Increased expression of CD38 on endothelial cells in SARS-CoV-2 infection in cynomolgus macaques.

SARS-CoV-2 infection causes activation of endothelial cells (ECs), leading to dysmorphology and dysfunction. To study the pathogenesis of endotheliopathy, the activation of ECs in lungs of cynomolgus macaques after SARS-CoV-2 infection and changes in nicotinamide adenine dinucleotide (NAD) metabolism in ECs were investigated, with a focus on the CD38 molecule, which degrades NAD in inflammatory responses after SARS-CoV-2 infection. Activation of ECs was seen from day 3 after SARS-CoV-2 infection in macaques, with increases of intravascular fibrin and NAD metabolism-associated enzymes including CD38. In vitro, upregulation of CD38 mRNA in human ECs was detected after interleukin 6 (IL-6) trans-signaling induction, which was increased in the infection. In the presence of IL-6 trans-signaling stimulation, however, CD38 mRNA silencing induced significant IL-6 mRNA upregulation in ECs and promoted EC apoptosis after stimulation. These results suggest that upregulation of CD38 in patients with COVID-19 has a protective role against IL-6 trans-signaling stimulation induced by SARS-CoV-2 infection.

SARS-CoV-2 induces inflammation and intracranial infection through the olfactory epithelium-olfactory bulb pathway in non-human primates.

We examined the histopathological changes in the olfactory mucosa of cynomolgus and rhesus macaque models of SARS-CoV-2 infection. SARS-CoV-2 infection induced severe inflammatory changes in the olfactory mucosa. A major histocompatibility complex (MHC) class II molecule, HLA-DR was expressed in macrophage and supporting cells, and melanocytes were increased in olfactory mucosa. Supporting cells and olfactory neurons were infected, and SARS-CoV-2 N protein was detected in the axons of olfactory neurons and in olfactory bulbs. Viral RNA was detected in olfactory bulbs and brain tissues. The olfactory epithelium-olfactory bulb pathway may be important as a route for intracranial infection by SARS-CoV-2.

Complement factor D targeting protects endotheliopathy in organoid and monkey models of COVID-19.

COVID-19 is linked to endotheliopathy and coagulopathy, which can result in multi-organ failure. The mechanisms causing endothelial damage due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remain elusive. Here, we developed an infection-competent human vascular organoid from pluripotent stem cells for modeling endotheliopathy. Longitudinal serum proteome analysis identified aberrant complement signature in critically ill patients driven by the amplification cycle regulated by complement factor B and D (CFD). This deviant complement pattern initiates endothelial damage, neutrophil activation, and thrombosis specific to organoid-derived human blood vessels, as verified through intravital imaging. We examined a new long-acting, pH-sensitive (acid-switched) antibody targeting CFD. In both human and macaque COVID-19 models, this long-acting anti-CFD monoclonal antibody mitigated abnormal complement activation, protected endothelial cells, and curtailed the innate immune response post-viral exposure. Collectively, our findings suggest that the complement alternative pathway exacerbates endothelial injury and inflammation. This underscores the potential of CFD-targeted therapeutics against severe viral-induced inflammathrombotic outcomes.

Suppression of viral RNA polymerase activity is necessary for persistent infection during the transformation of measles virus into SSPE virus.

