| 白銀 勇太（しろがねゆうた） | データ更新日：2023.12.04 |
講師 ／
医学研究院
基礎医学部門
病態制御学講座
| 1. | Ryuichi Takemoto, Yuichi Hirai, Shumpei Watanabe, Hidetaka Harada, Tateki Suzuki, Takao Hashiguchi, Yusuke Yanagi, Yuta Shirogane, Interaction of the Hemagglutinin Stalk Region with Cell Adhesion Molecule (CADM) 1 and CADM2 Mediates the Spread between Neurons and Neuropathogenicity of Measles Virus with a Hyperfusogenic Fusion Protein, Journal of Virology, 10.1128/jvi.00340-23, 2023.05. |
| 2. | Yuta Shirogane, Hidetaka Harada, Yuichi Hirai, Ryuichi Takemoto, Tateki Suzuki, Takao Hashiguchi, Yusuke Yanagi, Collective fusion activity determines neurotropism of an en bloc transmitted enveloped virus, Science Advances, 10.1126/sciadv.adf3731, 2023.01. |
| 3. | Ryuichi Takemoto, Tateki Suzuki, Takao Hashiguchi, Yusuke Yanagi, Yuta Shirogane, Short-stalk isoforms of CADM1 and CADM2 trigger neuropathogenic measles virus-mediated membrane fusion by interacting with the viral hemagglutinin, Journal of Virology, 10.1128/JVI.01949-21, 2022.01, Measles virus (MeV), an enveloped RNA virus in the family Paramyxoviridae, usually causes acute febrile illness with skin rash, but in rare cases persists in the brain, causing a progressive neurological disorder, subacute sclerosing panencephalitis (SSPE). MeV bears two envelope glycoproteins, the hemagglutinin (H) and fusion (F) proteins. The H protein possesses a head domain that initially mediates receptor binding and a stalk domain that subsequently transmits the fusion-triggering signal to the F protein. We have recently shown that cell adhesion molecule 1 (CADM1, also known as IGSF4A, Necl-2, SynCAM1) and CADM2 (also known as IGSF4D, Necl-3, SynCAM2) are host factors enabling cell-cell membrane fusion mediated by hyperfusogenic F proteins of neuropathogenic MeVs as well as MeV spread between neurons lacking the known receptors. CADM1 and CADM2 interact in cis with the H protein on the same cell membrane, triggering hyperfusogenic F protein-mediated membrane fusion. Multiple isoforms of CADM1 and CADM2 containing various lengths of their stalk regions are generated by alternative splicing. Here we show that only short-stalk isoforms of CADM1 and CADM2 predominantly expressed in the brain induce hyperfusogenic F protein-mediated membrane fusion. While the known receptors interact in trans with the H protein through its head domain, these isoforms can interact in cis even with the H protein lacking the head domain and trigger membrane fusion, presumably through its stalk domain. Thus, our results unveil a new mechanism of viral fusion triggering by host factors.. |
| 4. | Yinghong Xiao, Peter V. Lidsky, Yuta Shirogane, Ranen Aviner, Chien-Ting Wu, Weiyi Li, Weihao Zheng, Dale Talbot, Adam Catching, Gilad Doitsh, Weiheng Su, Colby E. Gekko, Arabinda Nayak, Joel D. Ernst, Leonid Brodsky, Elia Brodsky, Elsa Rousseau, Sara Capponi, Simone Bianco, Robert Nakamura, Peter K. Jackson, Judith Frydman, Raul Andino, A defective viral genome strategy elicits broad protective immunity against respiratory viruses, Cell, 10.1016/j.cell.2021.11.023, 184, 25, 6037-6051, 2021.12. |
| 5. | Yuta Shirogane, Ryuichi Takemoto, Tateki Suzuki, Tomonori Kameda, Kinichi Nakashima, Takao Hashiguchi, Yusuke Yanagi, CADM1 and CADM2 trigger neuropathogenic measles virus-mediated membrane fusion by acting in cis, Journal of Virology, 10.1128/JVI.00528-21, 2021.04. |
| 6. | Yuta Shirogane, Bert L. Semler, Elsa Rousseau, Jakub Voznica, Yinghong Xiao, Weiheng Su, Adam Catching, Zachary J. Whitfield, Igor M. Rouzine, Simone Bianco, Raul Andino, Experimental and mathematical insights on the interactions between poliovirus and a defective interfering genome, PLOS Pathogens, 10.1371/journal.ppat.1009277, 2021.09, During replication, RNA viruses accumulate genome alterations, such as mutations and deletions. The interactions between individual variants can determine the fitness of the virus population and, thus, the outcome of infection. To investigate the effects of defective interfering genomes (DI) on wild-type (WT) poliovirus replication, we developed an ordinary differential equation model. We experimentally determined virus and DI replication during co-infection, and use these data to infer model parameters. Our model predicts, and our experimental measurements confirm, that DI replication and encapsidation are the most important determinants for the outcome of the competition. WT replication inversely correlates with DI replication. Our model predicts that genome replication and effective DI genome encapsidation are critical to effectively inhibit WT production, but an equilibrium can be established which enables WT to replicate, albeit to reduce levels. . |
