九州大学 研究者情報
発表一覧
白銀 勇太(しろがね ゆうた) データ更新日:2021.12.21

助教 /  医学研究院 基礎医学部門 病態制御学講座


学会発表等
1. Ryuichi Takemoto, Tateki Suzuki, Takao Hashiguchi, Yusuke Yanagi, Yuta Shirogane, , Short-stalk isoforms of CADM1 and CADM2 induce membrane fusion mediated by hyperfusogenic fusion proteins of neuropathogenic measles viruses, 日本ウイルス学会学術集会, 2021.11, [Objectives]
Measles virus (MeV) may cause a progressive neurological disorder, subacute sclerosing panencephalitis (SSPE). MeV has the hemagglutinin (H) and fusion (F) proteins on its envelope. During viral entry, the H head domain binds to receptors, and then the H stalk domain transmits the fusion-triggering signal to the F. Recently, we have shown that cell adhesion molecule 1 (CADM1) and CADM2 are host factors enabling hyperfusogenic F protein (hF)-mediated membrane fusion by neuropathogenic MeVs. CADM1/2 interact in cis with the H on the same cell membrane, triggering membrane fusion. CADM1/2 exist in multiple splice isoforms containing various lengths of their stalks. This study investigated their differences in fusion-triggering, interaction with the H, and tissue distribution.
[Methods]
293FT cells were transfected with plasmids encoding H [the full length H, H(full), or the H lacking its head domain, H(headless), with or without a tetramerization domain (TD)], the hF, one of CADM1/2 isoforms, and EGFP, and observed under a fluorescence microscope 24-30 hours after transfection. Interactions of the isoforms with H were investigated by coimmunoprecipitation assay (Co-IP). Tissue distributions of the isoforms were obtained from the Genotype-Tissue Expression (GTEx) consortium using the GTEx Portal.
[Results]
Only short-stalk (SS) isoforms, which are predominantly expressed in the brain, induced syncytia, with either the H(full) or the H(headless)-TD. Co-IP showed that the SS isoforms interact with the H(headless), while long-stalk (LS) ones do not.
[Discussion]
These data suggest that structural changes in the CADM1/2 stalks might disrupt the essential site interacting with the H, or change their heights and make their binding sites apart from the corresponding binding site on the H. Notably, SS isoforms can interact in cis with the H(headless) and trigger fusion, independently of the H head domain. Thus, our results unveil a new mechanism of viral fusion triggering..
2. Yuta Shirogane, Ryuichi Takemoto, Tateki Suzuki, Tomonori Kameda, Kinichi Nakashima, Takao Hashiguchi, Yusuke Yanagi, Cell adhesion molecule (CADM) 1 and CADM2 enable measles virus spread in subacute sclerosing panencephalitis by cis-acting fusion triggering, 日本ウイルス学会学術集会, 2021.11, [Objectives]
Measles virus (MeV) may persist in the brain, causing subacute sclerosing panencephalitis (SSPE). MeV has the hemagglutinin (H) and fusion (F) proteins on its envelope. Upon receptor binding, the H triggers the F-mediated membrane fusion. Although MeV spreads in the brain by transsynaptic cell-to-cell fusion, neurons do not express the known receptors, signaling lymphocytic activation molecule family member 1 (SLAM) and nectin-4. We have recently shown that hyperfusogenic changes in the F enable MeV transmission between neurons. The structurally unstable hyperfusogenic F (hF) can be triggered even by the weak interaction between the SLAM/nectin-4-blind mutant H and its corresponding receptor. Here we investigated whether other SLAM/nectin family members, which may have structures closely related to the known receptors and weakly interact with the H, act as neuronal receptors.
[Methods]
Fusion-triggering by the SLAM/nectin family members, including CADM1/2, was evaluated by the dual split protein (DSP) assay as described previously (Shirogane et al., J Virol. 2020 Jan 6;94(2):e01727-19). Whether CADM1/2 act in trans (in cells different from those expressing the H and F) or in cis (in cells also expressing the H and F) was also examined. Interactions of CADM1/2 with H were investigated by coimmunoprecipitation (Co-IP). CADM1/2 knocked-down mouse primary neurons were infected with the recombinant MeV having hF, and the cells were observed under a fluorescence microscope 4 days after infection.
