研究者総覧

仲屋 友喜 (ナカヤ ユウキ)

  • 感染・免疫学講座(ウイルス学部門) 助教
Last Updated :2021/12/04

研究者情報

学位

  • 博士(医学)(2011年09月 京都大学)
  • 学士(獣医学)(2008年03月 岩手大学)

ホームページURL

ORCID ID

Researcher ID

  • AAD-1553-2020

J-Global ID

研究キーワード

  • 自然免疫   肝炎ウイルス   自己DNA   胎盤   内在性レトロウイルス   

研究分野

  • ライフサイエンス / ウイルス学
  • ライフサイエンス / 分子生物学
  • ライフサイエンス / 獣医学

経歴

  • 2021年04月 - 現在  自治医科大学医学部 感染・免疫学講座ウイルス学部門助教
  • 2019年04月 - 2021年03月  国立研究開発法人産業技術総合研究所(産総研)センシングシステム研究センター主任研究員
  • 2017年10月 - 2019年03月  産業技術総合研究所光センシンググループ産総研特別研究員
  • 2016年04月 - 2017年03月  イリノイ大学シカゴ校微生物免疫学部博士研究員
  • 2014年04月 - 2016年03月  ペンシルバニア大学ペレルマン医学校博士研究員
  • 2013年04月 - 2016年03月  日本学術振興会特別研究員(SPD)
  • 2011年11月 - 2013年03月  京都大学ウイルス研究所研究員(産官学連携)
  • 2011年10月 - 2011年10月  日本学術振興会特別研究員(PD)

学歴

  • 2008年04月 - 2011年09月   京都大学   大学院医学研究科   博士課程
  • 2002年04月 - 2008年03月   岩手大学   農学部獣医学科

研究活動情報

論文

  • Yuki Nakaya, Takashi Fukuda, Hiroki Ashiba, Masato Yasuura, Makoto Fujimaki
    BMC INFECTIOUS DISEASES 20 1 585  2020年08月 [査読有り][通常論文]
     
    BackgroundThe polymerase chain reaction (PCR) is commonly used to detect viral pathogens because of its high sensitivity and specificity. However, conventional PCR methods cannot determine virus infectivity. Virus infectivity is conventionally examined with methods such as the plaque assay, even though such assays require several days. Long-range reverse-transcription quantitative PCR (RT-qPCR) has previously been suggested for the rapid assessment of RNA virus infectivity where the loss of infectivity is attributable to genomic fragmentation.MethodsIAV was irradiated with 253.7nm ultraviolet (UV) rays to induce genomic strand breaks that were confirmed by a full-length RT-PCR assay. The IAV was then subjected to plaque assay, conventional RT-qPCR and long-range RT-qPCR to examine the relationship between infectious titer and copy number. A simple linear regression analysis was performed to examine the correlation between the results of these assays.ResultsA long-range RT-qPCR assay was developed and validated for influenza A virus (IAV). Although only a few minutes of UV irradiation was required to completely inactivate IAV, genomic RNA remained detectable by the conventional RT-qPCR and the full-length RT-PCR for NS of viral genome following inactivation. A long-range RT-qPCR assay was then designed using RT-priming at the 3 termini of each genomic segment and subsequent qPCR of the 5 ' regions. UV-mediated IAV inactivation was successfully analyzed by the long-range RT-qPCR assay especially when targeting PA of the viral genome. This was also supported by the regression analysis that the long-range RT-qPCR is highly correlated with plaque assay (Adjusted R-2=0.931, P=0.000066).Conclusions p id=Par This study suggests that IAV infectivity can be predicted without the infectivity assays. The rapid detection of pathogenic IAV has, therefore, been achieved with this sensing technology.
  • Waveguide-mode Sensor Chip with Si/SiO2/SiOx Structure
    Masato Yasuura, Koji Ueno, Hiroki Ashiba, Yuki Nakaya, and Makoto Fujimaki
    Sensors and Materials 32 4 1567 - 1576 2020年04月 [査読有り][通常論文]
  • Rie Nakaoka Miyaho, So Nakagawa, Akira Hashimoto-Gotoh, Yuki Nakaya, Sayumi Shimode, Shoichi Sakaguchi, Rokusuke Yoshikawa, Mahoko Ueda Takahashi, Takayuki Miyazawa
    Gene 690 137 - 137 2019年03月 [査読有り][通常論文]
  • Takahara Yusuke, Nakaya Yuki, Yasuura Masato, Ashiba Hiroki, Kumar Penmetcha KR, Fujimaki Makoto
    Sensors and Materials 31 1 79 - 87 2019年01月 [査読有り][通常論文]
  • Yuki Nakaya, Jingtao Lilue, Spyridon Stavrou, Eileen A. Moran, Susan R. Ross
    MBIO 8 4 e00944-17  2017年07月 [査読有り][通常論文]
     
