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PVRL4 (or nectin­4) is a promising therapeutic target since its upregulated expression is found in a wide range of human cancer types. Enfortumab vedotin, an antibody­drug conjugate targeting PVRL4, is clinically used for the treatment of urothelial bladder cancer. In addition, rMV­SLAMblind, a genetically engineered oncolytic measles virus, can infect cancer cells and induce apoptosis through interaction with PVRL4. Although PVRL4 transcript levels are elevated in breast, lung and ovarian cancer, the mechanisms of its upregulation have not yet been uncovered. To clarify the regulatory mechanisms of elevated PVRL4 expression in breast cancer cells, Assay for Transposase­Accessible Chromatin­sequencing and chromatin immunoprecipitation­sequencing (ChIP­seq) data were used to search for its regulatory regions. Using breast cancer cells, an enhancer region was ultimately identified. Additional analyses, including ChIP and reporter assays, demonstrated that FOS interacted with the PVRL4 enhancer region, and that alterations of the FOS­binding motifs in the enhancer region decreased reporter activity. Consistent with these data, exogenous expression of FOS enhanced the reporter activity and PVRL4 expression in breast cancer cells. Furthermore, RNA­seq analysis using breast cancer cells treated with PVRL4 small interfering RNA revealed its possible involvement in the cytokine response and immune system. These data suggested that FOS was involved, at least partly, in the regulation of PVRL4 expression in breast cancer cells, and that elevated PVRL4 expression may regulate the response of cancer cells to cytokines and the immune system.

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Canine primary lung cancer with metastasis has a poor prognosis with no effective treatment. We previously generated a recombinant measles virus (MV) that lost binding affinity to a principal receptor, SLAM, to eliminate its virulence as a new cancer treatment strategy. The virus, rMV-SLAMblind, targets nectin-4, recently listed as a tumor marker, and exerts antitumor activity against nectin-4-positive canine mammary cancer and urinary bladder transitional cell carcinoma cells. However, the effectivity of rMV-SLAMblind for other types of canine cancers is still unknown. Here we evaluated the antitumor effect of rMV-SLAMblind to canine lung cancer. Nectin-4 is expressed on three canine lung cancer cell lines (CLAC, AZACL1, AZACL2) and rMV-SLAMblind was able to infect these cell lines. CLAC cells showed reduced cell viability after virus infection. In the CLAC xenograft nude mouse model, intratumoral administration of rMV-SLAMblind significantly suppressed tumor growth. In rMV-SLAMblind-treated mice, natural killer cells were activated, and Cxcl10 and Il12a levels were significantly increased in comparison with levels in the control group. In addition, the depletion of NK cells reduced the anti-tumor effect. To understand difference in efficacy among canine lung cancer cell lines, we compared virus growth and gene expression pattern after virus treatment in the three canine lung cancer cell lines; virus growth was highest in CLAC cells compared with the other cell lines and the induction of interferon (IFN)-beta and IFN-stimulated genes was at lower levels in CLAC cells. These results suggested that rMV-SLAMblind exhibits oncolytic effect against some canine lung cancer cells and the cellular response after the virus infection may influence its efficacy.

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Oncolytic virotherapy is a promising therapy for cancer. We previously established a recombinant measles virus (rMV-SLAMblind) that targets NECTIN4-expressing cancer cells and demonstrated its antitumor effects using a xenograft model in an immunodeficient mouse. In the current study, to investigate the immune response after rMV-SLAMblind therapy, we developed an immunocompetent cancer mouse model by introducing the NECTIN4 gene into mouse cancer cell lines. NECTIN4-expressing mouse cancer cells were successfully killed by rMV-SLAMblind in vitro. After transplantation of the NECTIN4-expressing tumor cells, rMV-SLAMblind significantly suppressed tumor growth in immunocompetent mice. Thus, this immunocompetent mouse cancer model could be a powerful tool in which to study the effect of rMV-SLAMblind therapy on the immune response. Using this model we found that rMV-SLAMblind elicited significant activation of natural killer cells, type 1 helper T cells and the tumor-specific CD8+ T-cell response in the tumor microenvironment. Immune cell depletion study revealed that CD8+ cells particularly played significant roles in the therapeutic efficacy of rMV-SLAMblind. Thus, rMV-SLAMblind exerts a therapeutic effect, not only directly by tumor cell killing, but also indirectly by efficient induction of antitumor immunity.

