in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin. IGR-OV1, SF268, H522, OVCAR5, HT29, UACC257, UACC62 and ts20 were provided by the Walter & Eliza Hall Institute and maintained in RPMI1640 supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin. The MEFs were obtained from E14. 5 embryos and maintained in DMEM supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin. JEV (AT31 strain), DENV (H241 strain) and ZIKV (MR766-NIID strain) were propagated in C6/36 cells. Infectious titers of JEV, DENV and ZIKV were determined by focus-forming assay. Mouse monoclonal antibodies against JEV/DENV/ZIKV NS3 (#578, #1791); [58] were used to visualize the focuses. BIM, BIM-BADBH3, BIM-NOXABH3 and BIM-4EBH3 [28] were amplified by Tks Gflex DNA Polymerase (Takara-Bio) and cloned into an FUIPW lentiviral transfer vector [67] by using an In-Fusion HD cloning Kit (Takara-Bio). The MCL1K/R gene was synthesized by Thermo Fisher Scientific (GeneArt Strings), amplified and cloned into an FUIPW vector or a pCAGGS vector [68]. The cDNA of D2GFP [46] was synthesized (Integrated DNA Technologies (IDT) ) and cloned into an FUIPW vector. pCAG-HA-Ubiquitin was previously described [69]. Plasmids encoding HA-tagged JEV Core and NS proteins (pCAGPM-HA-NS) were previously described [58,70]. Plasmids encoding JEV prM-E protein was previously described [71]. pcDNA3-DN-hCUL1-FLAG was obtained from Addgene (#15818). The pSPT18 was obtained from Dr. Kamitani. For the CRISPR/Cas9 system, the plasmids of pX330 (#42230) and pCAG EGxxFP (#50716) were obtained from Addgene. All sgRNAs are listed in Appendix S1 Table. The lentivirus packaging vectors, pMDLg/pRRE, pRSV-Rev and pCMV-VSV-G, were obtained from Addgene. The sequences of all plasmids used in this study were confirmed by using an ABI Prism 3130 genetic analyzer (Applied Biosystems). To generate lentiviruses, 293T cells were transfected with transfer vectors, pMDLg/pRRE, pRSV-Rev and pCMV-VSV-G, using polyethylenimine (PEI; MW: 25,000; Polysciences Inc.), and the culture supernatants collected at 3 days post-transfection were passed through a 0. 45-mm filter. Culture supernatants containing the lentiviruses were inoculated into target cells seeded on 6-well plates using Polybrene (Sigma) and were centrifuged at 2,500 rpm for 45 min at 32 °C. The following antibodies were used: anti-JEV/DENV/ZIKV NS3 monoclonal antibody (#578, #1791,1: 1000 dilution) [58], anti-ACTIN mouse monoclonal antibody (A2228; Sigma, 1: 5000 dilution), horseradish peroxidase-conjugated anti-FLAG mouse monoclonal antibody (clone M2; Sigma, 1: 1000 dilution), anti-HA rat monoclonal antibody (clone 3F10; Roche, 1: 1000 dilution), mouse anti-cytochrome c (BD 556433,1: 1000 dilution), rabbit polyclonal antibodies to MCL1 (sc-819; Santa Cruz, 1: 1000 dilution, 600-401-394; Rockland, 1: 5000 dilution), mouse monoclonal antibodies to BCLXL (BD 610747,1: 1000 dilution, BD 610212,1: 1000 dilution), BAK (8F8,1: 1000 dilution), BAX (21C10-23-8-3,1: 1000 dilution), CYCLIN D1 (BD 556470,1: 1000 dilution), FKBP8 [72] (1: 1000 dilution), MULE (NB100-652; Novus Biologicals, 1: 1000 dilution), p62 (M162-3; MBL, 1: 1000 dilution), p53 (1C12; Cell Signaling Technology, 1: 1000 dilution), ABT-737, ABT-199 and A-1331852 were synthesized by Walter & Eliza Hall Institute. ABT-263 was obtained from Synkinase. S63845 was obtained from Active Biochem. ALLN (A6185) and bafilomycin A1 (B1793) were obtained from Sigma, QVD-OPH (sc-222230) was obtained from Santa Cruz Biotechnology and Digitonin (#300410) was obtained from Calbiochem, E64d (4321-v), and pepstatin A (4397-v) were obtained from Peptide Institute, Inc. human IL-4 (200–04) and GM-CSF (300–03) were obtained from peprotech. Poly (I: C) (tlrl-pic) was obtained from