    Cytosolic DNAs derived from retrotransposons serve as pathogen-associated molecular patterns for pattern recognition receptors (PRRs) that stimulate the induction of interferons (IFNs) and other cytokines, leading to autoimmune disease. Cyclic GMP-AMP synthase is one PRR that senses retrotransposon DNA, activating type I IFN responses through the stimulator of IFN genes (STING). Absent in melanoma 2 (AIM2)-like receptors (ALRs) have also been implicated in these pathways. Here we show that the mouse ALR IFI205 senses cytosolic retrotransposon DNA independently of cyclic GMP-AMP production. AIM2 antagonizes IFI205-mediated IFN induction activity by sequestering it from STING. We also found that the complement of genes located in the ALR locus in C57BL/6 and AIM2 knockout mice are different and unique, which has implications for interpretation of the sensing of pathogens in different mouse strains. Our data suggest that members of the ALR family are critical to the host IFN response to endogenous DNA. IMPORTANCE Autoimmune diseases like Aicardi-Goutieres syndrome and lupus erythematosus arise when cells of the immune system become activated and attack host cells and tissues. We found that DNA generated by endogenous retroviruses and retroelements in inbred mice and mouse cells is recognized by several host proteins found in macrophages that are members of the ALR family and that these proteins both suppress and activate the pathways leading to the generation of cytokines and IFNs. We also show that there is great genetic diversity between different inbred mouse strains in the ALR genes, which might contribute to differential susceptibility to autoimmunity. Understanding how immune cells become activated is important to the control of disease.
  • Yuki Nakaya, Spyridon Stavrou, Kristin Blouch, Peter Tattersall, Susan R. Ross
    JOURNAL OF VIROLOGY 90 17 8005 - 8012 2016年09月 [査読有り][通常論文]
     
    APOBEC3 knockout and human APOBEC3A and -3G transgenic mice were tested for their ability to be infected by the herpesviruses herpes simplex virus 1 and murine herpesvirus 68 and the parvovirus minute virus of mice (MVM). Knockout, APOBEC3A and APOBEC3G transgenic, and wild-type mice were equally infected by the herpesviruses, while APOBEC3A but not mouse APOBEC3 conferred resistance to MVM. No viruses showed evidence of cytidine deamination by mouse or human APOBEC3s. These data suggest that in vitro studies implicating APOBEC3 proteins in virus resistance may not reflect their role in vivo. IMPORTANCE It is well established that APOBEC3 proteins in different species are a critical component of the host antiretroviral defense. Whether these proteins also function to inhibit other viruses is not clear. There have been a number of in vitro studies suggesting that different APOBEC3 proteins restrict herpesviruses and parvoviruses, among others, but whether they also work in vivo has not been demonstrated. Our studies looking at the role of mouse and human APOBEC3 proteins in transgenic and knockout mouse models of viral infection suggest that these restriction factors are not broadly antiviral and demonstrate the importance of testing their activity in vivo.
  • Katsuo Koshi, Yuki Nakaya, Keiichiro Kizaki, Toshina Ishiguro-Oonuma, Takayuki Miyazawa, Thomas E. Spencer, Kazuyoshi Hashizume
    ANIMAL SCIENCE JOURNAL 87 3 419 - 422 2016年03月 [査読有り][通常論文]
     