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In general, in mammalian cells, cytosolic DNA viruses are sensed by cyclic GMP-AMP synthase (cGAS), and RNA viruses are recognized by retinoic acid-inducible gene I (RIG-I)-like receptors, triggering a series of downstream innate antiviral signaling steps in the host. We previously reported that measles virus (MeV), which possesses an RNA genome, induces rapid antiviral responses, followed by comprehensive downregulation of host gene expression in epithelial cells. Interestingly, gene ontology analysis indicated that genes encoding mitochondrial proteins are enriched among the list of downregulated genes. To evaluate mitochondrial stress after MeV infection, we first observed the mitochondrial morphology of infected cells and found that significantly elongated mitochondrial networks with a hyperfused phenotype were formed. In addition, an increased amount of mitochondrial DNA (mtDNA) in the cytosol was detected during progression of infection. Based on these results, we show that cytosolic mtDNA released from hyperfused mitochondria during MeV infection is captured by cGAS and causes consequent priming of the DNA sensing pathway in addition to canonical RNA sensing. We also ascertained the contribution of cGAS to the in vivo pathogenicity of MeV. In addition, we found that other viruses that induce downregulation of mitochondrial biogenesis as seen for MeV cause similar mitochondrial hyperfusion and cytosolic mtDNA-priming antiviral responses. These findings indicate that the mtDNA-activated cGAS pathway is critical for full innate control of certain viruses, including RNA viruses that cause mitochondrial stress.

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The prognosis of canine transitional cell carcinoma (TCC) of urinary bladder is generally poor because it is difficult to diagnose at early stages and conventional therapies, such as surgical resection and/or chemotherapy, are often not curative treatments. Based on our previous report that recombinant measles virus (rMV-SLAMblind) therapy could be a new treatment for canine mammary tumor, the applicability of rMV-SLAMblind in canine urinary bladder TCC was examined in this study. A canine TCC cell line was established from a TCC patient dog by transplanting a piece of the tumor mass into an immunodeficient mouse and then isolating the primary TCC cells from the grown tumor mass. The primary cultured cells, named TCC-NU1, express nectin-4, a receptor for rMV-SLAMblind infection. The rMV-SLAMblind infected TCC-NU1 cells, and dose-dependently showed cell cytotoxicity. Moreover, intratumoral administration of rMV-SLAMblind in a xenograft model bearing TCC-NU1 cells significantly suppressed the tumor growth reducing the endpoint mass of tumors in treated mice compared to control mice. These results suggest that virotherapy with rMV-SLAMblind be a new candidate therapy for canine TCC.

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One of the most refractory breast cancer types is triple negative (TN) breast cancer, in which cells are resistant to both hormone and Herceptin treatments and, thus, often cause recurrence and metastasis. Effective treatments are needed to treat TN breast cancer. We previously demonstrated that rMV-SLAMblind, a recombinant measles virus, showed anti-tumor activity against breast cancer cells. Here, we examined whether rMV-SLAMblind is effective for treating TN breast cancer. Nectin-4, a receptor for rMV-SLAMblind, was expressed on the surface of 75% of the analyzed TN breast cancer cell lines. rMV-SLAMblind infected the nectin-4-expressing TN breast cancer cell lines, and significantly decreased the viability in half of the analyzed cell lines in vitro. Additionally, intratumoral injection of rMV-SLAMblind suppressed tumor growth in xenografts of MDA-MB-468 and HCC70 cells. To assess treatment for metastatic breast cancer, we performed intravenous administration of the luciferase-expressing-rMV-SLAMblind to MDA xenografted mice. Virus replicated in the tumor and resulted in significant suppression of the tumor growth. The safety of the virus was tested by its intravenous injection into healthy cynomolgus monkeys, which did not cause any measles-like symptoms. These results suggest that rMV-SLAMblind is a promising candidate as a therapeutic agent for treating metastatic and/or TN type breast cancer.