invivogen. Knockout Huh7 cell lines were established by using CRISPR/Cas9 as described previously [73]. The plasmids of pX330 (#42230) and pCAG EGxxFP (#50716) were obtained from Addgene. The target sequences of each gene are summarized in S1 Table. Genomic DNAs of Huh7 were amplified and cloned into pCAG EGxxFP. Huh7 cells were transfected with pX330 and pCAG EGxxFP and incubated for 1 week. GFP-positive Huh7 cells were sorted by FACS and formed a single colony. Gene deficiency was confirmed by sequencing and Western blotting. Cell lysates were prepared by incubation in Onyx lysis buffer consisting of 20 mM Tris-HCl (pH 7. 4), 135 mM NaCl, 1% Triton X-100,1% glycerol and protease inhibitor cocktail tablets (Roche Molecular Biochemicals) for 15 min at 4°C. Cell lysates were centrifuged at 13,000 rpm for 5 min at 4°C after sonication. Protein concentrations of supernatants of cell lysates were determined by using Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad). The supernatants were incubated with sample buffer at 95°C for 5 min. The samples (50 μg of proteins) were resolved by SDS-PAGE (Novex, Life Technologies) and transferred onto nitrocellulose membranes (iBlot, Life Technologies). These membranes were blocked with PBS containing 5% skim milk, and then incubated with primary antibody at 4°C overnight. After washing, the membrane was reacted with HRP-conjugated secondary antibody at room temperature for 2 h. The immune complexes were visualized with Super Signal West Femto substrate (Pierce) and detected by an LAS-3000 image analyzer system (Fujifilm). Total RNA was subjected to electrophoresis in 1. 2% agarose-formaldehyde gel and morpholinepropanesulfonic acid buffer. rRNA was visualized by ethidium bromide staining, and electrophoresed RNA was transferred onto a positively charged nylon membrane (Cat No. 11 417 240 001; Roche). Full-length MCL1 cDNA was amplified and cloned into pSPT18 digested by EcoRI using an In-Fusion HD cloning kit (Clontech), an RNA probe was synthesized by using a digoxigenin (DIG) RNA labeling kit (Roche), and hybridization and detection were performed by using a DIG Northern Starter kit (Roche) according to the manufacturer’s protocols. The signals were detected by an LAS-3000 image analyzer system (Fujifilm). Huh7 cells (2x104 cells) were seeded on 12-well plates (Greiner) and incubated for 1 day. JEV, DENV or ZIKV were infected for 2 h and the culture medium was changed. At each time point, supernatants containing cells and adherent cells were collected and stained with 5 μg/mL of propidium iodide (PI, P4170; Sigma). Cell viability was determined by flow cytometry analyses (BD) and FlowJo software (FlowJo, LLC). Huh7 and BAX/BAKDKO (#29 and #47) cells were infected with JEV (MOI = 5) and incubated at 37°C for 2 days. Caspase 3/7 activity was determined by using Caspase 3/7Glo (Promega) according to the manufacturer’s protocol. Cells (2x105) seeded on 6-well plates (Greiner) were infected with lentiviruses expressing BIM, BIM-BADBH3, BIM-NOXABH3 and BIM-4E at one day after incubation, expanded into 10 cm dishes (Greiner) at 2 days post-infection, and cultured with medium containing 1 μg/ml of puromycin for 1 week with a change of culture medium every 2 days. The remaining cells were washed with PBS, incubated with 5 mL of Giemsa’s azur-eosin-methylene blue solution (Merck Milipore: 109204) for 1 h at room temperature, and washed in water. 293T cells (2x106) seeded on 10 cm dishes were transfected with 1. 0 μg of either pCAGGS MCL1 or pCAGGS MCL1K/R and 0. 05 μg of HA-ubiquitin plasmid by using PEI after 12 h of incubation. After 2 days, cells were treated with proteasome inhibitor (20 μM, ALLN) for 6 h and lysed with buffer A (50 mM Tris-HCl [pH 7. 