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NAKANISHI Akira

    Department of Genetic Engineering　Professor
Last Updated :2024/04/25

Researcher Information

Degree

  • Doctor of Agriculture（Kyoto University）

J-Global ID

Research Interests

  • 感染   カプシド   ウイルス   DDS   遺伝子治療   遺伝子導入   Infection   Capsid   Virus   Gene Therapy   

Research Areas

  • Life sciences / Virology
  • Life sciences / Pharmacology / Blood Brain Barrier
  • Life sciences / Genomics

Academic & Professional Experience

  • 2015/04 - 2019/03  国立長寿医療研究センターラジオアイソトープ管理室室長
  • 2010/04 - 2019/03  国立長寿医療研究センター老化制御研究部 遺伝子治療研究室室長
  • 2005/06 - 2010/03  国立長寿医療センター研究所老化制御研究部 遺伝子治療研究室室長
  • 2003/04 - 2005/06  Tokyo Institute of TechnologyBioscience and Biotechnology, Biological InformationCOE助手
  • 1997/04 - 2003/03  University of California, Los AngelesMolecular Biology Institute and Department of Molecular, Cell, and Developmental BiologyAssistant Researcher
  • 1993/04 - 1997/03  株式会社 明治乳業ヘルスサイエンス研究所研究員
  • 1992/08 - 1993/03  University of California, Los AngelesMolecular Biology Institute and Department of Molecular, Cell, and Developmental BiologyPost Doctoral Fellow
  • 1991/04 - 1993/03  京都大学農学研究科家畜繁殖学教室畜産学専攻家畜繁殖学教室日本学術振興会 特別研究員
  • 1990/04 - 1991/03  京都大学農学研究科畜産学専攻家畜繁殖学教室研修員

Education

  •        - 1990/03  Kyoto University  Graduate School of Agriculture
  •        - 1987/03  Kyoto University  Graduate School of Agriculture
  •        - 1985/03  Kyoto University  Faculty of Agriculture  畜産学科

Association Memberships

  • American Society of Microbiology   American Society for Gene Therapy   American Society of Virology   日本ウイルス学会   日本分子生物学会   

Published Papers

  • Mami Matsuda; Tian-Cheng Li; Akira Nakanishi; Kazuo Nakamichi; Makoto Saito; Tadaki Suzuki; Tomokazu Matsuura; Masamichi Muramatsu; Tetsuro Suzuki; Yoshiharu Miura; Ryosuke Suzuki
    Diagnostics MDPI AG 14 (3) 311 - 311 2024/01 [Refereed]
     
    Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease of the central nervous system (CNS) caused by reactivation of dormant JC polyomavirus (JCPyV). PML was mainly observed in immunocompromised individuals, such as HIV-positive patients, autoimmune disease patients, and cancer patients. Given that the presence of anti-JCPyV antibodies in serum is a risk indicator for PML development, it is essential to monitor anti-JCPyV antibody levels. In the present study, we established reporter-based single-infection neutralization assays for JCPyV and the genetically similar BK polyoma virus (BKPyV). We then confirmed the lack of cross-reactivity between the two viruses using test sera obtained from mice immunized with plasmids encoding the JCPyV or BKPyV capsid. Next, we compared neutralization antibody titers in sera from healthy donors, patients with multiple sclerosis (MS), and HIV-positive patients using an in-house enzyme-linked immunosorbent assay (ELISA) with JCPyV-like particles (virus-like particles; VLPs). A positive correlation was demonstrated between the neutralization titer (75% infectious concentration; IC75) against JCPyV and the antibody titer obtained by VLP-based JCPyV ELISA. This assay system may be applied to detect antibodies against other PyVs by generation of pseudoviruses using the respective capsid expression plasmids, and is expected to contribute to the surveillance of PyV as well as basic research on these viruses.
  • Ryoka Ishiyama; Kazuhiro Yoshida; Kazuki Oikawa; Reiko Takai-Todaka; Akiko Kato; Kumiko Kanamori; Akira Nakanishi; Kei Haga; Kazuhiko Katayama
    Journal of Virology American Society for Microbiology 0022-538X 2024/01 [Refereed]
     
    ABSTRACT Human norovirus (HuNoV) causes gastroenteritis, a disease with no effective therapy or vaccine, and does not grow well in culture. Murine norovirus (MNV) easily replicates in cell cultures and small animals and has often been used as a model to elucidate the structural and functional characteristics of HuNoV. An MNV plasmid-based reverse genetics system was developed to produce the modified recombinant virus. In this study, we attempted to construct the recombinant virus by integrating a foreign gene into MNV ORF3, which encodes the minor structural protein VP2. Deletion of VP2 expression abolished infectious particles from MNV cDNA clones, and supplying exogenous VP2 to the cells rescued the infectivity of cDNA clones without VP2 expression. In addition, the coding sequence of C-terminal ORF3 was essential for cDNA clones compensated with VP2 to produce infectious particles. Furthermore, the recombinant virus with exogenous reporter genes in place of the dispensable region of ORF3 was propagated when VP2 was constitutively supplied. Our findings indicate that foreign genes can be transduced into the norovirus ORF3 region when VP2 is supplied and that successive propagation of modified recombinant norovirus could lead to the development of norovirus-based vaccines or therapeutics. IMPORTANCE In this study, we revealed that some of the coding regions of ORF3 could be replaced by a foreign gene and infectious virus could be produced when VP2 was supplied. Propagation of this virus depended on VP2 being supplied in trans , indicating that this virus could infect only once. Our findings help to elucidate the functions of VP2 in the virus lifecycle and to develop other caliciviral vectors for recombinant attenuated live enteric virus vaccines or therapeutics tools.
  • Kei Haga; Reiko Takai-Todaka; Akiko Kato; Akira Nakanishi; Kazuhiko Katayama
    BioRxiv Cold Spring Harbor Laboratory 2022/11 
    Summary Human astrovirus (HAstV) is a global cause of gastroenteritis in infants, the elderly, and immunocompromised people. However, its infection mechanism is not fully understood, with its functional receptor not yet discovered. Here, we identify neonatal Fc receptor (FcRn) as a functional receptor for HAstV (mamastrovirus 1) using genome-wide CRISPR-Cas9 library screening in Caco2 cells. Deletion ofFCGRTorB2M, which encode subunits of FcRn, rendered Caco2 cells and intestinal organoid cells unsusceptible to HAstV. We also show that human FcRn expression renders non-permissive MDCK cells susceptible and that FcRn directly binds HAstV spike protein. Thus, our findings provide insight into the entry mechanism of HAstV.
  • Isobe T; Tange S; Tasaki H; Kanamori K; Kato A; Nakanishi A
    Journal of General Virology Microbiology Society 100 (5) 778 - 792 0022-1317 2019/05 [Refereed]
  • 片山 和彦; 芳賀 慧; 藤本 陽; 戸高 玲子; 村上 耕介; 村田 和義; 中西 章
    感染制御と予防衛生 (株)メディカルレビュー社 1 (1) 4 - 11 2433-4030 2017/09 [Invited]
  • Takuya Kitamoto; Reiko Takai-Todaka; Akiko Kato; Kumiko Kanamori; Hirotaka Takagi; Kazuhiro Yoshida; Kazuhiko Katayama; Akira Nakanishi
    FRONTIERS IN MICROBIOLOGY FRONTIERS MEDIA SA 8 1091  1664-302X 2017/06 [Refereed]
     