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The revision of the sub-order Microchiroptera is one of the most intriguing outcomes in recent mammalian molecular phylogeny. The unexpected sister-taxon relationship between rhinolophoid microbats and megabats, with the exclusion of other microbats, suggests that megabats arose in a relatively short period of time from a microbat-like ancestor. In order to understand the genetic mechanism underlying adaptive evolution in megabats, we determined the whole-genome sequences of two rousette megabats, Leschenault's rousette (Rousettus leschenaultia) and the Egyptian fruit bat (R. aegyptiacus). The sequences were compared with those of 22 other mammals, including nine bats, available in the database. We identified that megabat genomes are distinct in that they have extremely low activity of SINE retrotranspositions, expansion of two chemosensory gene families, including the trace amine receptor (TAAR) and olfactory receptor (OR), and elevation of the dN/dS ratio in genes for immunity and protein catabolism. The adaptive signatures discovered in the genomes of megabats may provide crucial insight into their distinct evolution, including key processes such as virus resistance, loss of echolocation, and frugivorous feeding.

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The tail domain of the measles virus (MeV) N protein is typically phosphorylated at S479 and S510. However, the protein kinase responsible for this phosphorylation has not been identified. To identify the protein kinase responsible, we conducted an in vitro kinase assay in the presence of various protein kinase inhibitors. Phosphorylation of S479 and S510 was suppressed in the presence of SP600125. We demonstrated that purified PIM 3 kinase, which is sensitive to SP600125, successfully phosphorylated both phosphorylation sites. Inhibitors of PIM kinase, CX6258 and LY294002, also suppressed phosphorylation of the N protein. These findings indicate that PIM 3 kinase is associated with the tail domain of the N protein and that PIM 3 kinase regulates N protein phosphorylation.

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Mammary gland cancer is the most common cancer occurring in women globally. Incidences of this cancer in Japan are on the increase. Annually, more than 70,000 new cases are recorded in Japan and about 1.7 million in the world. Many cases are still difficult to cure completely, and animal models are required for the characterization of the biology, therapeutic strategy, and preventive measures for spontaneous mammary tumor. The mouse model used currently has some limitations owing to structural differences between mouse and human mammary glands. Tupaia belangeri (tree shrew), which belongs to the Tupaiidae family, shows relatively high genetic homology and structural similarity to human mammary glands. Here, we characterized the spontaneous mammary tumors in 61 female tree shrews of different ages. The incidence rate was 24.6% (15/61), and the rate of simultaneous or metachronous multiplex tumors was 60% (9/15). From the incidence pattern, some cases seemed to be of familial mammary gland tumor, as the offspring of female tree shrews No. 3 and 9 and male tree shrew No. 11 showed a high incidence rate, of 73.3% (11/15). Average incidence age for tumor development was 2 years and 3 months, and the earliest was 10 months. Histochemical analysis indicated that spontaneous mammary gland tumors in the tree shrew show the features of intraductal papillary adenomas (22 cases), except 2 tubulopapillary carcinoma cases (No. 75 and 131). All the cases were positive for the progesterone receptor, whereas 91.3% were positive for the estrogen receptor, and 4.3% were HER-2 positive. We have also confirmed the expression of nectin-4 in some mammary tumor cells. Additionally, we subjected tree shrews to cytodiagnosis or X-ray CT. Thus, the findings of this study highlight the potential of the tree shrew as a valuable new animal model for mammary gland tumor study.

