ZAPS is a potent stimulator of signaling mediated by the RNA helicase RIG-I during antiviral responses.

The poly(ADP-ribose) polymerases (PARPs) participate in many biological and pathological processes. Here we report that the PARP-13 shorter isoform (ZAPS), rather than the full-length protein (ZAP), was selectively induced by 5'-triphosphate-modified RNA (3pRNA) and functioned as a potent stimulator of interferon responses in human cells mediated by the RNA helicase RIG-I. ZAPS associated with RIG-I to promote the oligomerization and ATPase activity of RIG-I, which led to robust activation of IRF3 and NF-κB transcription factors. Disruption of the gene encoding ZAPS resulted in impaired induction of interferon-α (IFN-α), IFN-ß and other cytokines after viral infection. These results indicate that ZAPS is a key regulator of RIG-I signaling during the innate antiviral immune response, which suggests its possible use as a therapeutic target for viral control.

Programmed Cell Death in the Pathogenesis of Influenza.

Influenza is a respiratory disease induced by infection by the influenza virus, which is a member of Orthomyxoviridae family. This infectious disease has serious impacts on public health systems and results in considerable mortality and economic costs throughout the world. Based on several experimental studies, massive host immune reaction is associated with the disease severity of influenza. Programmed cell death is typically induced during virus infection as a consequence of host immune reaction to limit virus spread by eliminating niches for virus propagation without causing inflammation. However, in some viral infectious diseases, such as influenza, in the process of immune reaction, aberrant induction of programmed cell death disturbs the maintenance of organ function. Current reports show that there are different types of programmed cell death that vary in terms of molecular mechanisms and/or associations with inflammation. In addition, these novel types of programmed cell death are associated with pathogenesis rather than suppressing virus propagation in the disease course. Here, we review our current understanding of mechanisms of programmed cell death in the pathogenesis of influenza.

Relevance of signaling molecules for apoptosis induction on influenza A virus replication.

Apoptosis is an important mechanism to maintain homeostasis in mammals, and disruption of the apoptosis regulation mechanism triggers a range of diseases, such as cancer, autoimmune diseases, and developmental disorders. The severity of influenza A virus (IAV) infection is also closely related to dysfunction of apoptosis regulation. In the virus infected cells, the functions of various host cellular molecules involved in regulation of induction of apoptosis are modulated by IAV proteins to enable effective virus replication. The modulation of the intracellular signaling pathway inducing apoptosis by the IAV infection also affects extracellular mechanisms controlling apoptosis, and triggers abnormal host responses related to the disease severity of IAV infections. This review focuses on apoptosis related molecules involved in IAV replication and pathogenicity, the strategy of the virus propagation through the regulation of apoptosis is also discussed.

Osteopontin modulates the generation of memory CD8+ T cells during influenza virus infection.

The adaptive immune system generates memory cells, which induce a rapid and robust immune response following secondary Ag encounter. Memory CD8(+) T cells are a critical component of protective immunity against infections and cancers. Therefore, understanding the mechanism whereby memory CD8(+) T cells are generated and maintained is important for inducing effective memory CD8(+) T cell response. Recent studies have demonstrated that the inflammatory cytokine IL-12 favors the generation of terminal effector CD8(+) T cells rather than memory precursor effector CD8(+) T cells by regulating the expression of the transcription factor T-bet. In this study, we report that the inflammatory cytokine osteopontin (Opn) modulates memory CD8(+) T cell generation during influenza virus infection. Although Opn wild-type and Opn knockout (KO) mice had similar numbers of virus-specific effector CD8(+) T cells, virus-specific effector CD8(+) T cells generated in Opn KO mice showed low levels of T-bet expression and an increased memory precursor cell population compared with cells generated in Opn wild-type mice. This resulted in the persistently increased number of memory CD8(+) T cells in Opn KO mice. Studies with bone marrow-derived dendritic cells demonstrated that Opn deficiency in bone marrow-derived dendritic cells results in low levels of IL-12 production in response to the stimulation with influenza virus. Thus, we hypothesize that Opn modulates the generation of memory precursor effector CD8(+) T cells by regulating cytokine milieu during the acute phase of virus infection. This finding may provide new insight into the role of Opn in adaptive immune response.

Osteopontin is critical to determine symptom severity of influenza through the regulation of NK cell population.