    Laboratory adaptation of viruses is an essential technique for basic virology research, including the generation of attenuated vaccine strains, although the principles of cell adaptation remain largely unknown. Deep sequencing of murine norovirus (MuNoV) S7 during serial passages in RAW264.7 cells showed that the frequencies of viral variants were altered more dynamically than previously reported. Serial passages of the virus following two different multiplicity of infections gave rise to distinct haplotypes, implying that multiple cell-adaptable sequences were present in the founder population. Nucleotide variants lost during passage were assembled into a viral genome representative of that prior to cell adaptation, which was unable to generate viral particles upon infection in cultured cells. In addition, presence of the reconstructed genome interfered with production of infectious particles from viruses that were fully adapted to in vitro culture. Although the key nucleotide changes dictating cell adaptation of MuNoV S7 viral infection are yet to be elucidated, our results revealed the elaborate interplay among selected sequences of viral variants better adapted to propagation in cell culture. Such knowledge will be instrumental in understanding the processes necessary for the laboratory adaptation of viruses, especially to those without relevant cell culture systems.
  • Mizutani T; Sayama Y; Nakanishi A; Ochiai H; Sakai K; Wakabayashi K; Tanaka N; Miura E; Oba M; Kurane I; Saijo M; Morikawa S; Ono SI
    Virology Elsevier BV 499 399 - 400 0042-6822 2016/12 [Refereed]
  • Kei Haga; Akira Fujimoto; Reiko Takai-Todaka; Motohiro Miki; Yen Hai Doan; Kosuke Murakami; Masaru Yokoyama; Kazuyoshi Murata; Akira Nakanishi; Kazuhiko Katayama
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA NATL ACAD SCIENCES 113 (41) E6248 - E6255 0027-8424 2016/10 [Refereed]
     
    Norovirus is the leading cause of acute gastroenteritis worldwide. Since the discovery of human norovirus (HuNoV), an efficient and reproducible norovirus replication system has not been established in cultured cells. Although limited amounts of virus particles can be produced when the HuNoV genome is directly transfected into cells, the HuNoV cycle of infection has not been successfully reproduced in any currently available cell-culture system. Those results imply that the identification of a functional cell-surface receptor for norovirus might be the key to establishing a norovirus culture system. Using a genome-wide CRISPR/Cas9 guide RNA library, we identified murine CD300lf and CD300ld as functional receptors for murine norovirus (MNV). The treatment of susceptible cells with polyclonal antibody against CD300lf significantly reduced the production of viral progeny. Additionally, ectopic CD300lf expression in nonsusceptible cell lines derived from other animal species enabled MNV infection and progeny production, suggesting that CD300lf has potential for dictating MNV host tropism. Furthermore, CD300ld, which has an amino acid sequence in the N-terminal region of its extracellular domain that is highly homologous to that of CD300lf, also functions as a receptor for MNV. Our results indicate that direct interaction of MNV with two cell-surface molecules, CD300lf and CD300ld, dictates permissive noroviral infection.
  • Benoit Chapellier; Shoichiro Tange; Hidetaka Tasaki; Kazuhiro Yoshida; Yan Zhou; Naomi Sakon; Kazuhiko Katayama; Akira Nakanishi
    MICROBIOLOGY AND IMMUNOLOGY WILEY-BLACKWELL 59 (10) 586 - 596 0385-5600 2015/10 [Refereed]
     
    A plasmid-based reverse genetics system for human astrovirus type 1 (HAstV1) is examined. Upon transfection into 293T cells, the plasmid vector, which harbors a HAstV1 expression cassette, expressed astroviral RNA that appeared to be capable of viral RNA replication, as indicated by the production of subgenomic RNA and capsid protein expression irrespective of the heterologous 5 ends of the transcribed RNA. Particles infectious to Caco-2 cells were made in this system; however, their infectivity was much lower than would be expected from the amount of particles apparently produced. Using Huh-7 cells as the transfection host with the aim of improving viral capsid processing for virion maturation partially restored the efficiency of infectious particle formation. Our results support the possibility that the DNA transfection process induces a cellular response that targets late, but not early, stages of HAstV1 infection.
  • Kazuhiko Katayama; Kosuke Murakami; Tyler M. Sharp; Susana Guix; Tomoichiro Oka; Reiko Takai-Todaka; Akira Nakanishi; Sue E. Crawford; Robert L. Atmar; Mary K. Estes
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA NATL ACAD SCIENCES 111 (38) E4043 - E4052 0027-8424 2014/09 [Refereed]
     
    Human norovirus (HuNoV) is the leading cause of gastroenteritis worldwide. HuNoV replication studies have been hampered by the inability to grow the virus in cultured cells. The HuNoV genome is a positive-sense single-stranded RNA (ssRNA) molecule with three open reading frames (ORFs). We established a reverse genetics system driven by a mammalian promoter that functions without helper virus. The complete genome of the HuNoV genogroup II.3 U201 strain was cloned downstream of an elongation factor-1 alpha (EF-1 alpha) mammalian promoter. Cells transfected with plasmid containing the full-length genome (pHuNoV(U201F)) expressed the ORF1 polyprotein, which was cleaved by the viral protease to produce the mature nonstructural viral proteins, and the capsid proteins. Progeny virus produced from the transfected cells contained the complete NoV genomic RNA (VP1, VP2, and VPg) and exhibited the same density in isopycnic cesium chloride gradients as native infectious NoV particles from a patient's stool. This system also was applied to drive murine NoV RNA replication and produced infectious progeny virions. A GFP reporter construct containing the GFP gene in ORF1 produced complete virions that contain VPg-linked RNA. RNA from virions containing the encapsidated GFP-genomic RNA was successfully transfected back into cells producing fluorescent puncta, indicating that the encapsidated RNA is replication-competent. The EF-1 alpha mammalian promoter expression system provides the first reverse genetics system, to our knowledge, generalizable for human and animal NoVs that does not require a helper virus. Establishing a complete reverse genetics system expressed from cDNA for HuNoVs now allows the manipulation of the viral genome and production of reporter virions.
  • Shoichiro Tange; Yan Zhou; Yuko Nagakui-Noguchi; Takeshi Imai; Akira Nakanishi
    VIROLOGY JOURNAL BIOMED CENTRAL LTD 10 153  1743-422X 2013/05 [Refereed]
     
    Background: Upon initial contact with a virus, host cells activate a series of cellular signaling cascades that facilitate viral entry and viral propagation within the cell. Little is known about how the human astrovirus (HAstV) exploits signaling cascades to establish an infection in host cells. Recent studies showed that activation of extracellular signal-regulated kinase 1/2 (ERK1/2) is important for HAstV infection, though the involvement of other signaling cascades remains unclear. Methods: A panel of kinase blockers was used to search for cellular signaling pathways important for HAstV1 infection. To determine their impact on the infectious process, we examined viral gene expression, RNA replication, and viral RNA and capsid protein release from host cells. Results: Inhibitors of phosphoinositide 3-kinase (PI3K) activation interfered with the infection, independent of their effect on ERK 1/2 activation. Activation of the PI3K signaling cascade occurred at an early phase of the infection, judging from the timeframe of Akt phosphorylation. PI3K inhibition at early times, but not at later times, blocked viral gene expression. However, inhibiting the downstream targets of PI3K activation, Akt and Rac1, did not block infection. Inhibition of protein kinase A (PKA) activation was found to block a later phase of HAstV1 production. Conclusions: Our results reveal a previously unknown, essential role of PI3K in the life cycle of HAstV1. PI3K participates in the early stage of infection, possibly during the viral entry process. Our results also reveal the role of PKA in viral production.
  • Marika Watanabe; Ellen Phamduong; Chu-Han Huang; Noriko Itoh; Janie Bernal; Akira Nakanishi; Kathleen Rundell; Ole Gjoerup; Harumi Kasamatsu
    JOURNAL OF VIROLOGY AMER SOC MICROBIOLOGY 87 (9) 5053 - 5064 0022-538X 2013/05 [Refereed]
     