    Endogenous retroviruses present in the genomes take a specific role in placental formation in various vertebrates, including bovine and sheep. Fematrin-1, which is the envelope (Env) protein of bovine endogenous retrovirus found in bovine placenta, is involved in the formation of fetomaternal hybrid cells in cattle placenta. This study was conducted to clarify whether fematrin-1 possesses fusogenic activity in trophoblast cells. Another question is whether Env proteins only have species-specific activity or not. For this, fematrin-1 gene was transfected in ovine trophoblast cells, and we examined fusogenic activity with Cos-7 cells. Although fematrin-1 fusogenic activity was detected in both neutral and acidic pH conditions, acidic condition significantly enhanced it. These activities were rather weaker than those of vesicular stomatitis virus G protein as a positive control. However, the ratio of fematrin-1 and vesicular stomatitis virus G protein fusion index was confirmed similar to those in the previous reports. Some fusion cells showed multinucleate cells. These results imply that fematrin-1 is involved in the formation of trophoblast hybrid cells even in different species trophoblastic cells.
  • Rie Nakaoka Miyaho, So Nakagawa, Akira Hashimoto-Gotoh, Yuki Nakaya, Sayumi Shimode, Shoichi Sakaguchi, Rokusuke Yoshikawa, Mahoko Ueda Takahashi, Takayuki Miyazawa
    Gene 567 2 189 - 95 2015年08月 [査読有り][通常論文]
     
    Retroviral vectors are used for gene transduction into cells and have been applied to gene therapy. Retroviral vectors using envelope protein (Env) of RD-114 virus, a feline endogenous retrovirus, have been used for gene transduction. In this study, we investigated the susceptibility to RD-114 Env-pseudotyped virus in twelve domestic animals including cattle, sheep, horse, pig, dog, cat, ferret, mink, rabbit, rat, mouse, and quail. Comparison of nucleotide sequences of ASCT2 (SLC1A5), a receptor of RD-114 virus, in 10 mammalian and 2 avian species revealed that insertion and deletion events at the region C of ASCT2 where RD-114 viral Env interacts occurred independently in the mouse and rat lineage and in the chicken and quail lineage. By the pseudotype virus infection assay, we found that RD-114 Env-pseudotyped virus could efficiently infect all cell lines except those from mouse and rat. Furthermore, we confirmed that bovine ASCT2 (bASCT2) functions as a receptor for RD-114 virus infection. We also investigated bASCT2 mRNA expression in cattle tissues and found that it is expressed in various tissues including lung, spleen and kidney. These results indicate that retrovirus vectors with RD-114 virus Env can be used for gene therapy in large domestic animals in addition to companion animals such as cat and dog.
  • Yuki Nakaya, Takayuki Miyazawa
    VIRUSES-BASEL 7 6 2928 - 2942 2015年06月 [査読有り][招待有り]
     
    Recent developments in genome sequencing techniques have led to the identification of huge numbers of endogenous retroviruses (ERV) in various mammals. ERVs, which occupy 8%-13% of mammalian genomes, are believed to affect mammalian evolution and biological diversity. Although the functional significance of most ERVs remains to be elucidated, several ERVs are thought to have pivotal roles in host physiology. We and other groups recently identified ERV envelope proteins (e.g., Fematrin-1, Syncytin-Rum1, endogenous Jaagsiekte sheep retrovirus Env) that may determine the morphogenesis of the unique fused trophoblast cells, termed trinucleate cells and syncytial plaques, found in ruminant placentas; however, there are still a number of outstanding issues with regard to the role of ERVs that remain to be resolved. Here, we review what is known about how these ERVs have contributed to the development of ruminant-specific trophoblast cells.
  • Yuki Nakaya, Takayuki Miyazawa
    JOURNAL OF VIROLOGY 88 12 6896 - 6905 2014年06月 [査読有り][通常論文]
     