Osteopontin (OPN) is involved in exacerbating various inflammatory diseases. A severe pulmonary inflammation is frequently found in lethal influenza A virus (IAV) infection. However, the function of OPN against the infection was poorly understood. Here, we demonstrate an importance of OPN on immune response and disease severity after IAV infection. We found that the expression level of OPN was increased in mice infected with IAV. The OPN knockout (KO) mice exhibited a severe pathological phenotype and the survival rate decreased after the lethal IAV infection, compared to the wild type mice, while the survival rate increased in OPN transgenic (Tg) mice. The population of natural killer (NK) cells significantly decreased in OPN KO mice at day 5 after the infection, whereas, it increased in OPN Tg mice. These results suggest that OPN plays an important role in host defense against IAV infection through the regulation of NK cell population.

Influenza A virus polymerase inhibits type I interferon induction by binding to interferon beta promoter stimulator 1.

Type I interferons (IFNs) are known to be critical factors in the activation of host antiviral responses and are also important in protection from influenza A virus infection. Especially, the RIG-I- and IPS-1-mediated intracellular type I IFN-inducing pathway is essential in the activation of antiviral responses in cells infected by influenza A virus. Previously, it has been reported that influenza A virus NS1 is involved in the inhibition of this pathway. We show in this report that the influenza A virus utilizes another critical inhibitory mechanism in this pathway. In fact, the viral polymerase complex exhibited an inhibitory activity on IFNß promoter activation mediated by RIG-I and IPS-1, and this activity was not competitive with the function of NS1. Co-immunoprecipitation analysis revealed that each polymerase subunit bound to IPS-1 in mammalian cells, and each subunit inhibited the activation of IFNß promoter by IPS-1 independently. In addition, by a combinational expression of each polymerase subunit, IPS-1-induced activation of IFNß promoter was more efficiently inhibited by the expression of PB2 or PB2-containing complex. Moreover, the expression of PB2 inhibited the transcription of the endogenous IFNß gene induced after influenza A virus infection. These findings demonstrate that the viral polymerase plays an important role for regulating host anti-viral response through the binding to IPS-1 and inhibition of IFNß production.

Requirement for Siva-1 for replication of influenza A virus through apoptosis induction.

Infection with influenza A virus causes acute respiratory tract infections in humans and may lead to lethal diseases including pneumonia. Identifying host factors that are involved in the severity of infectious diseases caused by influenza A virus is considered important for the prevention and treatment of these viral infections. This report demonstrated that Siva-1 is crucial for the induction of apoptosis caused by infection with influenza A virus and is involved in virus replication. Susceptibility to apoptosis induced by influenza A virus infection was increased in human lung-derived A549 cells, which stably express Siva-1. In addition, induction of apoptosis after influenza A virus infection was strongly inhibited by knockdown of Siva-1 expression. Furthermore, the replication of influenza A virus was significantly suppressed in A549 cells in which Siva-1 expression was inhibited and the effect of Siva-1 knockdown was eliminated by treatment with Z-VAD-FMK. These findings suggest that the caspase-dependent pathway for induction of apoptosis is involved in Siva-1-mediated influenza A virus replication.

Paradoxical effects of chondroitin sulfate-E on Japanese encephalitis viral infection.

Glycosaminoglycans (GAGs) have diverse functions in the body and are involved in viral infection. The purpose of this study was to evaluate the possible roles of the E-disaccharide units GlcAß1-3GalNAc(4,6-O-disulfate) of chondroitin sulfate (CS), a GAG involved in neuritogenesis and neuronal migration, in Japanese encephalitis virus (JEV) infection. Soluble CS-E (sCS-E) derived from squid cartilage inhibited JEV infection in African green monkey kidney-derived Vero cells and baby hamster kidney-derived BHK cells by interfering with viral attachment. In contrast, sCS-E enhanced viral infection in the mouse neuroblastoma cell line Neuro-2a, despite the fact that viral attachment to Neuro-2a cells was inhibited by sCS-E. This enhancement effect in Neuro-2a cells seemed to be related to increased viral RNA replication and was also observed in a rat infection model in which intracerebral coadministration of sCS-E with JEV in 17-day-old rats resulted in higher brain viral loads than in rats infected without sCS-E administration. These results show the paradoxical effects of sCS-E on JEV infection in different cell types and indicate that potential use of sCS-E as an antiviral agent against JEV infection should be approached with caution considering its effects in the neuron, the major target of JEV.

Alpha9 integrin and its ligands constitute critical joint microenvironments for development of autoimmune arthritis.