Subacute sclerosing panencephalitis (SSPE) is a fatal neurodegenerative disease caused by measles virus (MV), which typically develops 7 to 10 years after acute measles. During the incubation period, MV establishes a persistent infection in the brain and accumulates mutations that generate neuropathogenic SSPE virus. The neuropathogenicity is closely associated with enhanced propagation mediated by cell-to-cell fusion in the brain, which is principally regulated by hyperfusogenic mutations of the viral F protein. The molecular mechanisms underlying establishment and maintenance of persistent infection are unclear because it is impractical to isolate viruses before the appearance of clinical signs. In this study, we found that the L and P proteins, components of viral RNA-dependent RNA polymerase (RdRp), of an SSPE virus Kobe-1 strain did not promote but rather attenuated viral neuropathogenicity. Viral RdRp activity corresponded to F protein expression; the suppression of RdRp activity in the Kobe-1 strain because of mutations in the L and P proteins led to restriction of the F protein level, thereby reducing cell-to-cell fusion mediated propagation in neuronal cells and decreasing neuropathogenicity. Therefore, the L and P proteins of Kobe-1 did not contribute to progression of SSPE. Three mutations in the L protein strongly suppressed RdRp activity. Recombinant MV harboring the three mutations limited viral spread in neuronal cells while preventing the release of infectious progeny particles; these changes could support persistent infection by enabling host immune escape and preventing host cell lysis. Therefore, the suppression of RdRp activity is necessary for the persistent infection of the parental MV on the way to transform into Kobe-1 SSPE virus. Because mutations in the genome of an SSPE virus reflect the process of SSPE development, mutation analysis will provide insight into the mechanisms underlying persistent infection.

Alterations in Cytoskeleton and Mitochondria in the Development and Reversal of Steatosis in Human Hepatocytes.

BACKGROUND & AIMS: Alterations in mitochondrial morphology and function and increased oxidative stresses in hepatocytes are well established in nonalcoholic fatty liver disease (NAFLD). Patients can undergo lifestyle changes, especially in earlier NAFLD stages, to reverse disease-induced phenotypes on a gross level. Yet, little is known about whether mitochondrial function and injuries recover upon reversal. Thus, we elucidated this question and interplays between the cytoskeletal network and mitochondria in the development and reversal of steatosis. METHODS: We cultured primary human hepatocytes stably for 2 weeks and used free fatty acid supplementation to induce steatosis over 7 days and reversed steatosis by free fatty acid withdrawal over the next 7 days. We assessed cytoskeletal and mitochondrial morphologies using immunocytochemistry and confocal microscopy. We evaluated mitochondrial respiration and function via the Seahorse analyzer, in which we fully optimized reagent dosing specifically for human hepatocytes. RESULTS: During early steatosis, intracellular lipid droplets displaced microtubules altering mitochondrial distribution, and disrupted the F-actin network, leading to loss of bile canaliculi in steatotic hepatocytes. Basal mitochondrial respiration, maximum respiratory capacity, and resistance to H2O2-induced cell death also increased as an adaptative response. Upon reversal of steatosis, F-actin and bile canaliculi were restored in hepatocytes. Nevertheless, we observed an increase in elongated mitochondrial branches accompanied by decreases in α-tubulin expression, mitochondrial proton leak, and susceptibility to H2O2-induced cell death. CONCLUSIONS: Despite the restoration of cytoskeletons morphologically upon reversal of steatosis, the mitochondria in hepatocytes were impaired owing to early adaptative respiratory increase. Hepatocytes thus were highly predisposed to H2O2-induced cell death. These results indicate the persistence of potential health risks for recovering NAFLD patients.

Sesamin Metabolites Suppress the Induction of Cellular Senescence.

Cellular senescence induces inflammation and is now considered one of the causes of organismal aging. Accumulating evidence indicates that age-related deterioration of mitochondrial function leads to an increase in reactive oxygen species (ROS) and DNA damage, which in turn causes cellular senescence. Thus, it is important to maintain mitochondrial function and suppress oxidative stress in order to inhibit the accumulation of senescent cells. Sesamin and its isomer episesamin are types of lignans found in sesame oil, and after being metabolized in the liver, their metabolites have been reported to exhibit antioxidant properties. However, their effects on cellular senescence remain unknown. In this study, the effects of sesamin, episesamin, and their metabolites SC1 and EC1-2 on replicative senescence were evaluated using human diploid lung fibroblasts, and TIG-3 cells. The results showed that sesamin and episesamin treatment had no effect on proliferative capacity compared to the untreated late passage group, whereas SC1 and EC1-2 treatment improved proliferative capacity and mitigated DNA damage of TIG-3 cells. Furthermore, other cellular senescence markers, such as senescence-associated secretory phenotype (SASP), mitochondria-derived ROS, and mitochondrial function (ROS/ATP ratio) were also reduced by SC1 and EC1-2 treatment. These results suggest that SC1 and EC1-2 can maintain proper mitochondrial function and suppress the induction of cellular senescence.