    The folding and pentamer assembly of the simian virus 40 (SV40) major capsid protein Vp1, which take place in the infected cytoplasm, have been shown to progress through disulfide-bonded Vp1 folding intermediates. In this report, we further demonstrate the existence of another category of Vp1 folding or assembly intermediates: the nonreducible, covalently modified mdVp1s. These species were present in COS-7 cells that expressed a recombinant SV40 Vp1, Vp1 Delta C, through plasmid transfection. The mdVp1s persisted under cell and lysate treatment and SDS-PAGE conditions that are expected to have suppressed the formation of artifactual disulfide cross-links. As shown through a pulse-chase analysis, the mdVp1s were derived from the newly synthesized Vp1 Delta C in the same time frame as Vp1's folding and oligomerization. The apparent covalent modifications occurred in the cytoplasm within the core region of Vp1 and depended on the coexpression of the SV40 large T antigen (LT) in the cells. Analogous covalently modified species were found with the expression of recombinant polyomavirus Vp1s and human papillomavirus L1s in COS-7 cells. Furthermore, the mdVp1s formed multiprotein complexes with LT, Hsp70, and Hsp40, and a fraction of the largest mdVp1, md4, was disulfide linked to the unmodified Vp1 Delta C. Both mdVp1 formation and most of the multiprotein complex formation were blocked by a Vp1 folding mutation, C87A-C254A. Our observations are consistent with a role for LT in facilitating the folding process of SV40 Vp1 by stimulating certain covalent modifications of Vp1 or by recruiting certain cellular proteins.
  • Roberta Antonia Diotti; Akira Nakanishi; Nicola Clementi; Nicasio Mancini; Elena Criscuolo; Laura Solforosi; Massimo Clementi
    CLINICAL & DEVELOPMENTAL IMMUNOLOGY HINDAWI PUBLISHING CORPORATION 2013 1740-2522 2013 [Refereed]
     
    Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease of the central nervous system (CNS), observed in immunodeficient patients and caused by JC virus ((JCV), also called JC polyomavirus (JCPyV)). After the HIV pandemic and the introduction of immunomodulatory therapy, the PML incidence significantly increased. The correlation between the use of natalizumab, a drug used in multiple sclerosis (MS), and the PML development of particular relevance. The high incidence of PML in natalizumab-treated patients has highlighted the importance of two factors: the need of PML risk stratification among natalizumab-treated patients and the need of effective therapeutic options. In this review, we discuss these two needs under the light of the major viral models of PML etiopathogenesis.
  • Shoichiro Tange; Takeshi Imai; Akira Nakanishi
    VIRUS RESEARCH ELSEVIER SCIENCE BV 157 (1) 116 - 120 0168-1702 2011/04 [Refereed]
     
    On reexamination of temperature-sensitive D-type (tsD) mutants of simian virus 40 (SV40), we found that the tsD222 mutant is identical to the VP2 M228I mutant, which is defective in VP4 expression, at the nucleotide level. Although a previous study reported that lack of VP4 caused defects in viral dissemination in BSC-1 cells, this mutant showed a temperature-sensitive growth defect in CV-1 cells. tsD101:VP3 Q113K and tsD202:VP3 P108S exhibited a growth phenotype similar to that of tsD222, and they retained the VP4 open reading frame (ORF). These three mutants did not complement each other, suggesting that their defects were functionally indistinguishable. Transduction of the SV40 vector expressing wild-type VP4 in tsD222-infected cells did not ameliorate the growth defect at the non-permissive temperature. The results indicate that tsD mutation in minor capsid proteins has a more profound impact on viral propagation, and that lack of VP4 ORF seems to have little influence on viral growth. (C) 2011 Elsevier B.V. All rights reserved.
  • Tetsuya Mizutani; Yusuke Sayama; Akira Nakanishi; Hideharu Ochiai; Kouji Sakai; Kouji Wakabayashi; Nozomi Tanaka; Emi Miura; Mami Oba; Ichiro Kurane; Masayuki Saijo; Shigeru Morikawa; Shin-ichi Ono
    VIROLOGY ACADEMIC PRESS INC ELSEVIER SCIENCE 412 (1) 179 - 187 0042-6822 2011/03 [Refereed]
     
    Economic loss due to viral endothelial cell necrosis of eel (VECNE) of Anguilla japonica is a serious problem for the cultured Japanese eel market. However, the viral genome responsible for VECNE is unknown. We recently developed a rapid determination system for viral nucleic acid sequences (RDV) to determine viral genome sequences. In this study, viral DNA fragments were obtained using RDV, and approximately 15-kbp circular full genome sequences were determined using a next-generation sequencing system, overlapping PCR, and Southern blot analysis. One open reading frame (ORF) was homologous to the large T-antigen of polyomavirus; other ORFs have no homology with any nucleic or amino acid sequences of polyomavirus. Therefore, as this DNA virus might comprise a novel virus family, we provisionally named it Japanese eel endothelial cells-infecting virus (JEECV). JEECV was detected in both naturally and experimentally infected eels, suggesting that JEECV potentially causes VECNE. (c) 2011 Elsevier Inc. All rights reserved.
  • Benoit Chapellier; Naoya Maekawa; Masaki Hiramoto; Takeshi Imai; Akira Nakanishi
    JOURNAL OF GENE MEDICINE JOHN WILEY & SONS LTD 11 (12) 1156 - 1156 1099-498X 2009/12
  • Akira Nakanishi; Benoit Chapellier; Naoya Maekawa; Masaki Hiramoto; Takeshi Kuge; Ryo-u Takahashi; Hiroshi Handa; Takeshi Imai
    Virology ACADEMIC PRESS INC ELSEVIER SCIENCE 379 (1) 110 - 7 0042-6822 2008/09 [Refereed]
     
    Polyomaviral vectors are generated by transfecting 293T cells with three sets of DNAs: DNA for the expression of simian virus 40 (SV40) T antigen; DNA for the expression of SV40 capsid proteins, and vector DNA harboring a reporter gene expression cassette carrying a SV40 origin. The vector DNA harbors a minimal sequence originating from SV40, and thus can carry a longer transgene. Moreover, the viable recombinants are not detectable in the vector preparation, and the vectors can transduce the DNA with efficiency similar to that of virions. Vector particles bearing capsid proteins of BK virus, JC virus, and B-lymphotropic papovavirus instead of SV40 were prepared, and they exhibited differential efficiency of gene transduction to the target cells. This method can be used to develop a surrogate system to study the functions of capsid proteins of polyomaviruses and to generate a set of polyomaviral vectors targeted at specific cell types.
  • Akira Nakanishi; Noriko Itoh; Peggy P Li; Hiroshi Handa; Robert C Liddington; Harumi Kasamatsu
    Journal of virology AMER SOC MICROBIOLOGY 81 (8) 3778 - 85 0022-538X 2007/04 [Refereed]
     