4], 150 mM NaCl, 1 mM EDTA, 1% Triton X-100,1% sodium deoxycholate, 1% SDS) supplemented with 1 mM sodium fluoride and protease inhibitor cocktail tablets (Roche) on ice for 30 min and sonicated. Cell lysates (200 μl) were diluted with buffer A lacking SDS, incubated with anti-HA antibody (HA. 11,2 μl per sample; Covance) at 4 °C for 90 min and then further incubated with Protein G Sepharose 4B (GE Healthcare) at 4 °C for 90 min. The beads were washed five times in buffer A lacking SDS and eluted in sample buffer after incubation at 95°C for 5 min. Denatured samples were resolved by SDS-PAGE and immunoblotting. Huh7 and BAX/BAKDKO (#47) cells seeded on 10 cm dishes were infected with JEV (MOI = 5) and treated with ABT-737 (1μM) for 1 h at 37°C. Cells (1. 5 x106) were pelleted at 2 days post-infection, resuspended in 100 μl of permeabilization buffer consisting of 250 mM sucrose, 1 mM EDTA, 1 mM EGTA, 5 mM MgCl2,100 mM KCl, 20 mM Hepes, and 0. 025% of digitomin (#300410; Calbiochem) supplemented with protease inhibitor cocktail tablets, and incubated on ice for 5 min. Cell lysates were centrifuged by 13,000 rpm at 4°C for 5 min and the supernatants were collected as a soluble fraction. Pellets were resuspended with Onyx lysis buffer and then incubated on ice for 30 min, and the supernatants were collected as a pellet fraction. Both lysates were incubated with sample buffer at 95°C for 5 min, and denatured samples were resolved by SDS-PAGE and immunoblotting. Huh7 and BCLXKO Huh7 cells seeded onto 12-well or 24-well plates were inoculated with serially diluted virus and incubated for 2 h. The supernatants were removed and culture medium containing 1% methylcellulose was overlaid onto the cells. After 2 days of incubation, cells were fixed with 4% paraformaldehyde and visualized by staining with methylene blue (Sigma) or Giemsa’s azur-eosin-methylene blue solution (Merck Milipore: 109204) for 1 h at room temperature, and washed in water. Huh7 cells or JEV-infected cells were seeded on 12-well plates and washed twice with DMEM (deficient in methionine and cysteine; Gibco 21013024), then incubated with [35S] methionine and [35S] cysteine (EasyTag Express35S Protein labeling mix, Perkin Elmer) in DMEM without methionine and supplemented with 10% dialyzed FBS, 100 U/ml penicillin and 100 μg/ml streptomycin for 15 min at 1,2 and 3 days post-infection. Labeled cells were washed three times with cold PBS, then lysed with 200 μl/well radioimmunoprecipitation (RIPA) buffer, and the cell lysates were centrifuged at 13,000 rpm for 10 min at 4°C. Protein concentrations were determined by using Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad). Clear-sol (900 μl; Nakalai Tesque) was mixed with each supernatant (100 μl), and measured with a liquid scintillation counter (LS6500; Beckman Coulter). For detection of synthesis of MCL1 protein, Huh7 cells were seeded on 10 cm dishes, infected with JEV (MOI = 5) and incubated for 2days. These cells were washed twice with DMEM (deficient in methionine and cysteine; Gibco 21013024), then incubated with [35S] methionine and [35S] cysteine (EasyTag Express35S Protein labeling mix, Perkin Elmer) in DMEM without methionine and supplemented with 10% dialyzed FBS, 100 U/ml penicillin and 100 μg/ml streptomycin for 15 min at 37°C. Cell lysates were prepared by incubation in Onyx lysis buffer for 15 min at 4°C. Cell lysates were centrifuged at 13,000 rpm for 5 min at 4°C. Protein concentrations were determined by using Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad). Same amounts of cell lysates were incubated with 2μL of MCL1 antibody at 4°C for 90 min and then further incubated with Protein G Sepharose 4B (GE Healthcare) at 4°C for 90 min. The beads were washed three times