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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. |
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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.. |
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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. |
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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. |
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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.. |
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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, 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.. |
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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, 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.. |
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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, 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.. |
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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, 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.. |
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Yuta Shirogane, Shumpei Watanabe, Yusuke Yanagi, Cooperation between different RNA virus genomes produces a new phenotype, Nature Communications, 10.1038/ncomms2252, 3, 2012.12, 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.. |
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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, 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.. |
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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, 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.. |