    We investigated the roles of simian virus 40 capsid proteins in the viral life cycle by analyzing point mutants in Vp1 and Vp2/3, as well as a deletion mutant lacking the Vp2/3 coding sequence. The Vp1 mutants (V243E and L245E) and the Vp2/3 mutants (F157E-I158E and P164R-G165E-G166R) were previously shown to be defective in Vp1-Vp2/3 interaction and to be noninfectious or poorly infectious, respectively. Here, we show that all these point mutants form stable particles following DNA transfection into cells. The Vp2/3-mutant particles contained very low levels of Vp2/3, whereas the Vp1 mutant particles contained no detectable Vp2/3. As expected, the deletion mutant also formed particles that were noninfectious. We further characterized the two Vp1 point mutants and the deletion mutant. All three mutant particles comprised Vp1 and histone-associated viral DNA, and all were able to enter cells. However, the mutant complexes failed to associate with host importins (owing to the loss of the Vp2/3 nuclear localization signal), and the mutant viral DNAs prematurely dissociated from the Vp1s, suggesting that the nucleocapsids did not enter the nucleus. Consistently, all three mutant particles failed to express large T antigen. Together, our results demonstrate unequivocally that Vp2/3 is dispensable for the formation of nucleocapsids. Further, the nucleocapsids' ability to enter cells implies that Vp1 contains the major determinants for cell attachment and entry. We propose that the major role of Vp2/3 in infectivity is to mediate the nuclear entry of viral DNA.
  • Akira Nakanishi; Peggy P Li; Qiumin Qu; Qumber H Jafri; Harumi Kasamatsu
    Virus research ELSEVIER SCIENCE BV 124 (1-2) 226 - 30 0168-1702 2007/03 [Refereed]
     
    To establish viral infection, SV40 must expose nuclear localization signals (NLSs) that are internal in the virion architecture in order to enter the nucleus via interaction with the host's nuclear import machinery, which includes importin alpha and importin beta. The time course for SV40 association with the importins in infected cells was examined. The viral DNA associated with importin alpha by 1.5h post infection, before associating with the importin beta nuclear import receptor, by 3h post infection. Only a small fraction of cell-internalized SV40 that contained viral DNA was bound by the two importins. This fraction, termed "nuclear entry-competent SV40," was slightly smaller than the virion but, importantly, was larger than the viral chromatin and contained both Vp1 and Vp3. Furthermore, the internalized viral DNA in either anti-importin or anti-Vp3 immune complexes was sensitive to DNase I, whereas the viral DNA in mature virions was resistant. All these results suggest that once SV40 enters the cytoplasm, it undergoes an architectural modification that exposes the virion's NLSs for nuclear entry.
  • Naoki Yokoyama; Masa-aki Kawano; Hiroko Tsukamoto; Teruya Enomoto; Takamasa Inoue; Ryou-u Takahashi; Akira Nakanishi; Takeshi Imai; Tadashi Wada; Hiroshi Handa
    Journal of biochemistry JAPANESE BIOCHEMICAL SOC 141 (2) 279 - 86 0021-924X 2007/02 [Refereed]
     
    Virus-like particles (VLPs), a promising next-generation drug delivery vehicle, can be formed in vitro using a recombinant viral capsid protein VP1 from SV40. Seventy-two VP1 pentamers interconnect to form the T = 7d lattice of SV40 capsids, through three types of C-terminal interactions, alpha-alpha'-alpha'', beta-beta' and gamma-gamma. These appear to require VP1 conformational switch, which involve in particular the region from amino acids 301-312 (herein Region I). Here we show that progressive deletions from the C-terminus of VP1, up to 34 amino acids, cause size and shape variations in the resulting VLPs, including tubular formation, whereas deletions beyond 34 amino acids simply blocked VP1 self-assembly. Mutants carrying in Region I point mutations predicted to disrupt alpha-alpha'-alpha''-type and/or beta-beta'-type interactions formed small VLPs resembling T = 1 symmetry. Chimeric VP1, in which Region I of SV40 VP1 was substituted with the homologous region from VP1 of other polyomaviruses, assembled only into small VLPs. Together, our results show the importance of the integrity of VP1 C-terminal region and the specific amino acid sequences within Region I in the assembly of normal VLPs. By understanding how to alter VLP sizes and shapes contributes to the development of drug delivery systems using VLPs.
  • Akira Nakanishi; Akiko Nakamura; Robert Liddington; Harumi Kasamatsu
    Journal of virology AMER SOC MICROBIOLOGY 80 (18) 8891 - 8 0022-538X 2006/09 [Refereed]
     
    Interaction of simian virus 40 (SV40) major capsid protein Vp1 with the minor capsid proteins Vp2 and Vp3 is an integral aspect of the SV40 architecture. Two Vp3 sequence elements mediate Vp1 pentamer binding in vitro, Vp3 residues 155 to 190, or D1, and Vp3 residues 222 to 234, or D2. Of the two, D1 but not D2 was necessary and sufficient to direct the interaction with Vp1 in vivo. Rational mutagenesis of Vp3 residues (Phe157, Ile158, Pro164, Gly165, Gly166, Leu177, and Leu181) or Vp1 residues (Val243 and Leu245), based on a structural model of the SV40 Vp1 pentamer complexed with Vp3 D1, was carried out to disrupt the interaction between Vp1 and Vp3 and to study the consequences of these mutations for viral viability. Altering these residues to bulky, charged residues blocked the interaction in vitro. When these alterations were introduced into the viral genome, they reduced viral viability. Mutants with alterations in Vp1 Val243, Leu245, or both to glutamate were nearly nonviable, whereas those with Vp3 alterations reduced, but did not eliminate, viability. Our results defined the residues of Vp1 and the minor capsid proteins that are essential for both the interaction of the capsid proteins and viral viability in permissive cells.
  • Masa-aki Kawano; Takamasa Inoue; Hiroko Tsukamoto; Tatsuya Takaya; Teruya Enomoto; Ryou-u Takahashi; Naoki Yokoyama; Noriaki Yamamoto; Akira Nakanishi; Takeshi Imai; Tadashi Wada; Kohsuke Kataoka; Hiroshi Handa
    The Journal of biological chemistry AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC 281 (15) 10164 - 73 0021-9258 2006/04 [Refereed]
     
    The SV40 capsid is composed primarily of 72 pentamers of the VP1 major capsid protein. Although the capsid also contains the minor capsid protein VP2 and its amino-terminally truncated form VP3, their roles in capsid assembly remain unknown. An in vitro assembly system was used to investigate the role of VP2 in the assembly of recombinant VP1 pentamers. Under physiological salt and pH conditions, VP1 alone remained dissociated, and at pH 5.0, it assembled into tubular structures. A stoichiometric amount of VP2 allowed the assembly of VP1 pentamers into spherical particles in a pH range of 7.0 to 4.0. Electron microscopy observation, sucrose gradient sedimentation analysis, and antibody accessibility tests showed that VP2 is incorporated into VP1 particles. The functional domains of VP2 important for VP1 binding and for enhancing VP1 assembly were further explored with a series of VP2 deletion mutants. VP3 also enhanced VP1 assembly, and a region common to VP2 and VP3 (amino acids 119-272) was required to promote VP1 pentamer assembly. These results are relevant for controlling recombinant capsid formation in vitro, which is potentially useful for the in vitro development of SV40 virus vectors.
  • Peggy P Li; Akira Nakanishi; Vanessa Fontanes; Harumi Kasamatsu
    Journal of virology AMER SOC MICROBIOLOGY 79 (6) 3859 - 64 0022-538X 2005/03 [Refereed]
     