| 7. | Ayako Ueo, Marie Kubota, Yuta Shirogane, Shinji Ohno, Takao Hashiguchi, Yusuke Yanagi, Lysosome-associated membrane proteins support the furin-mediated processing of the mumps virus fusion protein, Journal of Virology, 2020.06, Mumps virus (MuV), an enveloped RNA virus of the Paramyxoviridae family and the causative agent of mumps, affects the salivary glands and other glandular tissues as well as the central nervous system. The virus enters the cell by inducing the fusion of its envelope with the plasma membrane of the target cell. Membrane fusion is mediated by MuV envelope proteins: the hemagglutininneuraminidase and fusion (F) protein. Cleavage of the MuV F protein (MuV-F) into two subunits by the cellular protease furin is a prerequisite for fusion and virus infectivity. Here, we show that 293T (a derivative of HEK293) cells do not produce syncytia upon expression of MuV envelope proteins or MuV infection. This failure is caused by the inefficient MuV-F cleavage despite the presence of functional furin in 293T cells. An expression cloning strategy revealed that overexpression of lysosome-associated membrane proteins (LAMPs) confers on 293T cells the ability to produce syncytia upon expression of MuV envelope proteins. The LAMP family comprises the ubiquitously expressed LAMP1 and LAMP2, the interferon-stimulated gene product LAMP3, and the cell type-specific proteins. The expression level of the LAMP3 gene, but not of LAMP1 and LAMP2 genes, differed markedly between 293T and HEK293 cells. Overexpression of LAMP1, LAMP2, or LAMP3 allowed 293T cells to process MuV-F efficiently. Furthermore, these LAMPs were found to interact with both MuV-F and furin. Our results indicate that LAMPs support the furin-mediated cleavage of MuV-F and that, among them, LAMP3 may be critical for the process, at least in certain cells. IMPORTANCE The cellular protease furin mediates proteolytic cleavage of many host and pathogen proteins and plays an important role in viral envelope glycoprotein maturation. MuV, an enveloped RNA virus of the Paramyxoviridae family and an important human pathogen, enters the cell through the fusion of its envelope with the plasma membrane of the target cell. Membrane fusion is mediated by the viral attachment protein and the F protein. Cleavage of MuV-F into two subunits by furin is a prerequisite for fusion and virus infectivity. Here, we show that LAMPs support the furin-mediated cleavage of MuV-F. Expression levels of LAMPs affect the processing of MuV-F and MuV-mediated membrane fusion. Among LAMPs, the interferon-stimulated gene product LAMP3 is most critical in certain cells. Our study provides potential targets for anti-MuV therapeutics.. |
| 8. | Yuta Shirogane, Takao Hashiguchi, Yusuke Yanagi, Weak cis and trans interactions of the hemagglutinin with receptors trigger fusion proteins of neuropathogenic measles virus isolates, Journal of virology, 10.1128/JVI.01727-19, 94, 2, 2020.01, [URL], Measles virus (MeV) is an enveloped RNA virus bearing two envelope glycoproteins, the hemagglutinin (H) and fusion (F) proteins. Upon receptor binding, the H protein triggers conformational changes of the F protein, causing membrane fusion and subsequent virus entry. MeV may persist in the brain, infecting neurons and causing fatal subacute sclerosing panencephalitis (SSPE). Since neurons do not express either of the MeV receptors, signaling lymphocytic activation molecule (SLAM; also called CD150) and nectin-4, how MeV propagates in neurons is unknown. Recent studies have shown that specific substitutions in the F protein found in MeV isolates from SSPE patients are critical for MeV neuropathogenicity by rendering the protein unstable and hyperfusogenic. Recombinant MeVs possessing the F proteins with such substitutions can spread in primary human neurons and in the brains of mice and hamsters and induce cell-cell fusion in cells lacking SLAM and nectin-4. Here, we show that receptor-blind mutant H proteins that have decreased binding affinities to receptors can support membrane fusion mediated by hyperfusogenic mutant F proteins, but not the wild-type F protein, in cells expressing