Osteopontin is critically involved in rheumatoid arthritis; however, the molecular cross-talk between osteopontin and joint cell components that leads to the inflammatory joint destruction is largely unknown. We found that not only osteopontin but also tenascin-C and their common receptor, alpha(9) integrin, are expressed at arthritic joints. The local production of osteopontin and tenascin-C is mainly due to synovial fibroblasts and, to a lesser extent, synovial macrophages. Synovial fibroblasts and macrophages express alpha(9) integrin, and autocrine and paracrine interactions of alpha(9) integrin on synovial fibroblasts and macrophages and its ligands contribute differently to the production of proinflammatory cytokines and chemokines. alpha(9) integrin is also involved in the recruitment and accumulation of inflammatory cells. Inhibition of alpha(9) integrin function with an anti-alpha(9) integrin Ab significantly reduces the production of arthrogenic cytokines and chemokines and ameliorates ongoing arthritis. Thus, we identified alpha(9) integrin as a critical intrinsic regulator that controls the development of autoimmune arthritis.

Identification of DELE, a novel DAP3-binding protein which is crucial for death receptor-mediated apoptosis induction.

Death associated protein 3 (DAP3) is known to be a highly conserved protein, and is responsible for regulating apoptosis induced by various stimuli. To understand the molecular mechanism of how DAP3 induces apoptosis, we performed yeast two-hybrid screening, and identified a novel DAP3-binding protein termed death ligand signal enhancer (DELE). In this report, we show that DELE actually binds to DAP3 in mammalian cells. We found that the cells stably expressing DELE are susceptible to apoptosis induction by the stimulation of TNF-α and TRAIL. In addition, knockdown of DELE expression rescued the HeLa cells from apoptosis induction by these stimuli. Moreover, activation of caspase-3, caspase-8 and caspase-9 induced by stimulation of TNF-α, anti-Fas or TRAIL was significantly inhibited by the knockdown of DELE expression. These results demonstrated the biological significance of DELE for apoptosis signal mediated by death receptors.

Characterization of the interaction of influenza virus NS1 with Akt.

Avian influenza viruses belong to the genus influenza A virus of the family Orthomyxoviridae. The influenza virus consists of eight segmented minus stranded RNA that encode 11 known proteins. Among the 11 viral proteins, NS1 (non-structural protein 1, encoded on segment 8) has been implicated in the regulation of several important intra-cellular functions. In this report, we investigated the functional interaction of NS1 with serine threonine kinase Akt, a core intra-cellular survival regulator. In co-immunoprecipitation assays and GST pull-down assays, NS1 directly interacted with Akt. The interaction was mediated primarily through the Akt-PH (Pleckstrin Homology) domain and the RNA-binding domain of NS1. NS1 preferentially interacted with phosphorylated Akt, but not with non-phosphorylated Akt. Functionally, the NS1-Akt interaction enhanced Akt activity both in the intra-cellular context and in in vitro Akt kinase assays. Confocal microscopic analysis revealed that phosphorylated Akt interacted with NS1 during the interphase of the cell cycle predominantly within the nucleus. Finally, mass spectrometric analysis demonstrated the position at Thr215 of NS1 protein is primary phosphorylation target site through Akt activation. The results together supported the functional importance of influenza virus NS1 with Akt, a core intra-cellular survival regulator.

Critical function of death-associated protein 3 in T cell receptor-mediated apoptosis induction.

Death-associated protein 3 (DAP3) is crucial for promoting apoptosis induced by various stimulations. This report demonstrates that DAP3 is also important for T cell receptor (TCR)-mediated apoptosis induction in immature thymocytes. Enforced expression of DAP3 accelerated the negative selection in developing thymocytes, using the reaggregate thymus organ culture system. In addition, expression of DAP3 accelerated TCR-mediated apoptosis induction in DO11.10 cells. We also demonstrated that DAP3 translocates into the nucleus during TCR-mediated apoptosis in a Nur77 dependent manner. It is concluded that DAP3 is critical for TCR-mediated induction of apoptosis at the downstream of Nur77.

Retraction Note: Silencing of Glutathione S-Transferase Pi Inhibits Cancer Cell Growth via Oxidative Stress Induced by Mitochondria Dysfunction.

Editor's Note: this Article has been retracted; the Retraction Note is available at https://www.nature.com/articles/s41598-020-77205-9.

IAN family critically regulates survival and development of T lymphocytes.

The IAN (immune-associated nucleotide-binding protein) family is a family of functionally uncharacterized GTP-binding proteins expressed in vertebrate immune cells and in plant cells during antibacterial responses. Here we show that all eight IAN family genes encoded in a single cluster of mouse genome are predominantly expressed in lymphocytes, and that the expression of IAN1, IAN4, and IAN5 is significantly elevated upon thymic selection of T lymphocytes. Gain-of-function experiments show that the premature overexpression of IAN1 kills immature thymocytes, whereas short hairpin RNA-mediated loss-of-function studies show that IAN4 supports positive selection. The knockdown of IAN5 perturbs the optimal generation of CD4/CD8 double-positive thymocytes and reduces the survival of mature T lymphocytes. We also show evidence suggesting that IAN4 and IAN5 are associated with anti-apoptotic proteins Bcl-2 and Bcl-xL, whereas IAN1 is associated with pro-apoptotic Bax. Thus, the IAN family is a novel family of T cell-receptor-responsive proteins that critically regulate thymic development and survival of T lymphocytes and that potentially exert regulatory functions through the association with Bcl-2 family proteins.