An attenuated vaccinia vaccine encoding the severe acute respiratory syndrome coronavirus-2 spike protein elicits broad and durable immune responses, and protects cynomolgus macaques and human angiotensin-converting enzyme 2 transgenic mice from severe acute respiratory syndrome coronavirus-2 and its variants.

As long as the coronavirus disease-2019 (COVID-19) pandemic continues, new variants of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) with altered antigenicity will emerge. The development of vaccines that elicit robust, broad, and durable protection against SARS-CoV-2 variants is urgently required. We have developed a vaccine consisting of the attenuated vaccinia virus Dairen-I (DIs) strain platform carrying the SARS-CoV-2 S gene (rDIs-S). rDIs-S induced neutralizing antibody and T-lymphocyte responses in cynomolgus macaques and human angiotensin-converting enzyme 2 (hACE2) transgenic mice, and the mouse model showed broad protection against SARS-CoV-2 isolates ranging from the early-pandemic strain (WK-521) to the recent Omicron BA.1 variant (TY38-873). Using a tandem mass tag (TMT)-based quantitative proteomic analysis of lung homogenates from hACE2 transgenic mice, we found that, among mice subjected to challenge infection with WK-521, vaccination with rDIs-S prevented protein expression related to the severe pathogenic effects of SARS-CoV-2 infection (tissue destruction, inflammation, coagulation, fibrosis, and angiogenesis) and restored protein expression related to immune responses (antigen presentation and cellular response to stress). Furthermore, long-term studies in mice showed that vaccination with rDIs-S maintains S protein-specific antibody titers for at least 6 months after a first vaccination. Thus, rDIs-S appears to provide broad and durable protective immunity against SARS-CoV-2, including current variants such as Omicron BA.1 and possibly future variants.

Potent SARS-CoV-2 neutralizing antibodies with therapeutic effects in two animal models.

The use of therapeutic neutralizing antibodies against SARS-CoV-2 infection has been highly effective. However, there remain few practical antibodies against viruses that are acquiring mutations. In this study, we created 494 monoclonal antibodies from patients with COVID-19-convalescent, and identified antibodies that exhibited the comparable neutralizing ability to clinically used antibodies in the neutralization assay using pseudovirus and authentic virus including variants of concerns. These antibodies have different profiles against various mutations, which were confirmed by cell-based assay and cryo-electron microscopy. To prevent antibody-dependent enhancement, N297A modification was introduced. Our antibodies showed a reduction of lung viral RNAs by therapeutic administration in a hamster model. In addition, an antibody cocktail consisting of three antibodies was also administered therapeutically to a macaque model, which resulted in reduced viral titers of swabs and lungs and reduced lung tissue damage scores. These results showed that our antibodies have sufficient antiviral activity as therapeutic candidates.

Human metapneumovirus M2-2 protein inhibits RIG-I signaling by preventing TRIM25-mediated RIG-I ubiquitination.