    Transient disulfide bonding occurs during the intracellular folding and pentamerization of simian virus 40 (SV40) major capsid protein Vp1 (P. P. Li, A. Nakanishi, S. W. Clark, and H. Kasamatsu, Proc. Natl. Acad. Sci. USA 99:1353-1358, 2002). We investigated the requirement for Vp1 cysteine pairs during SV40 infection. Our analysis identified three Vp1 double-cysteine mutant combinations that abolished viability as assayed by plaque formation. Mutating the Cys49-Cys87 pair or the Cys87-Cys254 pair led to ineffective nuclear localization and diminished accumulation of the mutant Vp1s, and the defect extended in a dominant-negative manner to the wild-type minor capsid proteins Vp2/3 and an affinity-tagged recombinant Vp1 expressed in the same cells. Mutating the Cys87-Cys207 pair preserved the nuclear localization and normal accumulation of the capsid proteins but diminished the production of virus-like particles. Our results are consistent with a role for Cys49, Cys87, and Cys254 in the folding and cytoplasmic-nuclear trafficking of Vp1 and with a role for Cys87 and Cys207 in the assembly of infectious particles. These findings suggest that transient disulfide bond formation between certain Vp1 cysteine residues functions at two stages of SV40 infection: during Vp1 folding and oligomerization in the cytoplasm and during virion assembly in the nucleus.
  • Song-Iee Han; Masa-Aki Kawano; Ken-Ichiro Ishizu; Hajime Watanabe; Makoto Hasegawa; Shin-Nosuke Kanesashi; Yang-Su Kim; Akira Nakanishi; Kohsuke Kataoka; Hiroshi Handa
    Virology ACADEMIC PRESS INC ELSEVIER SCIENCE 320 (1) 144 - 55 0042-6822 2004/03 [Refereed]
     
    Rep78/68 proteins of adeno-associated virus type 2 (AAV-2) are involved in many aspects of the viral life cycle, including replication, gene expression, and site-specific integration. To understand the molecular mechanisms of the actions of Rep proteins, we searched for Rep68-interacting cellular proteins by utilizing a one-step affinity purification technique and identified two members of 14-3-3 proteins (14-3-3 epsilon and gamma). We found that phosphorylation of 535Ser at the carboxy terminus of Rep68 was critical for its association with 14-3-3. The association of 14-3-3 proteins to Rep68 resulted in reduction of the affinity of Rep68 for DNA. Furthermore, genome DNA replication of a recombinant mutant virus carrying a phosphorylation-deficient Rep68 (Ser535Ala) was more efficient than that of the wild-type virus. These results suggest that phosphorylation of Rep68 and subsequent association with 14-3-3 proteins regulates Rep-mediated functions during the AAV life cycle.
  • PP Li; A Naknanishi; MA Tran; KI Ishizu; M Kawano; M Phillips; H Handa; RC Liddington; H Kasamatsu
    JOURNAL OF VIROLOGY AMER SOC MICROBIOLOGY 77 (13) 7527 - 7538 0022-538X 2003/07 [Refereed]
     
    For polyomaviruses, calcium ions are known to be essential for virion integrity and for the assembly of capsid structures. To define the role of calcium ions in the life cycle of the virus, we analyzed simian virus 40 (SV40) mutants in which structurally deduced calcium-binding amino acids of Vp1 were mutated singly and in combination. Our study provides evidence that calcium ions mediate not only virion assembly but also the initial infection processes of cell entry and nuclear entry. Mutations at Glu48, Glu157, Glu160, Glu216, and/or Glu330 are correlated with different extents of packaging defects. The low packaging ability of mutant E216R suggests the need to position the Glu216 side chain for proper virion formation. All other mutants selected for further analysis produced virus-like particles (VLPs) but were poorly infectious. The VLPs of mutant E330K could not attach to or enter the cell, and mutant E157A-E160A and E216K VLPs entered the cell but failed to enter the nucleus, apparently as a result of premature VLP dissociation. Our results show that five of the seven acidic side chains at the two calcium-binding sites-Glu48 and Glu330 (site 1), Glu157 and Glu160 (site 2), and Glu216 (both sites)-are important for SV40 infection. We propose that calcium coordination imparts not only stability but also structural flexibility to the virion, allowing the acquisition or loss of the ion at the two sites to control virion formation in the nucleus, as well as virion structural alterations at the cell surface and in the cytoplasm early during infection.
  • 中西 章
    蛋白質・核酸・酵素 共立出版 48 (5) 621 - 630 0039-9450 2003 [Invited]
  • Akira Nakanishi; Dorothy Shum; Hiroshi Morioka; Eiko Otsuka; Harumi Kasamatsu
    Journal of virology AMER SOC MICROBIOLOGY 76 (18) 9368 - 77 0022-538X 2002/09 [Refereed]
     
    For nuclear entry of large nucleoprotein complexes, it is thought that one key nuclear localization signal (NLS) of a protein component becomes exposed to mediate importin recognition. We show that the nuclear entry of simian virus 40 involves a dynamic interplay between two distinct interiorly situated capsid NLSs, the Vp1 NLS and the Vp3 NLS, and the selective exposure and importin recognition of the Vp3 NLS. The Vp3 NLS-null mutants assembled normally into virion-like particles (VLP) in mutant DNA-transfected cells. When used to infect a new host, the null VLP entered the cell normally but was impaired in viral DNA nuclear entry due to a lack of recognition by the importin alpha 2/beta heterodimer, leading to reduced viability. Both Vp3 and Vp1 NLSs directed importin interaction in vitro, but the Vp1 NLS, which overlaps the Vp1 DNA binding domain, did not bind importins in the presence of DNA. The results suggest that certain canonical NLSs within a nucleoprotein complex, such as the Vp1 NLS, can be masked from functioning by binding to the nucleic acid component and that the availability of an NLS that is not masked and can become exposed for importin binding, such as the Vp3 NLS, is a general feature of the nuclear entry of the nucleoprotein complexes, including those of other animal viruses.
  • Peggy P Li; Akira Nakanishi; Sean W Clark; Harumi Kasamatsu
    Proceedings of the National Academy of Sciences of the United States of America NATL ACAD SCIENCES 99 (3) 1353 - 8 0027-8424 2002/02 [Refereed]
     