the corresponding receptors. The results suggest that weak interactions of the H protein with certain molecules (putative neuron receptors) trigger hyperfusogenic F proteins in SSPE patients. Notably, where cell-cell contacts are ensured, the weak cis interaction of the H protein with SLAM on the same cell surface also could trigger hyperfusogenic F proteins. Some enveloped viruses may exploit such cis interactions with receptors to infect target cells, especially in cell-to-cell transmission. IMPORTANCE Measles virus (MeV) may persist in the brain, causing incurable subacute sclerosing panencephalitis (SSPE). Because neurons, the main target in SSPE, do not express receptors for wild-type (WT) MeV, how MeV propagates in the brain is a key question for the disease. Recent studies have demonstrated that specific substitutions in the MeV fusion (F) protein are critical for neuropathogenicity. Here, we show that weak cis and trans interactions of the MeV attachment protein with receptors that are not sufficient to trigger the WT MeV F protein can trigger the mutant F proteins from neuropathogenic MeV isolates. Our study not only provides an important clue to understand MeV neuropathogenicity but also reveals a novel viral strategy to expand cell tropism.. |
| 9. | Marie Kubota, Iori Okabe, Shin Ichi Nakakita, Ayako Ueo, Yuta Shirogane, Yusuke Yanagi, Takao Hashiguchi, Disruption of the dimer-dimer interaction of the mumps virus attachment protein head domain, aided by an anion located at the interface, compromises membrane fusion triggering, Journal of virology, 10.1128/JVI.01732-19, 94, 2, 2020.01, [URL], Mumps virus (MuV), an enveloped negative-strand RNA virus belonging to the family Paramyxoviridae, enters the host cell through membrane fusion mediated by two viral envelope proteins, an attachment protein hemagglutinin-neuraminidase (MuV-HN) and a fusion (F) protein. However, how the binding of MuV-HN to glycan receptors triggers membrane fusion is not well understood. The crystal structure of the MuV-HN head domain forms a tetramer (dimer of dimers) like other paramyxovirus attachment proteins. In the structure, a sulfate ion (SO42) was found at the interface between two dimers, which may be replaced by a hydrogen phosphate ion (HPO42) under physiological conditions. The anion is captured by the side chain of a positively charged arginine residue at position 139 of one monomer each from both dimers. Substitution of alanine or lysine for arginine at this position compromised the fusion support activity of MuV-HN without affecting its cell surface expression, glycan-receptor binding, and interaction with the F protein. Furthermore, the substitution appeared to affect the tetramer formation of the head domain as revealed by blue native-PAGE analysis. These results, together with our previous similar findings with the measles virus attachment protein head domain, suggest that the dimer-dimer interaction within the tetramer may play an important role in triggering membrane fusion during paramyxovirus entry. IMPORTANCE Despite the use of effective live vaccines, mumps outbreaks still occur worldwide. Mumps virus (MuV) infection typically causes flu-like symptoms and parotid gland swelling but sometimes leads to orchitis, oophoritis, and neurological complications, such as meningitis, encephalitis, and deafness. MuV enters the host cell through membrane fusion mediated by two viral proteins, a receptor-binding attachment protein, and a fusion protein, but its detailed mechanism is not fully understood. In this study, we show that the tetramer (dimer of dimers) formation of the MuV attachment protein head domain is supported by an anion located at the interface between two dimers and that the dimer-dimer interaction plays an important role in triggering the activation of the fusion protein and causing membrane fusion. These results not only further our understanding of MuV entry but provide useful information about a possible target for antiviral drugs.. |