Retinoic acid-inducible gene I (RIG-I) is a receptor that senses viral RNA and interacts with mitochondrial antiviral signaling (MAVS) protein, leading to the production of type I interferons and inflammatory cytokines to establish an antiviral state. This signaling axis is initiated by the K63-linked RIG-I ubiquitination, mediated by E3 ubiquitin ligases such as TRIM25. However, many viruses, including several members of the family Paramyxoviridae and human respiratory syncytial virus (HRSV), a member of the family Pneumoviridae, escape the immune system by targeting RIG-I/TRIM25 signaling. In this study, we screened human metapneumovirus (HMPV) open reading frames (ORFs) for their ability to block RIG-I signaling reconstituted in HEK293T cells by transfection with TRIM25 and RIG-I CARD (an N-terminal CARD domain that is constitutively active in RIG-I signaling). HMPV M2-2 was the most potent inhibitor of RIG-I/TRIM25-mediated interferon (IFN)-ß activation. M2-2 silencing induced the activation of transcription factors (IRF and NF-kB) downstream of RIG-I signaling in A549 cells. Moreover, M2-2 inhibited RIG-I ubiquitination and CARD-dependent interactions with MAVS. Immunoprecipitation revealed that M2-2 forms a stable complex with RIG-I CARD/TRIM25 via direct interaction with the SPRY domain of TRIM25. Similarly, HRSV NS1 also formed a stable complex with RIG-I CARD/TRIM25 and inhibited RIG-I ubiquitination. Notably, the inhibitory actions of HMPV M2-2 and HRSV NS1 are similar to those of V proteins of several members of the Paramyxoviridae family. In this study, we have identified a novel mechanism of immune escape by HMPV, similar to that of Pneumoviridae and Paramyxoviridae family members.

Middle East respiratory syndrome coronavirus ORF4b protein inhibits TLR7- and TLR9-dependent alpha interferon induction.

The Toll-like receptor (TLR)7- and TLR9-dependent signalling cascade is responsible for production of a large amount of alpha interferon by plasmacytoid dendritic cells upon viral infection. Here, we show that Middle East respiratory syndrome coronavirus (MERS-CoV) accessory protein ORF4b has the most potential among the MERS-CoV accessory proteins to inhibit the TLR7/9-signaling-dependent alpha interferon production. ORF4b protein, which has a bipartite nuclear localization signal, was found to bind to IKKα, a kinase responsible for phosphorylation of interferon regulatory factor (IRF)7. This interaction caused relocation of a large proportion of IKKα from the cytoplasm to the nucleus. Studies using ORF4b and IKKα mutants demonstrated that ORF4b protein inhibited IKKα-mediated IRF7 phosphorylation by sequestering IKKα in the nucleus and by impeding the phosphorylation process of cytoplasmic IKKα.

Upregulation of viral RNA polymerase activity promotes adaptation of SSPE virus to neuronal cells.

Subacute sclerosing panencephalitis (SSPE) is a rare progressive neurodegenerative disease caused by measles virus variants (SSPE viruses) that results in eventual death. Amino acid substitution(s) in the viral fusion (F) protein are key for viral propagation in the brain in a cell-to-cell manner, a specific trait of SSPE viruses, leading to neuropathogenicity. In this study, we passaged an SSPE virus in cultured human neuronal cells and isolated an adapted virus that propagated more efficiently in neuronal cells and exhibited increased cell-to-cell fusion. Contrary to our expectation, the virus harbored mutations in the large protein, a viral RNA-dependent RNA polymerase, and in the phosphoprotein, its co-factor, rather than in the F protein. Our results imply that upregulated RNA polymerase activity, which increases F protein expression and cell-to-cell fusion, could be a viral factor that provides a growth advantage and contributes to the adaptation of SSPE viruses to neuronal cells.

Molecular mechanisms underlying the promotion of wound repair by coenzyme Q10: PI3K/Akt signal activation via alterations to cell membrane domains.

Coenzyme Q10 (CoQ10) promotes wound healing in vitro and in vivo. However, the molecular mechanisms underlying the promoting effects of CoQ10 on wound repair remain unknown. In the present study, we investigated the molecular mechanisms through which CoQ10 induces wound repair using a cellular wound-healing model. CoQ10 promoted wound closure in a dose-dependent manner and wound-mediated cell polarization after wounding in HaCaT cells. A comparison with other CoQ homologs, benzoquinone derivatives, and polyisoprenyl compounds suggested that the whole structure of CoQ10 is required for potent wound repair. The phosphorylation of Akt after wounding and the plasma membrane translocation of Akt were elevated in CoQ10-treated cells. The promoting effect of CoQ10 on wound repair was abrogated by co-treatment with a phosphatidylinositol 3-kinase (PI3K) inhibitor. Immuno-histochemical and biochemical analyses showed that CoQ10 increased the localization of caveolin-1 (Cav-1) to the apical membrane domains of the cells and the Cav-1 content in the membrane-rich fractions. Depletion of Cav-1 suppressed CoQ10-mediated wound repair and PI3K/Akt signaling activation in HaCaT cells. These results indicated that CoQ10 increases the translocation of Cav-1 to the plasma membranes, activating the downstream PI3K/Akt signaling pathway, and resulting in wound closure in HaCaT cells.