[Results]
The DSP assay and Co-IP showed that CADM1/2 interact in cis, but not in trans, with the H, triggering the hF-mediated membrane fusion. Knockdown of CADM1/2 inhibited neuropathogenic MeV transmission between mouse primary neurons.
[Discussion]
This study unravels the molecular mechanism by which MeV spreads transsynaptically between neurons, thereby causing SSPE. Careful screening may lead to more examples of such “cis-acting fusion triggering” in other viruses..
3. 白銀勇太、橋口隆生、柳雄介, 一般的な受容体の条件に制約されない麻疹ウイルスの低親和性受容体による膜融合の誘導, 日本分子生物学会, 2019.12, 麻疹ウイルスは脂質二重膜に被覆されたウイルス(エンベロープウイルス)の一種であり、エンベロープ上に受容体結合蛋白(H蛋白)と膜融合蛋白(F蛋白)を持つ。麻疹ウイルスの宿主受容体として免疫系細胞に発現するSLAM(CD150)および上皮系細胞に発現するNectin-4が同定されている。受容体はウイルス侵入時にH蛋白に結合し、続いてF蛋白の構造変化が誘導されてエンベロープと細胞膜が融合する。よって受容体はウイルスの接着と膜融合誘導という二つの機能を担う。また感染細胞に発現するH、F蛋白と隣接する細胞に発現する受容体の相互作用により細胞間の膜融合が引き起こされる。
麻疹ウイルスは稀ではあるが中枢神経に持続感染が成立し、シナプスを介して感染を拡大させ、致死性の亜急性硬化性全脳炎の原因となる。脳内環境に適応した麻疹ウイルスはF遺伝子が変異しており既知の受容体を持たない神経細胞で増殖可能となっている。これは変異により低親和性の宿主分子でも受容体として利用可能になるからだと予測されている。
私たちはこの仮説を証明するためH蛋白に既知の受容体(SLAMおよびNectin-4)との親和性を低下させる変異を導入し、低親和性受容体による膜融合誘導のための条件について検討を行った。その結果、麻疹ウイルスのF遺伝子に変異が入ることにより、1.H蛋白と親和性の低い受容体が細胞融合をトリガーできるようになること、2.低親和性受容体はH蛋白との接着能を失うが膜融合の誘導は可能なこと、3.膜融合の誘導はH蛋白と同一細胞に(cisに)受容体が発現しても起こり得ること、の3点を発見した。
神経細胞はシナプスで細胞同士が密に接しており、神経細胞間のウイルス伝搬は受容体による接着が必ずしも求められない。よって麻疹ウイルスは脳内に適応すると一般的な受容体の概念に縛られない増殖スタイルを確立していくと考えられる。本研究はウイルスの中枢神経病原性の分子メカニズムに迫るとともに、ウイルスの新たな進化可能性を提示する。
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4. Yuta Shirogane, Tateki Suzuki, Takao Hashiguchi, Ryuichi Takemoto, Yusuke Yanagi, A hyperfusogenic mutation in the measles virus fusion gene rescues the defect of receptor-blind mutations in the hemagglutinin gene, 日本ウイルス学会学術集会, 2019.10, [Objectives]
Measles virus (MV) is an enveloped RNA virus causing measles. MV has two envelope glycoproteins, the hemagglutinin (H) and fusion (F) protein. Upon binding to a host cell receptor, the H protein triggers the conformational changes of the F protein, resulting in membrane fusion. Two receptors for MV, signaling lymphocytic activation molecule (SLAM) and nectin-4, have been identified. In this study, we investigated whether a defect in one of the three steps of MV-mediated membrane fusion (the H protein-receptor interaction, F protein triggering by the H protein, and conformational changes of the F protein) can be compensated by a change in another step.
[Methods]
293FT cells were transfected with plasmids encoding the H, F, receptor, and EGFP genes to examine their fusogenicity. Plasmids encoding three types of H genes (wild-type H, SLAM-blind H(R533A), and nectin-4-blind H(Y543S)), plasmids encoding two types of F genes (wild-type F and F(T461I)), and plasmids encoding two receptors (SLAM and nectin-4), were used in different combinations.
[Results] Co-expression of wild-type H and wild-type F proteins with SLAM or nectin-4 induced syncytium formation. The H(R533A) and H(Y543S) mutant proteins did not support membrane fusion when expressed with SLAM and nectin-4, respectively. The F (T461I) protein has been shown to be destabilized and hyperfusogenic. When this mutant F protein was used, syncytium formation was clearly observed even in combination with H(R533A) and SLAM, or H(Y543S) and nectin-4. When the receptors were not expressed in cells, any combination of the H and F proteins did not induce syncytium formation.
[Discussion]
These data indicate that a hyperfusogenic mutation in the F protein could allow the H protein to utilize as its receptor a host molecule with a lower binding affinity than the authentic receptor. MVs derived from SSPE patients also have hyperfusogenic F proteins. Presumably, such mutations in the F protein could broaden the range of receptor usage and enable the viruses to spread in the brain tissues lacking known receptors..
5. Yuta Shirogane, Yusuke Yanagi , Combinatorial minigenome assays reveal cooperative effects of RNA-dependent RNA polymerases between different mononegavirus families, 日本ウイルス学会学術集会, 2018.10.

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