| 10. | Naganori Nao, Ko Sato, Junya Yamagishi, Maino Tahara, Yuichiro Nakatsu, Fumio Seki, Hiroshi Katoh, Aiko Ohnuma, Yuta Shirogane, Masahiro Hayashi, Tamio Suzuki, Hideaki Kikuta, Hidekazu Nishimura, Makoto Takeda, Consensus and variations in cell line specificity among human metapneumovirus strains, PloS one, 10.1371/journal.pone.0215822, 14, 4, 2019.04, [URL], Human metapneumovirus (HMPV) has been a notable etiological agent of acute respiratory infection in humans, but it was not discovered until 2001, because HMPV replicates only in a limited number of cell lines and the cytopathic effect (CPE) is often mild. To promote the study of HMPV, several groups have generated green fluorescent protein (GFP)-expressing recombinant HMPV strains (HMPV GFP ). However, the growing evidence has complicated the understanding of cell line specificity of HMPV, because it seems to vary notably among HMPV strains. In addition, unique A2b clade HMPV strains with a 180-nucleotide duplication in the G gene (HMPV A2b 180nt-dup strains) have recently been detected. In this study, we reevaluated and compared the cell line specificity of clinical isolates of HMPV strains, including the novel HMPV A2b 180nt-dup strains, and six recombinant HMPV GFP strains, including the newly generated recombinant HMPV A2b 180nt-dup strain, MG0256-EGFP. Our data demonstrate that VeroE6 and LLC-MK2 cells generally showed the highest infectivity with any clinical isolates and recombinant HMPV GFP strains. Other human-derived cell lines (BEAS-2B, A549, HEK293, MNT-1, and HeLa cells) showed certain levels of infectivity with HMPV, but these were significantly lower than those of VeroE6 and LLC-MK2 cells. Also, the infectivity in these suboptimal cell lines varied greatly among HMPV strains. The variations were not directly related to HMPV genotypes, cell lines used for isolation and propagation, specific genome mutations, or nucleotide duplications in the G gene. Thus, these variations in suboptimal cell lines are likely intrinsic to particular HMPV strains. Progress of Research on Infectious Disease for Global Endemic (J-PRIDE and JP18fm0208005) and the Advanced Research and Development for Medical Innovation (AMED-CREST and JP18gm0910005) to M.Takeda.. |
| 11. | Yuta Shirogane, Shumpei Watanabe, Yusuke Yanagi, Cooperation between different variants A unique potential for virus evolution, Virus Research, 10.1016/j.virusres.2019.02.015, 264, 68-73, 2019.04, [URL], RNA viruses exist as quasispecies containing many variants within their populations because of the error prone nature of viral RNA-dependent RNA polymerases. Quasispecies are not a simple collection of individual variants. Instead, internal interactions among variants provide quasispecies with unique evolvability. An example is ‘cooperation’ between wild-type and defective measles viruses, in which co-existence of a wild-type and a mutant genome produces a new phenotype. Such internal interactions presuppose efficient co-transmission of multiple genomes to the same cell, which is achieved by polyploid virions of some virus families or by a high multiplicity of infection. Recent studies have revealed that multiple viral genomes can also be transmitted simultaneously (‘bloc transmission’) by other mechanisms, strengthening the concept of internal interactions among viral quasispecies. Elucidation of the mechanisms of virus evolution, including internal interactions and bloc transmission, may provide rational strategies to solve such important problems of virus infections as drug-resistance, immune evasion, and acquisition of the new tropism and host range.. |
| 12. | Shumpei Watanabe, Yuta Shirogane, Yuma Sato, Takao Hashiguchi, Yusuke Yanagi, New Insights into Measles Virus Brain Infections, Trends in Microbiology, 10.1016/j.tim.2018.08.010, 27, 2, 164-175, 2019.02, [URL], Measles virus (MeV) may persist in the brain, causing fatal neurodegenerative diseases, subacute sclerosing panencephalitis, and measles inclusion-body encephalitis. However, the mechanism of MeV propagation in the brain remains unexplained because human neurons affected by the diseases do not express the known receptors for MeV. Recent studies have revealed that certain changes in the ectodomain of the MeV fusion (F) protein play a key role in MeV spread in the brain. These changes destabilize the prefusion form of the F protein and render it hyperfusogenic, which in turn allows the virus to propagate in neurons. Based on crystal structures of the F protein, effective fusion inhibitors could be developed to treat these diseases.. |