SeV C Protein Plays a Role in Restricting Macrophage Phagocytosis by Limiting the Generation of Intracellular Double-Stranded RNA.

Macrophages play a central role in the innate immune response to respiratory viral infections through pro-inflammatory factor secretion and phagocytosis. However, as a countermeasure, viral pathogens have evolved virulence factors to antagonize macrophage function. In our recent in vitro analyses of murine macrophage cell lines, Sendai virus (SeV) accessory protein C inhibited the secretion of pro-inflammatory factors, and C gene-knockout SeV (SeVΔC) caused drastic morphological changes in RAW264.7 macrophages, similar to those observed after stimulation with Lipid A, a well-known activator of actin-rich membrane ruffle formation and phagocytosis. Hence, we sought to determine whether the C protein limits phagocytosis in SeV-infected macrophages through the suppression of membrane ruffling. Phagocytosis assays indicated an upregulation of phagocytosis in both SeVΔC-infected and Lipid A-stimulated macrophages, but not in SeV WT-infected cells. Further, the observed membrane ruffling was associated with phagocytosis. RIG-I is essential for Lipid A-induced phagocytosis; its deficiency inhibited SeVΔC-stimulated phagocytosis and ruffling, confirming the essential role of RIG-I. Moreover, treatment with interferon (IFN)-ß stimulation and neutralizing antibodies against IFN-ß suggested that SeVΔC-induced phagocytosis and ruffling occurred in an IFN-ß-independent manner. A newly isolated SeVΔC strain that does not generate dsRNA further highlighted the importance of dsRNA in the induction of phagocytosis and ruffling. Taken together, the current results suggest that SeV C protein might limit phagocytosis-associated membrane ruffling in an RIG-I-mediated but IFN-independent manner via limiting the generation of intracellular dsRNA.

M protein of subacute sclerosing panencephalitis virus, synergistically with the F protein, plays a crucial role in viral neuropathogenicity.

Subacute sclerosing panencephalitis (SSPE) is a rare fatal neurodegenerative disease caused by a measles virus (MV) variant, SSPE virus, that accumulates mutations during long-term persistent infection of the central nervous system (CNS). Clusters of mutations identified around the matrix (M) protein in many SSPE viruses suppress productive infectious particle release and accelerate cell-cell fusion, which are features of SSPE viruses. It was reported, however, that these defects of M protein function might not be correlated directly with promotion of neurovirulence, although they might enable establishment of persistent infection. Neuropathogenicity is closely related to the character of the viral fusion (F) protein, and amino acid substitution(s) in the F protein of some SSPE viruses confers F protein hyperfusogenicity, facilitating viral propagation in the CNS through cell-cell fusion and leading to neurovirulence. The F protein of an SSPE virus Kobe-1 strain, however, displayed only moderately enhanced fusion activity and required additional mutations in the M protein for neuropathogenicity in mice. We demonstrated here the mechanism for the M protein of the Kobe-1 strain supporting the fusion activity of the F protein and cooperatively inducing neurovirulence, even though each protein, independently, has no effect on virulence. The occurrence of SSPE has been estimated recently as one in several thousand in children who acquired measles under the age of 5 years, markedly higher than reported previously. The probability of a specific mutation (or mutations) occurring in the F protein conferring hyperfusogenicity and neuropathogenicity might not be sufficient to explain the high frequency of SSPE. The induction of neurovirulence by M protein synergistically with moderately fusogenic F protein could account for the high frequency of SSPE.