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Nipah virus (NiV) is a highly pathogenic paramyxovirus that causes lethal encephalitis in humans. We previously reported that the V protein, one of the three accessory proteins encoded by the P gene, is one of the key determinants of the pathogenesis of NiV in a hamster infection model. Satterfield B.A. et al. have also revealed that V protein is required for the pathogenicity of henipavirus in a ferret infection model. However, the complete functions of NiV V have not been clarified. In this study, we identified UBX domain-containing protein 1 (UBXN1), a negative regulator of RIG-I-like receptor signaling, as a host protein that interacts with NiV V. NiV V interacted with the UBX domain of UBXN1 via its proximal zinc-finger motif in the C-terminal domain. NiV V increased the level of UBXN1 protein by suppressing its proteolysis. Furthermore, NiV V suppressed RIG-I and MDA5-dependent interferon signaling by stabilizing UBXN1 and increasing the interaction between MAVS and UBXN1 in addition to directly interrupting the activation of MDA5. Our results suggest a novel molecular mechanism by which the induction of interferon is potentially suppressed by NiV V protein via UBXN1.

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Highly pathogenic avian influenza virus (HPAIV) is a serious threat not only to domestic fowls but also to humans. Vaccines inducing long-lasting immunity against HPAIV are required. In the present study, we generated recombinant measles virus (MV) expressing the hemagglutinin protein of HPAIV without the multibasic site necessary for its pathogenicity in chickens using the backbone of an MV vaccine strain (rMV-Ed-H5HA) or a wild-type MV-derived mutant (rMV-HL-Vko-H5HA). We examined protective efficacy of the candidate vaccines in the monkey infection model by the challenge with a HPAIV (H5N1). Cynomolgus monkeys inoculated with the candidate vaccines produced both anti-H5 HA and anti-MV antibodies. They recovered earlier from influenza symptoms than unvaccinated monkeys after the challenge with the HPAIV strain. Chest radiography and histopathological analyses confirmed less severe pneumonia in the vaccinated monkeys. Vaccination tended to suppress viral shedding and reduced the interleukin-6 levels in the lungs. Furthermore, the vaccination with rMV-Ed-H5HA of monkeys with pre-existing anti-MV immunity induced the production of anti-H5 HA antibodies. These results suggest that both candidate vaccines effectively reduce disease severity in naïve hosts, and that rMV-Ed-H5HA is a particularly good candidate vaccine against HPAIV infection.

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Henipaviruses, such as Nipah (NiV) and Hendra (HeV) viruses, are highly pathogenic zoonotic agents within the Paramyxoviridae family. The phosphoprotein (P) gene products of the paramyxoviruses have been well characterized for their interferon (IFN) antagonist activity and their contribution to viral pathogenicity. In this study, we demonstrated that the nucleoprotein (N) of henipaviruses also prevents the host IFN signaling response. Reporter assays demonstrated that the NiV and HeV N proteins (NiV-N and HeV-N, respectively) dose-dependently suppressed both type I and type II IFN responses and that the inhibitory effect was mediated by their core domains. Additionally, NiV-N prevented the nuclear transport of signal transducer and activator of transcription 1 (STAT1) and STAT2. However, NiV-N did not associate with Impα5, Impß1, or Ran, which are members of the nuclear transport system for STATs. Although P protein is known as a binding partner of N protein and actively retains N protein in the cytoplasm, the IFN antagonist activity of N protein was not abolished by the coexpression of P protein. This suggests that the IFN inhibition by N protein occurs in the cytoplasm. Furthermore, we demonstrated that the complex formation of STATs was hampered in the N protein-expressing cells. As a result, STAT nuclear accumulation was reduced, causing a subsequent downregulation of interferon-stimulated genes (ISGs) due to low promoter occupancy by STAT complexes. This novel route for preventing host IFN responses by henipavirus N proteins provides new insight into the pathogenesis of these viruses.IMPORTANCE Paramyxoviruses are well known for suppressing interferon (IFN)-mediated innate immunity with their phosphoprotein (P) gene products, and the henipaviruses also possess P, V, W, and C proteins for evading host antiviral responses. There are numerous studies providing evidence for the relationship between viral pathogenicity and antagonistic activities against IFN responses by P gene products. Meanwhile, little attention has been paid to the influence of nucleoprotein (N) on host innate immune responses. In this study, we demonstrated that both the NiV and HeV N proteins have antagonistic activity against the JAK/STAT signaling pathway by preventing the nucleocytoplasmic trafficking of STAT1 and STAT2. This inhibitory effect is due to an impairment of the ability of STATs to form complexes. These results provide new insight into the involvement of N protein in viral pathogenicity via its IFN antagonism.