| 13. | Takao Hashiguchi, Yoshinari Fukuda, Rei Matsuoka, Daisuke Kuroda, Marie Kubota, Yuta Shirogane, Shumpei Watanabe, Kouhei Tsumoto, Daisuke Kohda, Richard Karl Plemper, Yusuke Yanagi, Structures of the prefusion form of measles virus fusion protein in complex with inhibitors, Proceedings of the National Academy of Sciences of the United States of America, 10.1073/pnas.1718957115, 115, 10, 2496-2501, 2018.03, [URL], Measles virus (MeV), a major cause of childhood morbidity and mortality, is highly immunotropic and one of the most contagious pathogens. MeV may establish, albeit rarely, persistent infection in the central nervous system, causing fatal and intractable neurodegenerative diseases such as subacute sclerosing panencephalitis and measles inclusion body encephalitis. Recent studies have suggested that particular substitutions in the MeV fusion (F) protein are involved in the pathogenesis by destabilizing the F protein and endowing it with hyperfusogenicity. Here we show the crystal structures of the prefusion MeV-F alone and in complex with the small compound AS-48 or a fusion inhibitor peptide. Notably, these independently developed inhibitors bind the same hydrophobic pocket located at the region connecting the head and stalk of MeV-F, where a number of substitutions in MeV isolates from neurodegenerative diseases are also localized. Since these inhibitors could suppress membrane fusion mediated by most of the hyperfusogenic MeV-F mutants, the development of more effective inhibitors based on the structures may be warranted to treat MeV-induced neurodegenerative diseases.. |
| 14. | Shumpei Watanabe, Shinji Ohno, Yuta Shirogane, Satoshi Suzuki, Ritsuko Koga, Yusuke Yanagi, Measles virus mutants possessing the fusion protein with enhanced fusion activity spread effectively in neuronal cells, but not in other cells, without causing strong cytopathology, Journal of Virology, 10.1128/JVI.03346-14, 89, 5, 2710-2717, 2015.01, [URL], Subacute sclerosing panencephalitis (SSPE) is caused by persistent measles virus (MV) infection in the central nervous system (CNS). Since human neurons, its main target cells, do not express known MV receptors (signaling lymphocyte activation molecule [SLAM] and nectin 4), it remains to be understood how MV infects and spreads in them. We have recently reported that fusion-enhancing substitutions in the extracellular domain of the MV fusion (F) protein (T461I and S103I/N462S/N465S), which are found in multiple SSPE virus isolates, promote MV spread in human neuroblastoma cell lines and brains of suckling hamsters. In this study, we show that hyperfusogenic viruses with these substitutions also spread efficiently in human primary neuron cultures without inducing syncytia. These substitutions were found to destabilize the prefusion conformation of the F protein trimer, thereby enhancing fusion activity. However, these hyperfusogenic viruses exhibited stronger cytopathology and produced lower titers at later time points in SLAM- or nectin 4-expressing cells compared to the wild-type MV. Although these viruses spread efficiently in the brains of SLAM knock-in mice, they did not in the spleens. Taken together, the results suggest that enhanced fusion activity is beneficial for MV to spread in neuronal cells where no cytopathology occurs, but detrimental to other types of cells due to strong cytopathology. Acquisition of enhanced fusion activity through substitutions in the extracellular domain of the F protein may be crucial for MV's extensive spread in the CNS and development of SSPE.. |
| 15. | Shumpei Watanabe, Yuta Shirogane, Satoshi Suzuki, Satoshi Ikegame, Ritsuko Koga, Yusuke Yanagi, Mutant Fusion Proteins with Enhanced Fusion Activity Promote Measles Virus Spread in Human Neuronal Cells and Brains of Suckling Hamsters, Journal of Virology, 10.1128/JVI.02632-12, 87, 5, 2648-2659, 2013.03, [URL], Subacute sclerosing panencephalitis (SSPE) is a fatal degenerative disease caused by persistent measles virus (MV) infection in the central nervous system (CNS). From the genetic study of MV isolates obtained from SSPE patients, it is thought that defects of the matrix (M) protein play a crucial role in MV pathogenicity in the CNS. In this study, we report several notable mutations in the extracellular domain of the MV fusion (F) protein, including those found in multiple SSPE strains. The F proteins with these mutations induced syncytium formation in cells lacking SLAM and nectin 4 (receptors used by wild-type MV), including human neuronal cell lines, when expressed together with the attachment protein hemagglutinin. Moreover, recombinant viruses with these mutations exhibited neurovirulence in suckling hamsters, unlike the parental wild-type MV, and the mortality correlated with their fusion activity. In contrast, the recombinant MV lacking the M protein did not induce syncytia in cells lacking SLAM and nectin 4, although it formed larger syncytia in cells with either of the receptors. Since human neuronal cells are mainly SLAM and nectin 4 negative, fusion-enhancing mutations in the extracellular domain of the F protein may greatly contribute to MV spread via cell-to-cell fusion in the CNS, regardless of defects of the M protein.. |