Neutralizing antibody-dependent and -independent immune responses against SARS-CoV-2 in cynomolgus macaques.

We examined the pathogenicity of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in cynomolgus macaques for 28 days to establish an animal model of COVID-19 for the development of vaccines and antiviral drugs. Cynomolgus macaques infected with SARS-CoV-2 showed body temperature rises and X-ray radiographic pneumonia without life-threatening clinical signs of disease. A neutralizing antibody against SARS-CoV-2 and T-lymphocytes producing interferon (IFN)-γ specifically for SARS-CoV-2 N-protein were detected on day 14 in one of three macaques with viral pneumonia. In the other two macaques, in which a neutralizing antibody was not detected, T-lymphocytes producing IFN-γ specifically for SARS-CoV-2 N protein increased on day 7 to day 14, suggesting that not only a neutralizing antibody but also cellular immunity has a role in the elimination of SARS-CoV-2. Thus, because of similar symptoms to approximately 80% of patients, cynomolgus macaques are appropriate to extrapolate the efficacy of vaccines and antiviral drugs for humans.

Annexin A1 accounts for an anti-inflammatory binding target of sesamin metabolites.

Sesamin [(7α,7'α,8α,8'α)-3,4:3',4'-bis(methylenedioxy)-7,9':7',9-diepoxylignane] is a major lignan in sesame seeds. Sesamin is converted to the catechol metabolite, SC1 [(7α,7'α,8α,8'α)-3',4'-methylenedioxy-7,9':7',9-diepoxylignane-3,4-diol] with anti-inflammatory effects after oral administration. However, its molecular target remains unknown. Analysis using high-performance affinity nanobeads led to the identification of annexin A1 (ANX A1) as an SC1-binding protein. SC1 was found to bind to the annexin repeat 3 region of ANX A1 with a high-affinity constant (Kd = 2.77 µmol L-1). In U937 cells, SC1 exhibited an anti-inflammatory effect dependent on ANX A1. Furthermore, administration of sesamin or SC1 attenuated carbon tetrachloride-induced liver damage in mice and concurrently suppressed inflammatory responses dependent on ANX A1. The mechanism involved SC1-induced ANX A1 phosphorylation at serine 27 that facilitates extracellular ANX A1 release. Consequently, the ANX A1 released into the extracellular space suppressed the production of tumor necrosis factor α. This study demonstrates that ANX A1 acts as a pivotal target of sesamin metabolites to attenuate inflammatory responses.

Multiple-endpoint genotoxicity assay for colon carcinogen 1,2-dimethylhydrazine.

Human risk assessment of the toxic potency of chemicals typically includes genotoxicity assays for predicting carcinogenicity. Gene mutation frequency and chromosomal aberration are two major genotoxicity endpoints in standardized in vitro and in vivo assays. The weight-of-evidence approach in risk assessment is more focused on in vivo assay results; however, animal welfare considerations are aimed at the reduction, replacement, and refinement (3R's) of animal experiments, including a reduction in the number of experimental animals. Proposals to reduce experimental animals in genotoxicity testing include the incorporation of genotoxicity endpoint(s) into other toxicological studies and the combination of two or more assays detecting different genotoxicity endpoints in the same animals. In this study, we used 1,2-dimethylhydrazine as a model chemical of colon carcinogen to assess gene mutation frequency and chromosomal aberration in vivo simultaneously. Specifically, a gene mutation frequency assay was combined with a multiple-organ micronucleus test (peripheral blood, bone marrow, liver, and colon) in F344 gpt delta transgenic rats. Both gpt mutant frequency and micronucleated cell frequency significantly increased in colon and liver but not in bone marrow. Interestingly, we found that the colon carcinogen induced both gene mutations and micronuclei in the targeted colon tissue. Thus, we demonstrated that the mechanism of a carcinogen could be derived from an animal experiment using a lower number of experimental animals as currently recommended. Moreover, a significant increase in mutant frequency in colon and liver was already observed on the first day after treatment completion, as well as on the third day, which is the guideline-recommended period. Thus, this endpoint is compatible with other genotoxicity assays. We confirmed that performing the micronucleus assay in combination with a gene mutation assay in F344 gpt delta transgenic rats is useful to evaluate different genotoxic endpoints simultaneously in the same animals, which reduces the number of experimental animals.