with Onyx lysis buffer, boiled at 95°C for 5 min, and subjected to SDS-PAGE. The 35S-labeled proteins were visualized by using a Fujifilm bioimaging analyzer (FLA-7000, Fujifilm). RNA was extracted by using ISOGEN II (Nippon Gene), and cDNAs were synthesized from the total RNA using a High Capacity RNA-to-cDNA kit (Applied Biosystems) according to the manufacturer’s instructions. JEV RNA were quantified using a TaqMan RNA-to-Ct 1-Step Kit and the ViiA7 real-time PCR system (Life Technologies). The primers of JEV were 5′-GGGTCAAAGTCATTTCTGGTCCATA’ and 5′-TCCACGCTGCTCGAA’. The following probe were used: for JEV, 50-6-FAM/ATGACCTCG/ZEN/CTCTCCC/-30IABkFQ; . mRNAs of Il6, Tnfα, Ccl5, Cxcl10, Ccl2, Ifna, Ifnb and β-actin were used for quantification of mRNA expression by using a Power SYBR green RNA-to-Ct 1-Step kit and theViiA7 real-time PCR system (Life Technologies). The following primers were used: for Il6 5′-CCACTTCACAAGTCGGAGGCTTA-3′ and 5′-GCAAGTGCATCATCGTTGTTCATAC-3′ Tnfα 5′-CAGGAGGGAGAACAGAAACTCCA-3′ and 5′-CCTGGTTGGCTGCTTGCTT-3′, Ccl5 5′-AGATCTCTGCAGCTGCCCTCA-3′ and 5′-GGAGCACTTGCTGCTGGTGTAG-3′, Cxcl10 5′-ACACCAGCCTGGCTTCCATC-3′ and 5′-TTGGAGCTGGAGCTGCTTATAGTTG-3′, Ccl2 (5′-GCATCCACGTGTTGGCTCA-3′ and 5′-CTCCAGCCTACTCATTGGGATCA-3′, Ifna1 5′-AGCCTTGACACTCCTGGTACAAATG-3′ and 5′-TGGGTCAGCTCACTCAGGACA-3′ Ifnb1 5′-ACACCAGCCTGGCTTCCATC-3′ and 5′-TTGGAGCTGGAGCTGCTTATAGTTG-3′, β-actin 5′-TTGCTGACAGGATGCAGAAG-3′ and 5′-GTACTTGCGCTCAGGAGGAG- 3′. Human monocyte-derived DCs were generated from healthy human blood donors using Lymphocyte Separation Solution (nacalai tesque). CD14+ cells were isolated after Ficoll-Hypaque gradient centrifugation using MACS CD14+ magnetic beads with FcR Blocking Reagent (Miltenyi Biotech) as manufacturer’s instructions. These human monocytes (5x105 cells) were seeded on 24 well plates and differentiated into naive DCs by culturing for 4 days in differentiated medium, Iscove’s Modified Dulbecco’s Media containing 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin 10 μg/ml human IL-4 (PeproTech) and 50 μg/ml human granulocyte-macrophage colony-stimulating factor (GM-CSF) (PeproTech). MDDCs (4 x 105 cells) were infected with DENV (MOI = 3) for 60 min at 37°C. Cells were then pelleted at 2,000 rpm for 5 min and resuspended in 1000 μl of differentiated medium. Bclx+/- mice were generated as previously described [74] using a BDF1 genetic background. The pX330 containing an sgRNA against the mouse Bclx gene (S1 Table) was injected into mouse zygotes and transplanted into pseudopregnant female mice. The obtained mice (F1) were crossed with wild type mice and F2 mice were analyzed by sequencing. We obtained three Bclx+/- lines from different F1 mice and kept them by crossing with wild type mice. Peritoneal exudate cells (PECs) were isolated from C57BL/6 (Japan SLC, Inc.) mice intraperitoneally injected with 2 ml of 3% thioglycolate solution by rinsing the peritoneal cavity with 5 ml of PBS. Cells were pelleted by 1,500 rpm at 4 °C for 5 min and suspended in RPMI supplemented with 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin, and cultured on 10 cm culture dishes. After 2 h of incubation, adherent cells were collected and seeded in culture medium containing 10 ng/ml human IL-4 (PeproTech), then incubated overnight at 37 °C. Huh7 and BCLXKOHuh7 cells infected with JEV were incubated with pHrodo green (10 μM; Life Technologies) at 37 °C for 30 min and washed twice with culture medium. Labeled-cells were overlaid onto PECs and incubated at 37 °C for 45 min. PECs were washed with culture medium three times to remove non-adherent cells. The amounts of engulfed PECs were determined by FACS (Becton Dickinson). Labeled cells overlaid on PEC were incubated at 37 °C for 2 h and replaced with fresh medium. Infectious titers and cytokine production in the supernatants were determined after 24 h of incubation. The productions of mouse IL-6 (KMC0061, Novex), TNF-α (KMC3010, Novex), CCL5 (NMR00, R&D Systems), CCL2 (MJE00, R&D Systems) and CXCL10 (BMS6018, Thermo Fisher Scientific) were quantified by ELISA according to the manufacturer’s protocol. Three-week-old mice were anaesthetized by intraperitoneal administration of 0. 