Lactobacillus helveticus SBT2171 Induces A20 Expression via Toll-Like Receptor 2 Signaling and Inhibits the Lipopolysaccharide-Induced Activation of Nuclear Factor-kappa B and Mitogen-Activated Protein Kinases in Peritoneal Macrophages.

Lactobacillus helveticus SBT2171 (LH2171) has been reported to ameliorate the development of autoimmune diseases, such as collagen-induced arthritis and experimental autoimmune encephalitis in mice and inhibit interleukin (IL)-6 production in antigen-presenting cells in vitro. Regulation of cytokine production by antigen-presenting cells might be critical for the anti-inflammatory function of LH2171 in autoimmune diseases. However, the mechanism and contributing components of LH2171-mediated inhibition of IL-6 production are unclear. Here, we examined the anti-inflammatory effects of LH2171 in lipopolysaccharide (LPS)-stimulated peritoneal macrophages, as a model of antigen-presenting cells, necessary for the pathogenesis of autoimmune diseases. LH2171 significantly reduced LPS-induced expression and secretion of IL-6 and IL-1ß cytokines. It also inhibited activation of nuclear factor-kappa B and mitogen-activated protein kinases (NF-κB/MAPKs). Moreover, LH2171 induced gene expression of several negative regulators of NF-κB/MAPKs. Among these regulators, A20 was strongly up-regulated at the mRNA and protein levels upon LH2171 treatment. The cell wall fraction of LH2171 also demonstrated a similar increase in A20 gene expression and exerted an anti-inflammatory effect. These results suggest that the cell wall may be one of the anti-inflammatory components of LH2171. Since cell wall components of Gram-positive bacteria are recognized by toll-like receptor 2 (TLR2), we investigated whether the anti-inflammatory effect of LH2171 was mediated by TLR2 signaling. Specifically, LH2171-mediated IL-6 suppression and A20 upregulation in wild-type macrophages were reversed and significantly reduced in TLR2 knock-out macrophages. These results suggest that LH2171 induces A20 expression via TLR2 signaling, inhibiting the activation of NF-κB/MAPKs and cytokine production in antigen-presenting cells. This might contribute to the anti-inflammatory activity of LH2171 on autoimmune diseases.

Prevention of respiratory syncytial virus infection with probiotic lactic acid bacterium Lactobacillus gasseri SBT2055.

Lactobacillus gasseri SBT2055 (LG2055) is a probiotic lactic acid bacterium with multifunctional effects, including the prevention of influenza A virus infection in mice, reduction of adipocyte size in mice, and increased lifespan in C. elegans. We investigated whether LG2055 exhibits antiviral activity against respiratory syncytial virus (RSV), a global pathogen for which a preventive strategy is required. Following oral administration of LG2055 in mice, the RSV titre in the lung was significantly decreased, while body weight was not decreased after virus infection. Additionally, the elevated expression of pro-inflammatory cytokines in the lung upon RSV infection decreased after LG2055 administration. Moreover, interferon and interferon stimulated genes were upregulated by LG2055 treatment. Comparative cellular proteomic analysis revealed that SWI2/SNF2-related CREB-binding protein activator protein (SRCAP) was a candidate for the antiviral activity of LG2055 against RSV. There was a positive correlation between the inhibition of RSV replication and the suppression of SRCAP expression and RSV replication was suppressed by SRCAP silencing. Since SRCAP is a scaffold protein to which viral non-structural proteins bind, the downregulation of SRCAP induced by LG2055 could provide new insights about the inhibition of RSV replication. In summary, our study demonstrated that LG2055 has prophylactic potential against RSV infection.

Silencing of Glutathione S-Transferase Pi Inhibits Cancer Cell Growth via Oxidative Stress Induced by Mitochondria Dysfunction.