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In rare cases, measles virus (MV) in children leads to fatal neurological complications such as primary measles encephalitis, post-acute measles encephalitis, subacute sclerosing panencephalitis and measles inclusion-body encephalitis. To investigate the pathogenesis of MV-induced encephalitis, rodent brain-adapted MV strains CAM/RB and CAMR40 were generated. These strains acquired mutations to adapt to the rodent brain during 40 passages in rat brain. However, it is still unknown which genes confer the neurovirulence of MV. We previously established a rescue system for recombinant MVs possessing the backbone of wild-type strain HL, an avirulent strain in mice. In the present study, to identify the genes in CAMR40 that elicit neurovirulence, we generated chimeric recombinant MVs based on strain HL. As a result, recombinant wild-type MV in which the haemagglutinin (H) gene was substituted with that of CAMR40 caused a non-lethal mild disease in mice, while additional substitution of the HL phosphoprotein (P) gene with that of strain CAMR40 caused lethal severe neurological signs comparable to those of CAMR40. These results clearly indicated that, in addition to the H gene, the P gene is required for the neurovirulence of MV CAMR40.

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The regulation of transcription during Nipah virus (NiV) replication is poorly understood. Using a bicistronic minigenome system, we investigated the involvement of non-coding regions (NCRs) in the transcriptional re-initiation efficiency of NiV RNA polymerase. Reporter assays revealed that attenuation of NiV gene expression was not constant at each gene junction, and that the attenuating property was controlled by the 3' NCR. However, this regulation was independent of the gene-end, gene-start and intergenic regions. Northern blot analysis indicated that regulation of viral gene expression by the phosphoprotein (P) and large protein (L) 3' NCRs occurred at the transcription level. We identified uridine-rich tracts within the L 3' NCR that are similar to gene-end signals. These gene-end-like sequences were recognized as weak transcription termination signals by the viral RNA polymerase, thereby reducing downstream gene transcription. Thus, we suggest that NiV has a unique mechanism of transcriptional regulation.

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Natural infection with measles virus (MV) establishes lifelong immunity. Persistent infection with MV is likely involved in this phenomenon, as non-replicating protein antigens never induce such long-term immunity. Although MV establishes stable persistent infection in vitro and possibly in vivo, the mechanism by which this occurs is largely unknown. Here, we demonstrate that MV changes the infection mode from lytic to non-lytic and evades the innate immune response to establish persistent infection without viral genome mutation. We found that, in the persistent phase, the viral RNA level declined with the termination of interferon production and cell death. Our analysis of viral protein dynamics shows that during the establishment of persistent infection, the nucleoprotein level was sustained while the phosphoprotein and large protein levels declined. The ectopic expression of nucleoprotein suppressed viral replication, indicating that viral replication is self-regulated by nucleoprotein accumulation during persistent infection. The persistently infected cells were able to produce interferon in response to poly I:C stimulation, suggesting that MV does not interfere with host interferon responses in persistent infection. Our results may provide mechanistic insight into the persistent infection of this cytopathic RNA virus that induces lifelong immunity.