75 mg kg-1 medetomidine (Meiji Seika), 4 mg kg-1 midazolam (Sandoz) and 5 mg kg-1 butorphanol tartrate (Meiji Seika). JEV (2x106 FFU/50 μl) was injected subcutaneously into both rear footpads (4x106 FFU in total). JEV (1x105 FFU) was also intracerebrally injected. The survival of the mice was monitored for 25 days. To collect the brain tissues, mice were sacrificed at the end-point and then recorded as dead. Dosing of ABT-263 was performed as previously described [51,64]. Briefly, ABT-263 was formulated in 10% ethanol, 30% polyethylene glycol 400, and 60% Phosal 50 PG for dosing. The mice were dosed orally once a day for 2 to 8 days with vehicle or 50mg kg-1 ABT-263. All animal experiments conformed to the Guidelines for the Care and Use of Laboratory Animals, and were approved by the Institutional Committee of Laboratory Animal Experimentation (Research Institute for Microbial Diseases, Osaka University. Project number: H27-06-0). All healthy donors provided oral consent and were properly informed of the risks of the study. The statistical analyses were performed using GraphPad Prism (GraphPad Prism Software, Inc.). Significant differences were determined using Student’s t-test and are indicated with asterisks (*P<0. 05) and double asterisks (**P<0. 01) in each figure. Significant differences of in vivo survival data were determined using log-rank test and are indicated with asterisks (*P<0. 05) and double asterisks (**P<0. 01) in each figure.
summary: The genus Flavivirus including Japanese encephalitis virus, Dengue virus, and Zika virus all of which are mosquito-borne human pathogen and cause serious diseases in humans. Therefore, the development of effective vaccines and antivirals against several flaviviruses is still needed. BCL2 family proteins control mitochondria-mediated apoptosis to maintain cell homeostasis via the removal of damaged cells and pathogen-infected cells, deregulation of which leads to severe diseases including cancer and autoimmune diseases. Here, we showed that BCLXL is a critical cell survival factor during infection with flaviviruses, and that inhibition of BCLXL by treatment with BH3 mimetics restricts the production of infectious particles and the expression of chemokines in vitro and in vivo. Inhibition of BCLXL induces apoptosis in cells infected with flaviviruses and these cells are quickly removed by engulfment of phagocytes, which leads to inhibition of virus dissemination without any inflammatory reaction. Based on these data, BCLXL would appear to be a suitable target for the development of novel antivirals against a broad range of flavivirus infections.
keywords: cell death
  cell motility
  medicine and health sciences
  pathology and laboratory medicine
  molecular probe techniques
  pathogens
  cell processes
  microbiology
  immunoblotting
  cloning
  viruses
  protein expression
  rna viruses
  molecular biology techniques
  research and analysis methods
  medical microbiology
  microbial pathogens
  molecular biology
  phagocytosis
  chemotaxis
  molecular biology assays and analysis techniques
  gene expression and vector techniques
  cell biology
  flaviviruses
  apoptosis
  viral pathogens
  chemokines
  biology and life sciences
  organisms
title: Infection with flaviviruses requires BCLXL for cell survival