    Pentamer formation by Vp1, the major capsid protein of simian virus 40, requires an interdigitation of structural elements from the Vp1 monomers [Liddington, R. C., Yan, Y., Moulai, J., Sahli, R., Benjamin, T. L. & Harrison, S. C. (1991) Nature (London) 354, 278-284]. Our analyses reveal that disulfide-linked Vp1 homooligomers are present in the simian virus 40-infected cytoplasm and that they are derived from a 41-kDa monomeric intermediate containing an intrachain disulfide bond(s). The 41-kDa species, emerging within 5 min of pulse labeling with [(35)S]methionine, is converted into a 45-kDa, disulfide-free Vp1 monomer and disulfide-bonded dimers through pentamers. The covalent oligomer formation is blocked in the presence of a sulfhydryl-modifying reagent. We propose that there are two stages in this Vp1 disulfide bonding. First, the newly synthesized Vp1 monomers acquire intrachain bonds as they fold and begin to interact. Next, these bonds are replaced with intermolecular bonds as the monomers assemble into pentamers. This sequential appearance of transitory disulfide bonds is consistent with a role for sulfhydryl-disulfide redox reactions in the coordinate folding of Vp1 chains into pentamers. The cytoplasmic Vp1 does not colocalize with marker proteins of the endoplasmic reticulum. This paper demonstrates in vivo disulfide formations and exchanges coupled to the folding and oligomerization of a mammalian protein in the cytoplasm, outside the secretory pathway. Such disulfide dynamics may be a general phenomenon for other cysteine-bearing mammalian proteins that fold in the cytoplasm.
  • PP Li; A Nakanishi; D Shum; PCK Sun; AM Salazar; CF Fernandez; SW Chan; H Kasamatsu
    JOURNAL OF VIROLOGY AMER SOC MICROBIOLOGY 75 (16) 7321 - 7329 0022-538X 2001/08 [Refereed]
     
    A DNA-binding domain (DBD) was identified on simian virus 40 (SV40) major capsid protein Vp1, and the domain's function in the SV40 life cycle was examined. The DBD was mapped by assaying various recombinant Vp1 proteins for DNA binding in vitro. The carboxy-terminal 58-residue truncated Vp1 Delta C58 pentamer bound DNA,with a K-d of 1.8 X 10(-9) M in terms of the protein pentamer, while full-length Vp1 and carboxy-terminal-17-truncated Vp1 Delta C17 had comparable apparent K(d)s of 5.3 X 10(-9) to 7.3 X 10(-9) M in terms of the protein monomers. Previously identified on Vp1 was a nuclear localization signal (NLS) consisting of two N-terminal basic clusters, NLS1 (4-KRK-6) and NLS2 (15-KKPK-18). Vp1 Delta C58 pentamers harboring multiple-point mutations in NLS1 (NLSm1), NLS2 (NLSm2), or both basic clusters (NLSm1 . 2) had progressively decreased DNA-binding activity, down to 0.7% of the Vp1 Delta C58 level for NLSm1 . 2 Vp1. These data, along with those of N-terminally truncated proteins, placed the DBD in overlap with the bipartite NLS. The role of the Vp1 DBD during infection was investigated by taking advantage of NLS phenotypic complementation (N. Ishii, A. Nakanishi, M. Yamada, M. H. Macalalad, and H. Kasamatsu, J. Virol. 68:8209-8216, 1994), in which an NLS-defective Vp1 could localize to the nucleus in the presence of wild-type minor capsid proteins Vp2 and Vp3. This approach made it possible to dissect the role of the bifunctional Vp1 NLS-DBD in virion assembly in the nucleus. Mutants of the viable nonoverlaping SV40 (NO-SV40) DNA NLSm1, NLSm2, and NLSm1 - 2 replicated normally following transfection into host cells and produced capsid proteins at normal levels. All mutant Vp1s were able to interact with Vp3 in vitro. The mutants NLSm1 and NLSm1 - 2 were nonviable, and the mutant Vp1s unexpectedly failed to localize to the nucleus though Vp2 and Vp3 did, suggesting that the mutated NLS1 acted as a dominant signal for the cytoplasmic localization of Vp1. Mutant NLSm2, for which the mutant Vp1's nuclear localization defect was complemented by Vp2 and Vp3, displayed a 5,000-fold reduced viability. Analysis of NLSm2 DNA-transfected cell lysate revealed a 10-fold reduction in the level of DNase I-protected viral DNA, and yet virion-like particles were found among the DNase I-resistant material. Collective results support a role for Vp1 NLS2-DBD2 in the assembly of virion particles. The results also suggest that this determinant can function in the infection of new cells.
  • PP Li; A Nakanishi; MA Tran; AM Salazar; RC Liddington; H Kasamatsu
    JOURNAL OF VIROLOGY AMER SOC MICROBIOLOGY 74 (23) 11388 - 11393 0022-538X 2000/12 [Refereed]
     
    We have developed a new nonoverlapping infectious viral genome (NO-SV40) in order to facilitate structure-based analysis of the simian virus 40 (SV40) life cycle. We first tested the role of cysteine residues in the formation of infectious virions by individually mutating the seven cysteines in the major capsid protein, Vp1. All seven cysteine mutants-C9A, C49A, C87A, C104A, C207S, C254A, and C267L-retained viability. In the crystal structure of SV40, disulfide bridges are formed between certain Cys104 residues on neighboring pentamers. However, our results show that none of these disulfide bonds are required for virion infectivity in culture. We also introduced five different mutations into Cys254, the most strictly conserved cysteine across the polyomavirus family. We found that C254L, C254S, C254G, C254Q, and C254R mutants all showed greatly reduced (around 100,000-fold) plaque-forming ability. These mutants had no apparent defect in viral DNA replication. Mutant Vp1's, as well as wild-type Vp2/3, were mostly localized in the nucleus. Further analysis of the C254L mutant revealed that the mutant Vp1 was able to form pentamers in vitro. DNase I-resistant virion-like particles were present in NO-SV40-C254L-transfected cell lysate, but at about 1/18 the amount in wild-type-transfected lysate. An examination of the three-dimensional structure reveals that Cys254 is buried near the surface of Vp1, so that it cannot form disulfide bonds, and is not involved in intrapentamer interactions, consistent with the normal pentamer formation by the C254L mutant. It is, however, located at a critical junction between three pentamers, on a conserved loop (G2H) that packs against the dual interpentamer Ca2+-binding sites and the invading C-terminal helix of an adjacent pentamer. The substitution by the larger side chains is predicted to cause a localized shift in the G2H loop, which may disrupt Ca2+ ion coordination and the packing of the invading helix, consistent with the defect in virion assembly. Our experimental system thus allows dissection of structure-function relationships during the distinct steps of the SV40 life cycle.
  • A Nakanishi; LF Guan; RR Kane; H Kasamatsu; MF Hawthorne
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA NATL ACAD SCIENCES 96 (1) 238 - 241 0027-8424 1999/01 [Refereed]
     
    The viability of boron neutron capture therapy depends on the development of tumor-targeting agents that contain large numbers of boron-10 (B-10) atoms and are readily taken up by cells. Here we report on the selective uptake of homogeneous fluorescein-labeled nido-carboranyl oligomeric phosphate diesters (nido-OPDs) by the cell nucleus and their long-term retention after their delivery into the cytoplasm of TC7 cells by microinjection, All nido-OPDs accumulated in the cell nucleus within 2 h after microinjection. However, nido-OPDs in which the carborane cage was located on a side chain attached to the oligomeric backbone were redistributed between both the cytoplasm and nucleus after 24 h of incubation, whereas nido-OPDs in which the carborane cage was located along the oligomeric backbone remained primarily in the nucleus. Furthermore, cell-free incubation of digitonin-permeabilized TC7 cells with the nido-OPDs resulted in nuclear accumulation of the compounds, thus corroborating the microinjection studies. Our observation of fluorescence primarily located in the cell nucleus indicates that nuclear-specific uptake of sufficient amounts of 10B for effective boron neutron capture therapy (approximate to 10(8)-10(9) B-10 atoms/tumor cell) via nido-OPDs is achievable.
  • Annual Review of Microbiology 52 627 - 686 1998 [Invited]
  • 中西 章; 笠松 春美
    蛋白質 核酸 酵素 共立出版 42 (1) 12 - 21 0039-9450 1997 [Invited]
  • T Kuwana; K Hashimoto; A Nakanish; Y Yasuda; A Tajima; M Naito
    INTERNATIONAL JOURNAL OF DEVELOPMENTAL BIOLOGY UNIV BASQUE COUNTRY PRESS 40 (5) 1061 - 1064 0214-6282 1996/10 [Refereed]
     