| 16. | Mai Nakashima, Yuta Shirogane, Takao Hashiguchi, Yusuke Yanagi, Mutations in the putative dimer-dimer interfaces of the measles virus hemagglutinin head domain affect membrane fusion triggering, Journal of Biological Chemistry, 10.1074/jbc.M112.427609, 288, 12, 8085-8091, 2013.03, [URL], Measles virus (MV), an enveloped RNA virus belonging to the Paramyxoviridae family, enters the cell through membrane fusion mediated by two viral envelope proteins, an attachment protein hemagglutinin (H) and a fusion (F) protein. The crystal structure of the receptor-binding head domain of MV-H bound to its cellular receptor revealed that the MV-H head domain forms a tetrameric assembly (dimer of dimers), which occurs in two forms (forms I and II). In this study, we show that mutations in the putative dimer-dimer interface of the head domain in either form inhibit the ability of MV-H to support membrane fusion, without greatly affecting its cell surface expression, receptor binding, and interaction with the F protein. Notably, some anti-MV-H neutralizing monoclonal antibodies are directed to the region around the dimer-dimer interface in form I rather than receptor-binding sites. These observations suggest that the dimer-dimer interactions of the MV-H head domain, especially that in form I, contribute to triggering membrane fusion, and that conformational shift of head domain tetramers plays a role in the process. Furthermore, our results indicate that although the stalk and transmembrane regions may be mainly responsible for the tetramer formation of MV-H, the head domain alone can form tetramers, albeit at a low efficiency.. |
| 17. | Yuta Shirogane, Shumpei Watanabe, Yusuke Yanagi, Cooperation between different RNA virus genomes produces a new phenotype, Nature Communications, 10.1038/ncomms2252, 3, 2012.12, [URL], An RNA virus population generally evolves rapidly under selection pressure, because of high error rates of the viral RNA polymerase. Measles virus, an enveloped RNA virus, has a fusion protein mediating fusion of the viral envelope with the cell membrane. Here we observe that a non-fusogenic recombinant measles virus evolves, after passages, into mutant viruses which regain the ability to induce membrane fusion. Unexpectedly, we identify a mutant virus possessing two types of genomes within a single virion: one genome encoding the wild-type fusion protein, the other a mutant version with a single amino-acid substitution. Neither the wild-type nor mutant protein by itself is able to mediate membrane fusion, but both together exhibit enhanced fusion activity through hetero-oligomer formation. Our results reveal a molecular mechanism for the 'cooperation' between different RNA virus genomes, which may have implications in viral evolution and in the evolution of other macromolecules.. |
| 18. | Yuta Shirogane, Makoto Takeda, Maino Tahara, Satoshi Ikegame, Takanori Nakamura, Yusuke Yanagi, Epithelial-mesenchymal transition abolishes the susceptibility of polarized epithelial cell lines to measles virus, Journal of Biological Chemistry, 10.1074/jbc.M110.102590, 285, 27, 20882-20890, 2010.07, [URL], Measles virus (MV), an enveloped negative-strand RNA virus, remains a major cause of morbidity and mortality in developing countries. MV predominantly infects immune cells by using signaling lymphocyte activation molecule (SLAM; also called CD150) as a receptor, but it also infects polarized epithelial cells, forming tight junctions in a SLAM-independent manner. Although the ability of MV to infect polarized epithelial cells is thought to be important for its transmission, the epithelial cell receptor for MV has not been identified. A transcriptional repressor, Snail, induces epithelial-mesenchymal transition (EMT), in which epithelial cells lose epithelial cell phenotypes, such as adherens and tight junctions. In