Antitumor drug development based on the concept of intervening in the antioxidant system of cancer cells has been gaining increased interest. In this study, we propose a promising strategy for cancer treatment using modulation of oxidative stress by suppression of glutathione S-transferases (GSTs), a typical antioxidant enzyme. siRNA which can be applied to the development of nucleic acid drugs, enabling them to eliminate unwanted side effects, increase specificity, and avoid the problem of drug resistance, was employed for GSTP-silencing at the transcriptional level. The silencing of the pi class of GST (GSTP) that displayed the most characteristic expression profile in 13 kinds of cancer cell lines has shown significant impairment in the growth of cancer cells due to oxidative stress caused by excess ROS accumulation. Comparative proteomics between normal cells and GSTP-silenced pancreatic cancer cell PANC-1 suggested that GSTP-silencing facilitated the mitochondrial dysfunction. These findings show promise for the development of strategies toward cancer therapy based on the mechanism that allows genetic silencing of GSTP to promote oxidative stress through mitochondria dysfunction.

Mammalian dap3 is an essential gene required for mitochondrial homeostasis in vivo and contributing to the extrinsic pathway for apoptosis.

Death-associated protein-3 (DAP3) is a GTP binding protein previously implicated in both intramitochondrial protein synthesis and apoptosis. To explore the in vivo roles of DAP3, we generated and characterized DAP3-deficient mice. Homozygous dap3-/- embryos died at approximately day 9.5 in utero. The dap3-/- embryos and placentas were markedly shrunken. Embryos had arrested development, displaying severe growth restriction and lack of axial turning. Transmission electron microscopy analysis revealed abnormal, shrunken mitochondria with swollen crystae in dap3-/- embryos. Levels of cytochrome c oxidase-I, a protein encoded in the mitochondrial genome, were reduced in dap3-/- embryos, consistent with a role for DAP3 in intramitochondrial protein synthesis. A requirement for DAP3 in mitochondrial respiration was also revealed by oxygen consumption measurements using cultured cells treated with DAP3-specific small interfering RNA (siRNA). Studies of cultured cells from dap3-/- embryos confirmed a role in apoptosis induced by stimuli that trigger the extrinsic (TNFalpha, TRAIL, anti-Fas antibody) but not intrinsic (mitochondrial) cell death pathway. Thus, DAP3 joins a growing list of bifunctional proteins that play roles in normal mitochondrial physiology and in apoptosis.

A Sialylated Voltage-Dependent Ca2+ Channel Binds Hemagglutinin and Mediates Influenza A Virus Entry into Mammalian Cells.

Influenza A virus (IAV) infection is initiated by the attachment of the viral glycoprotein hemagglutinin (HA) to sialic acid on the host cell surface. However, the sialic acid-containing receptor crucial for IAV infection has remained unidentified. Here, we show that HA binds to the voltage-dependent Ca2+ channel Cav1.2 to trigger intracellular Ca2+ oscillations and subsequent IAV entry and replication. IAV entry was inhibited by Ca2+ channel blockers (CCBs) or by knockdown of Cav1.2. The CCB diltiazem also inhibited virus replication in vivo. Reintroduction of wild-type but not the glycosylation-deficient mutants of Cav1.2 restored Ca2+ oscillations and virus infection in Cav1.2-depleted cells, demonstrating the significance of Cav1.2 sialylation. Taken together, we identify Cav1.2 as a sialylated host cell surface receptor that binds HA and is critical for IAV entry.

LKB1 is crucial for TRAIL-mediated apoptosis induction in osteosarcoma.

BACKGROUND: Despite improvements in chemotherapy and surgery in the treatment of osteosarcoma, satisfactory results are still difficult to achieve. New therapeutic modalities need to be developed for the improvement of these treatments. TRAIL (TNF-related apoptosis inducing ligand) is known as a selective apoptosis inducer in most tumor cells, but not in normal cells. Therefore, TRAIL is a good candidate target for the treatment of tumors. However, sensitivity of osteosarcoma cells to TRAIL-induced apoptosis is lower than that of other types of tumor cells. Recently, DAP3 (death associated protein 3) was demonstrated to play a critical role in TRAIL-mediated apoptosis through activation of pro-caspase-8. Here, we found that LKB1, a serine/threonine kinase, expressed in bone and soft tissue sarcoma cells, associated with DAP3. We also demonstrated that expression of DAP3 induced apoptosis in osteosarcoma cells. Furthermore, expression of LKB1 induced apoptosis and co-expression of LKB1 with DAP3 strongly induced apoptosis in osteosarcoma cells. In addition, expression of LKB1 kinase dead mutant, LKB1 (K78M), inhibited DAP3-induced apoptosis in these cells. These results suggest that LKB1 is critical for TRAIL-induced apoptosis induction, cooperating with DAP3 in osteosarcoma cells. It is predicted that LKB1 and DAP3 could be critical target molecules for the treatment of osteosarcomas.