    The pH of the embryonic blood, one of the most important environmental factors for embryonic cells, was found to range from 8.1 to 8.5 in chick embryos until 108 h after incubation. Based on these results, the culture medium adjusted to pH 8.0 was used to culture embryonic chick and quail cells. They were easily subcultured for a long period of time at pH 8.0. This pH culture condition may have wide application for manipulating embryonic cells or tissues and establishing cell lines from avian embryos.
  • A Nakanishi; J Clever; M Yamada; PP Li; H Kasamatsu
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA NATL ACAD SCIENCES 93 (1) 96 - 100 0027-8424 1996/01 [Refereed]
     
    All animal DNA viruses except pox virus utilize the cell nucleus as the site for virus reproduction. Yet, a critical viral infection process, nuclear targeting of the viral genome, is poorly understood, The role of capsid proteins in nuclear targeting of simian virus 40 (SV40) DNA, which is assessed by the nuclear accumulation of large tumor (T) antigen, the initial sign of the infectious process, was tested by two independent approaches: antibody interception experiments and reconstitution experiments. When antibody against viral capsid protein Vp1 or Vp3 was introduced into the cytoplasm, the nuclear accumulation of T antigen was nor observed in cells either infected or cytoplasmically injected with virion. Nuclearly introduced anti-T-Vp3 IgG also showed the inhibitory effect. In the reconstitution experiments, SV40 DNA was allowed to interact with protein components of the virus, either empty particles or histones, and the resulting complexes were tested for the capability of protein components to target the DNA to the nucleus from cytoplasm as effectively as the targeting of DNA in the mature virion, In cells injected with empty particle-DNA, but not in minichromosome-injected cells, T antigen was observed as effectively as in SV40-injected cells, These results demonstrate that SV40 capsid proteins can facilitate transport of SV40 DNA into the nucleus and indicate that Vp3, one of the capsid proteins, accompanies SV40 DNA as it enters the nucleus during virus infection.
  • 和田志士; 澤田幸治; 和賀 祥; 中西 章
    蛋白質 核酸 酵素 共立出版 40 (7) 932 - 939 0039-9450 1995 [Invited]
  • N ISHII; A NAKANISHI; M YAMADA; MH MACALALAD; H KASAMATSU
    JOURNAL OF VIROLOGY AMER SOC MICROBIOLOGY 68 (12) 8209 - 8216 0022-538X 1994/12 [Refereed]
     
    Structural proteins of simian virus 40 (SV40), Vp2 and Vp3 (Vp2/3) and Vp1, carry individual nuclear targeting signals, Vp3(198-206) (Vp2(316-324)) and Vp1(1-8), respectively, which are encoded in different reading frames of an overlapping region of the genome. How signals coordinate nuclear targeting during virion morphogenesis was examined by using SV40 variants in which there is only one structural gene for Vp1 or Vp2/3, nuclear targeting-defective mutants thereof, Vp2/3(202T) and Vp1 Delta N5, or nonoverlapping SV40 variants in which the genes for Vp1 and Vp2/3 are separated, and mutant derivatives of the gene carrying either one or both mutations. Nuclear targeting was assessed immunocytochemically following nuclear microinjection of the variant DNAs. When Vp2/3 and Vp1 mutants with defects in the nuclear targeting signals were expressed individually, the mutant proteins localized mostly to the cytoplasm. However, when mutant Vp2/3(202T) was coexpressed in the same cell along with wild-type Vp1, the mutant protein was effectively targeted to the nucleus. Likewise, the Vp1 Delta N5 mutant protein was transported into the nucleus when wild-type Vp2/3 was expressed in the same cells. These results suggest that while Vp1 and Vp2/3 have independent nuclear targeting signals, additional signals, such as those defining protein-protein interactions, play a concerted role in nuclear localization along with the nuclear targeting signals of the individual proteins.
  • A NAKANISHI; A IRITANI
    MOLECULAR REPRODUCTION AND DEVELOPMENT WILEY-LISS 36 (2) 258 - 261 1040-452X 1993/10 [Invited]
  • A NAKANISHI; M MIYAKE; K UTSUMI; A IRITANI
    MOLECULAR REPRODUCTION AND DEVELOPMENT WILEY-LISS 28 (2) 131 - 135 1040-452X 1991/02 [Refereed]
     
    Fertilizing competency of multiple ovulated eggs in the domestic fowl was examined by fertilization in vitro and early development in culture. Normal laying hens (White Leghorn) were treated with 75 IU of PMSG for 7 days followed by injection of anterior pituitary extracts from chickens (CAPE). Ovulation began to occur 7.5 h after injection of CAPE. These hens ovulated 1-7 ova but some premature ovulation of GV stage ova were observed. In vitro fertilization of the multiple ovulated ova was examined by inseminating 10(6)-10(7) sperm onto the germinal disks in m-Ringer's solution. The gamete or zygote nuclei were detected by DNA specific fluorescence using DAPI (4',6' -diamidino-2-phenylindole) in the histological section prepared from the germinal disk. Process of fertilization was examined in the eggs incubated for 4 h after insemination in DMEM + liquid albumen at 41-degrees-C under the atmosphere of 5% CO2 in air. Fertilization rate of the total multiple ovulated eggs was 55% (11/20), in which 90% (9/10) and 10% (1/10) in the eggs recovered 7.5-8.5 h and 9.0-9.5 h after CAPE injection were obtained, respectively. Normal pronuclei were formed in five eggs of those recovered 7.5-8.5 h after CAPE injection. Early development after fertilization in vitro was also examined by incubation for 12 h in DMEM + liquid albumen at 41-degrees-C under the atomosphere of 5% CO2 in air. Although development in vitro was delayed compared to that in utero condition, normal development was observed in naturally and multiple ovulated eggs. The developmental rate of the multiple ovulated eggs was 67% (20/30) in total, in which 94% (15/16) and 5% (5/14) were obtained in the eggs recovered 7.5-8.5 h and 9.0-9.5 h after CAPE injection, respectively. Both fertilization and developmental rate of the eggs recovered 9.0-9.5 h after CAPE injection were significantly lower (P < 0.01) than those recovered 7.5-8.5 h after CAPE injection or naturally ovulated. These results suggested that multiple ovulated fowl eggs recovered 7.5-8.5 h after CAPE injection had normal fertilizing and developmental ability in vitro.
  • A NAKANISHI; K UTSUMI; A IRITANI
    MOLECULAR REPRODUCTION AND DEVELOPMENT WILEY-LISS 26 (3) 217 - 221 1040-452X 1990/07 [Refereed]