this study, EMTwas induced by expressing Snail in a lung adenocarcinoma cell line, II-18, which is highly susceptible to wild-type MV. Snail-expressing II-18 cells lost adherens and tight junctions. Microarray analysis confirmed the induction of EMT in II-18 cells and suggested a novel function of Snail in protein degradation and distribution. Importantly, wild-type MV no longer entered EMT-induced II-18 cells, suggesting that the epithelial cell receptor is down-regulated by the induction of EMT. Other polarized cell lines, NCI-H358 and HT-29, also lost susceptibility to wild-type MV when EMT was induced. However, the complete formation of tight junctions rather reduced MV entry into HT-29 cells. Taken together, these data suggest that the unidentified epithelial cell receptor forMVis involved in the formation of epithelial intercellular junctions.. |
| 19. | Masaharu Iwasaki, Makoto Takeda, Yuta Shirogane, Yuichiro Nakatsu, Takanori Nakamura, Yusuke Yanagi, The matrix protein of measles virus regulates viral RNA synthesis and assembly by interacting with the nucleocapsid protein (Journal of Virology (2009) 83, 20, (10374-10383)), Journal of Virology, 10.1128/JVI.02186-09, 84, 1, 2010.01, [URL]. |
| 20. | Masaharu Iwasaki, Makoto Takeda, Yuta Shirogane, Yuichiro Nakatsu, Takanori Nakamura, Yusuke Yanagi, The matrix protein of measles virus regulates viral RNA synthesis and assembly by interacting with the nucleocapsid protein, Journal of Virology, 10.1128/JVI.01056-09, 83, 20, 10374-10383, 2009.10, [URL], The genome of measles virus (MV) is encapsidated by the nucleocapsid (N) protein and associates with RNA-dependent RNA polymerase to form the ribonucleoprotein complex. The matrix (M) protein is believed to play an important role in MV assembly by linking the ribonucleoprotein complex with envelope glycoproteins. Analyses using a yeast two-hybrid system and coimmunoprecipitation in mammalian cells revealed that the M protein interacts with the N protein and that two leucine residues at the carboxyl terminus of the N protein (L523 and L524) are critical for the interaction. In MV minigenome reporter gene assays, theMprotein inhibited viral RNA synthesis only when it was able to interact with the N protein. The N protein colocalized with the M protein at the plasma membrane when the proteins were coexpressed in plasmid-transfected or MV-infected cells. In contrast, the N protein formed small dots in the perinuclear area when it was expressed without the M protein, or it was incapable of interacting with the M protein. Furthermore, a recombinant MV possessing a mutant N protein incapable of interacting with the M protein grew much less efficiently than the parental virus. Since the M protein has an intrinsic ability to associate with the plasma membrane, it may retain the ribonucleoprotein complex at the plasma membrane by binding to the N protein, thereby stopping viral RNA synthesis and promoting viral particle production. Consequently, our results indicate that the M protein regulates MV RNA synthesis and assembly via its interaction with the N protein.. |
| 21. | Makoto Takeda, Shinji Ohno, Maino Tahara, Hiroki Takeuchi, Yuta Shirogane, Hirofumi Ohmura, Takafumi Nakamura, Yusuke Yanagi, Measles viruses possessing the polymerase protein genes of the Edmonston vaccine strain exhibit attenuated gene expression and growth in cultured cells and SLAM knock-in mice, Journal of Virology, 10.1128/JVI.00867-08, 82, 23, 11979-11984, 2008.12, [URL], Live attenuated vaccines against measles have been developed through adaptation of clinical isolates of measles virus (MV) in various cultured cells. Analyses using recombinant MVs with chimeric genomes between wild-type and Edmonston vaccine strains indicated that viruses possessing the polymerase protein genes of the Edmonston strain exhibited attenuated viral gene expression and growth in cultured cells as well as in mice expressing an MV receptor, signaling lymphocyte activation molecule, regardless of whether the virus genome had the wild-type or vaccine-type promoter sequence. These data demonstrate that the polymerase protein genes of the Edmonston strain contribute to its attenuated phenotype.. |