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UNLABELLED: In the current study, we generated recombinant chimeric canine distemper viruses (CDVs) by replacing the hemagglutinin (H) and/or phosphoprotein (P) gene in an avirulent strain expressing enhanced green fluorescent protein (EGFP) with those of a mouse-adapted neurovirulent strain. An in vitro experimental infection indicated that the chimeric CDVs possessing the H gene derived from the mouse-adapted CDV acquired infectivity for neural cells. These cells lack the CDV receptors that have been identified to date (SLAM and nectin-4), indicating that the H protein defines infectivity in various cell lines. The recombinant viruses were administered intracerebrally to 1-week-old mice. Fatal neurological signs of disease were observed only with a recombinant CDV that possessed both the H and P genes of the mouse-adapted strain, similar to the parental mouse-adapted strain, suggesting that both genes are important to drive virulence of CDV in mice. Using this recombinant CDV, we traced the intracerebral propagation of CDV by detecting EGFP. Widespread infection was observed in the cerebral hemispheres and brainstems of the infected mice. In addition, EGFP fluorescence in the brain slices demonstrated a sequential infectious progression in the central nervous system: CDV primarily infected the neuroependymal cells lining the ventricular wall and the neurons of the hippocampus and cortex adjacent to the ventricle, and it then progressed to an extensive infection of the brain surface, followed by the parenchyma and cortex. In the hippocampal formation, CDV spread in a unidirectional retrograde pattern along neuronal processes in the hippocampal formation from the CA1 region to the CA3 region and the dentate gyrus. Our mouse model demonstrated that the main target cells of CDV are neurons in the acute phase and that the virus spreads via neuronal transmission pathways in the hippocampal formation. IMPORTANCE: CDV is the etiological agent of distemper in dogs and other carnivores, and in many respects, the pathogenesis of CDV infection in animals resembles that of measles virus infection in humans. We successfully generated a recombinant CDV containing the H and P genes from a mouse-adapted neurovirulent strain and expressing EGFP. The recombinant CDV exhibited severe neurovirulence with high mortality, comparable to the parental mouse-adapted strain. The mouse-infectious model could become a useful tool for analyzing CDV infection of the central nervous system subsequent to passing through the blood-cerebrospinal fluid barrier and infectious progression in the target cells in acute disease.

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Pancreatic cancer is one of the most intractable cancers and has a devastating prognosis; over the past three decades the 5-year survival rate has been <10%. Therefore, development of a novel anticancer treatment for pancreatic cancer is a matter of urgency. We previously developed an oncolytic recombinant measles virus (MV), rMV-SLAMblind, that had lost the ability to bind to its principal receptor, signaling lymphocyte activity molecule (SLAM), but which selectively infected and efficiently killed nectin-4-expressing breast and lung cancer cells. In this study, we analyzed the antitumor effect of this virus against pancreatic cancer. Nectin-4 was expressed on the surface of 4/16 tested pancreatic cancer cell lines, which were efficiently infected and killed by rMV-SLAMblind in vitro. The intratumoral inoculation of rMV-SLAMblind suppressed the growth of KLM1 and Capan-2 cells xenografted in SCID mice. The sequence analysis of MV isolated from the tumor revealed that the designed mutation in the H protein of rMV-SLAMblind had been stably maintained for 47 days after the last inoculation. These results suggest that rMV-SLAMblind is a promising candidate for the novel treatment of pancreatic cancer.

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Nipah virus (NiV) causes severe encephalitis in humans, with high mortality. NiV nonstructural C protein (NiV-C) is essential for its pathogenicity, but its functions are unclear. In this study, we focused on NiV-C trafficking in cells and found that it localizes predominantly in the cytoplasm but partly in the nucleus. An analysis of NiV-C mutants showed that amino acids 2, 21-24 and 110-139 of NiV-C are important for its localization in the cytoplasm. Inhibitor treatment indicates that the nuclear export determinant is not a classical CRM1-dependent nuclear export signal. We also determined that amino acids 60-75 and 72-75 were important for nuclear localization of NiV-C. Furthermore, NiV-C mutants that had lost their capacity for nuclear localization inhibited the interferon (IFN) response more strongly than complete NiV-C. These results indicate that the IFN-antagonist activity of NiV-C occurs in the cytoplasm.

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Nipah virus belongs to the genus Henipavirus in the family Paramyxoviridae, and its RNA genome is larger than those of other paramyxoviruses because it has long untranslated regions (UTRs) in each gene. However, the functions of these UTRs are not fully understood. In this study, we investigated the functions of the 5' UTRs and found that the 5' UTR of the M gene upregulated the translation of a reporter gene. Using an RNA pull-down assay, we showed that eukaryotic elongation factor 1-beta (EEF1B2) interacts with nucleotides 81-100 of the M 5' UTR and specifically enhances its translation efficiency. Our results suggest that the M 5' UTR promotes the production of M protein and viral budding by recruiting EEF1B2.

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