Books etc

  • 技術情報協会 (Joint work8章3節 ウイルス構造タンパク質の改変とその機能解析)技術情報協会 2023/01 9784861049361 580p
  • NAKANISHI Akira (Joint workAstrovirus)診断と治療社 2019/09
  • 中西 章 (Contributor８章1節 1節 認知症治療薬の血液脳関門薬剤通過のメカニズムと評価)技術情報協会 2019/04
  • 片山和彦; 芳賀慧; 藤本陽; 戸高玲子; 村上耕介; 村田和義; 中西 章 (Joint workノロウイルス研究の最近の知見)メディカルレビュー社 2017/09
  • 小関弘恵知; 中西 章 (Joint work血液脳関門の薬物透過性の評価法 p477-483)技術情報協会 2017
  • Kasamatsu H; Nakanishi A; Liddington RC; Sawa H (ContributorStructural and functional studies of polyomavirus late gene productsIn 'Molecular Biology of Tumor Virus Gene Products’)Research Signpost Kerala, India 2010
  • Frontiers in Neutron Capture Therapy, Vol.2,
    Mark B. Smuckler; Satish S. Jalisatgi; Lufeng Guan; Akira Nakanishi; Harumi Kasamatsu; M. Frederick Hawthorne (ContributorBoron-Rich Oligomeric Phosphate Diesters that Target the Cell Nucleus In Vitro)Kluwer Academic/Plenum Publishers: New York, New York, 1103 2001
  • CAN (Nup214)
    ノックアウトマウスデータブック Molecular Medicine 増刊号 （中山書店） 1997
  • 新しい遺伝子工学 半田 宏編著 4章 遺伝子の機能を調べる 4.1及び4.2
    昭晃堂

Conference Activities & Talks

Industrial Property Rights

  • 特願2021-126962:ヒトアストロウイルスの感染レセプターとその応用  2021年/08/02
    片山和彦, 中西 章, 芳賀 慧, 戸高玲子, 小池紫文, 工藤栞  国立研究開発法人 国立長寿医療研究センター、 学校法人 北里研究所
  • 特願2019-188238:弱毒化生ウイルスワクチン、その有効成分、有効成分の生産方法  2019年/10/11
    片山和彦, 戸高玲子, 中西 章, 及川和樹, 石川涼翔  学校法人北里研究所、学校法人近畿大学
  • 特願2005-39102:改変されたカプシドタンパク質及びその使用  2005年/02/16
    半田宏, 中西章, 金刺新之介, 高橋陵宇  財団法人理工学振興会
  • 特開2010-207246:生理的条件化でのウイルス粒子様構造体及びその形成方法  
    半田宏, 中西章, 川野雅章  財団法人理工学振興会
  • 特許6180400:鳥類由来の細胞の培養方法および該培養方法によって得られた細胞系  
    桑名貴, 橋本光一郎, 中西 章  明治乳業株式会社

Research Grants & Projects

  • 下痢症ウイルス感染症の予防法、治療法の開発に向けた細胞生物学的、構造生物学的研究
    日本医療研究開発機構：感染症実用化事業 新興・再興感染症に対する革新的医薬品等開発推進研究事業
    Date (from‐to) : 2023/04 -2026/03 
    Author : 中西 章
  • 日本医療研究開発機構：感染症実用化事業 新興・再興感染症に対する革新的医薬品等開発推進研究事業
    Date (from‐to) : 2020/04 -2023/03 
    Author : 中西 章
  • 日本医療研究開発機構：感染症実用化事業 新興・再興感染症に対する革新的医薬品等開発推進研究事業
    Date (from‐to) : 2017/04 -2020/03 
    Author : 中西 章
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
    Date (from‐to) : 2015/04 -2019/03 
    Author : KATAYAMA KAZUHIKO
     
    Human norovirus (HNV) is the leading cause of acute gastroenteritis worldwide. Since the discovery of HNV, an efficient and reproducible norovirus replication system has not been established in cultured cells. Those results imply that the identification of a functional cell-surface receptor for norovirus might be the key to establishing a norovirus culture system. Using a genome-wide CRISPR/Cas9 guide RNA library, we identified murine CD300lf and CD300ld as functional receptors for murine norovirus (MNV). The treatment of susceptible cells with polyclonal antibody against CD300lf significantly reduced the production of viral progeny. Furthermore, CD300ld, which has an amino acid sequence in the N-terminal region of its extracellular domain that is highly homologous to that of CD300lf, also functions as a receptor for MNV.
  • 日本医療研究開発機構：感染症実用化事業 新興・再興感染症に対する革新的医薬品等開発推進研究事業
    Date (from‐to) : 2016/04 -2019/03 
    Author : 中西 章
  • 新興・再興感染症に対する革新的医薬品等開発推進
    Date (from‐to) : 2014/04 -2017/03 
    Author : 中西 章
  • 新型インフルエンザ等新興・再興感染症研究事業
    Date (from‐to) : 2013/04 -2016/03 
    Author : 中西 章
  • 長寿科学研究者支援事業
    Date (from‐to) : 2011/04 -2013/03 
    Author : 中西 章
  • 共同研究
    Date (from‐to) : 2010/04 -2013/03 
    Author : 中西 章
     
    予防診断・治療法のない、新興・再興感染症の原因ウイルスについての実験技術の確立
  • 委託研究
    Date (from‐to) : 2005 -2011 
    Author : 中西 章
  • 保健医療分野における基礎研究推進事業
    Date (from‐to) : 2007 -2009 
    Author : 中西 章
     
    高い遺伝子導入能をもつ種々のウイルスカプシドの機能解析から、導入過程に不可欠な機能ドメインの単離そしてその応用をめざす。
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2005 -2006 
    Author : NAKANISHI Akira
     
    Upon viral entry into the host cell disassembly of the capsid and subsequent release of the viral genome is the essential steps for SV40 infection, though mechanism underlies of which is poorly defined. The viral disassembly, normally induced by cellular cue, was not properly regulated when the viral particles lack the minor capsid proteins, Vp2 and Vp3 (collectively Vp2/3) : the Vp2/3-less particles prematurely released the viral DNA upon cell entry (Nakanishi et al. J.Virol. 2007). In order to examine the contribution of Vp1 and Vp2/3 interaction on the regulation of viral disassembly, mutations were introduced to the residues involving Vp1 and Vp2/3 binding, and analyzed their impact on viral infectious cycle. The mutations are designed to (1) alter the ionic interaction interface between Vp1 and Vp2/3, Vp1 K116R and Vp3 Q174D, and to (2) form disulfide bond between Vp1 and Vp2/3, Vp1 V230C-Vp3 E159C and Vp1 E257C-Vp3 Q174C. In addition (3) a mutant combined with three distinct temperature sensitive mutations (tsDs), each reported to exhibit 'uncoating defect', Vp3 P103S-M110I-Q113K, was also included in the analysis. Upon transfection of the mutant viral DNA, capsid protein expression and viral DNA replication takes place normally, and all the mutants form particles with similar composition with that of wild-type virion. However, the mutants, except Vp1 K116R, were severely defective in their ability to form plaques at 37C or at the restrictive temperature (Vp3 P103S-M110I-Q113K). The mutant particles are apparently be able to enter the cells, interact with importin alpha/beta heterodimer, though either defective or delayed in initiating viral early gene expression, indicating that the defect of the mutants lies after interaction with nuclear import machinery of the viral particles. The results imply that major conformational change of viral capsid involving shift of Vp1-Vp2/3 interaction occurs at the later phase of cell entry processes, including the post-nuclear entry events, during the infection.

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  • University of California, Los Angeles Molecular Biology Institute, and Department of Molecular Cell and Developmental Biology Assistant Researcher 1997 - 2003 University of California, Los Angeles Molecular Biology Institute Post-doctoral fellow 1992 - 1994

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