| 22. | Yuta Shirogane, Makoto Takeda, Masaharu Iwasaki, Nobuhisa Ishiguro, Hiroki Takeuchi, Yuichiro Nakatsu, Maino Tahara, Hideaki Kikuta, Yusuke Yanagi, Efficient multiplication of human metapneumovirus in vero cells expressing the transmembrane serine protease TMPRSS2, Journal of Virology, 10.1128/JVI.00676-08, 82, 17, 8942-8946, 2008.09, [URL], Human metapneumovirus (HMPV) is a major causative agent of severe bronchiolitis and pneumonia. Its fusion (F) protein must be cleaved by host proteases to cause membrane fusion, a critical step for virus infection. By generating Vero cells constitutively expressing the transmembrane serine protease TMPRSS2 and green fluorescent protein-expressing recombinant HMPV, we show that TMPRSS2, which is expressed in the human lung epithelium, cleaves the HMPV F protein efficiently and supports HMPV multiplication. The results indicate that TMPRSS2 is a possible candidate protease involved in the development of lower respiratory tract illness in HMPV-infected patients.. |
| 23. | Yuichiro Nakatsu, Makoto Takeda, Shinji Ohno, Yuta Shirogane, Masaharu Iwasaki, Yusuke Yanagi, Measles virus circumvents the host interferon response by different actions of the C and V proteins, Journal of Virology, 10.1128/JVI.00108-08, 82, 17, 8296-8306, 2008.09, [URL], Measles is an acute febrile infectious disease with high morbidity and mortality. The genome of measles virus (MV), the causative agent, encodes two accessory products, V and C proteins, that play important roles in MV virulence. The V but not the C protein of the IC-B strain (a well-characterized virulent strain of MV) has been shown to block the Jak/Stat signaling pathway and counteract the cellular interferon (IFN) response. We have recently shown that a recombinant IC-B strain that lacks C protein expression replicates poorly in certain cell lines, and its growth defect is related to translational inhibition and strong IFN induction. Here, we show that the V protein of the MV IC-B strain also blocks the IFN induction pathway mediated by the melanoma differentiation-associated gene 5 product, thus actively interfering with the host IFN response at two different steps. On the other hand, the C protein per se possesses no activity to block the IFN induction pathway. Our data indicate that the C protein acts as a regulator of viral RNA synthesis, thereby acting indirectly to suppress IFN induction. Since recombinant MVs with C protein defective in modulating viral RNA synthesis or lacking C protein expression strongly stimulate IFN production, in spite of V protein production, both the C and V proteins must be required for MV to fully circumvent the host IFN response.. |
| 24. | Maino Tahara, Makoto Takeda, Yuta Shirogane, Takao Hashiguchi, Shinji Ohno, Yusuke Yanagi, Measles virus infects both polarized epithelial and immune cells by using distinctive receptor-binding sites on its hemagglutinin, Journal of Virology, 10.1128/JVI.02691-07, 82, 9, 4630-4637, 2008.05, [URL], Measles is one of the most contagious human infectious diseases and remains a major cause of childhood morbidity and mortality worldwide. The signaling lymphocyte activation molecule (SLAM), also called CD150, is a cellular receptor for measles virus (MV), presumably accounting for its tropism for immune cells and its immunosuppressive properties. On the other hand, pathological studies have shown that MV also infects epithelial cells at a later stage of infection, although its mechanism has so far been unknown. In this study, we show that wild-type MV can infect and produce syncytia in human polarized epithelial cell lines independently of SLAM and CD46 (a receptor for the vaccine strains of MV). Progeny viral particles are released exclusively from the apical surface of these polarized epithelial cell lines. We have also identified amino acid residues on the MV attachment protein that are likely to interact with a putative receptor on epithelial cells. All of these residues have aromatic side chains and may form a receptor-binding pocket located in a different position from the putative SLAM- and CD46-binding sites on the MV attachment protein. Thus, our results indicate that MV has an intrinsic ability to infect both polarized epithelial and immune cells by using distinctive receptor-binding sites on the attachment protein corresponding to each of their respective receptors. The ability of MV to infect polarized epithelial cells and its exclusive release from the apical surface may facilitate its efficient transmission via aerosol droplets, resulting in its highly contagious nature.. |
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