Target-specific micronucleus induction by colon carcinogens: 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 1,2-dimethylhydrazine.

Genotoxicity occurring at the target organs of carcinogenesis is important for understanding the mechanisms of chemical carcinogenicity and also for setting of threshold estimation. In vivo gene mutations have been evaluated by transgenic animal models in which any organ can be targeted; however, the methodologies that have been applied to assess chromosomal aberrations including micronucleus induction, are organ restricted, (often to bone marrow hematopoietic cells, as a common example). For food and food-related chemicals, the digestive tract is the important target organ as it is the organ of first contact. In the present study, we used 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 1,2-dimethylhydrazine (DMH) as model chemicals of carcinogens primarily targeting the colon. We evaluated the applicability of colon cells and hepatocytes, together with bone marrow cells, in the micronucleus assay. Both model chemicals induced micronuclei in the colon, which is the target organ of these carcinogens, after short- and long-term treatment(s). The results demonstrate the target specificity of micronucleus induction and the assay using organs other than bone marrow will play an important role in understanding the mechanism of carcinogenicity and predicting new carcinogenic agents.

Respirovirus C protein inhibits activation of type I interferon receptor-associated kinases to block JAK-STAT signaling.

Respirovirus C protein blocks the type I interferon (IFN)-stimulated activation of the JAK-STAT pathway. It has been reported that C protein inhibits IFN-α-stimulated tyrosine phosphorylation of STATs, but the underlying mechanism is poorly understood. Here, we show that the C protein of Sendai virus (SeV), a member of the Respirovirus genus, binds to the IFN receptor subunit IFN-α/ß receptor subunit (IFNAR)2 and inhibits IFN-α-stimulated tyrosine phosphorylation of the upstream receptor-associated kinases, JAK1 and TYK2. Analysis of various SeV C mutant (Cm) proteins demonstrates the importance of the inhibitory effect on receptor-associated kinase phosphorylation for blockade of JAK-STAT signaling. Furthermore, this inhibitory effect and the IFNAR2 binding capacity are observed for all the respirovirus C proteins examined. Our results suggest that respirovirus C protein inhibits activation of the receptor-associated kinases JAK1 and TYK2 possibly through interaction with IFNAR2.

Oncogenic role of TYRO3 receptor tyrosine kinase in the progression of pancreatic cancer.

The expression and functions of TYRO3, a member of the TAM receptor tyrosine kinase family, in pancreatic cancer (PC) have not been specifically elucidated. In this study, we confirmed TYRO3 expression in five human PC cell lines (PANC-1, MIA PaCa-2, BxPC-3, AsPC-1, and PK-9) using Western blotting. TYRO3 silencing and overexpression studies have revealed that TYRO3 promotes cell proliferation and invasion in PC via phosphorylation of protein kinase B (Akt) and extracellular signal-regulated kinase (ERK). Using a mouse xenograft model, we showed that tumor growth was significantly suppressed in mice subcutaneously inoculated with TYRO3-knockdown PC cells compared with mice inoculated with control PC cells. Furthermore, TYRO3 expression was examined in PC tissues obtained from 106 patients who underwent pancreatic resection for invasive ductal carcinoma through immunohistochemical staining. TYRO3-positive patients had poor prognoses for overall survival and disease-specific survival compared with TYRO3-negative patients. Multivariate analysis revealed that TYRO3 expression is an independent prognostic factor for overall survival. Our study demonstrates the critical role of TYRO3 in PC progression through Akt and ERK activation and suggests TYRO3 as a novel promising target for therapeutic strategies against PC.