    Endogenous retroviruses (ERVs) are the remnants of retroviral infection of ancestral germ cells. Mutations introduced into ERVs halt the production of infectious agents, but their effects on the function of retroviral proteins are not fully understood. Retroviral envelope glycoproteins (Envs) are utilized in membrane fusion during viral entry, and we recently identified intact coding sequences for bovine endogenous retrovirus K1 (BERV-K1) and BERV-K2 Envs. Amino acid sequences of BERV-K1 Env (also called Fematrin-1) and BERV-K2 Env are similar, and both viruses are classified in the genus Betaretrovirus. While Fematrin-1 plays an important role in cell-to-cell fusion in bovine placenta, the BERV-K2 envelope gene is marginally expressed in vivo, and its recombinant Env protein is defective in membrane fusion due to inefficient cleavage of surface (SU) and transmembrane subunits. Here, we conducted chimeric analyses of Fematrin-1 and BERV-K2 Envs and revealed that defective maturation of BERV-K2 Env contributed to failed intracellular trafficking. Fluorescence microscopy and flow cytometric analysis suggested that in contrast to Fematrin-1 Env, BERV-K2 Env could not be transported from the endoplasmic reticulum to the trans-Golgi network, where cellular proteases required for processing retroviral Envs are localized. We also identified that one of the responsive regions of this phenomenon resided within a 65-amino-acid region of BERV-K2 SU. This is the first report to identify that retroviral Env SU is involved in the regulation of intracellular trafficking, and it may help to elucidate the maturation process of Fematrin-1 and other related Envs.
  • Yuki Nakaya, Katsuo Koshi, So Nakagawa, Kazuyoshi Hashizume, Takayuki Miyazawa
    Journal of Virology 87 19 10563 - 10572 2013年 [査読有り][通常論文]
     
    During placentation, mammals employ different strategies for nourishing and supporting fetuses. Members of the Bovidae family, consisting of cloven-hoofed ruminants, utilize multiple maternal attachment points on the placenta, known as cotyledons, and hybrid cells, named trinucleate cells or syncytial plaques, made up of a fusion of fetal trophoblasts and maternal endometrial cells to provide essential hormones and maintain long gestation periods. These hybrid cells are unique to the Bovidae, as fetomaternal borders are clearly separated by syncytiotrophoblasts or epithelial cells in the placenta of other mammals. Recently, it was reported that Syncytin-Rum1 was inserted into ruminant genomes, including cattle and sheep, and was possibly involved in fetomaternal cell-to-cell fusion in both species. However, Syncytin-Rum1 alone is insufficient to explain the morphological diversity of the fetomaternal hybrids between Bovinae and Caprinae (i.e., trinucleate cells in Bovinae and syncytial plaques in Caprinae). Here we report that the bovine endogenous retrovirus K1 (BERV-K1) envelope, which we term Fematrin- 1, was specifically expressed in binucleated trophoblasts throughout gestation in cattle and induced fusion with bovine endometrial cells in vitro at a significantly higher level than Syncytin-Rum1 under physiological conditions. Fematrin-1 was found to be integrated into intron 18 of FAT tumor suppressor homolog 2 (FAT2) about 18.3 to 25.4 million years ago and has been subject to purifying selection through the evolution of Bovinae. Phylogenetically, Fematrin-1 is distinct from Syncytin genes found in other mammalian species that form syncytiotrophoblasts. Our results suggest that the newly acquired endogenous retroelement has contributed to generating placentation diversity through ruminant evolution. © 2013, American Society for Microbiology.
  • So Nakagawa, Hanako Bai, Toshihiro Sakurai, Yuki Nakaya, Toshihiro Konno, Takayuki Miyazawa, Takashi Gojobori, Kazuhiko Imakawa
    GENOME BIOLOGY AND EVOLUTION 5 2 296 - 306 2013年 [査読有り][通常論文]
     
    In evolution of mammals, some of essential genes for placental development are known to be of retroviral origin, as syncytin-1 derived from an envelope (env) gene of an endogenous retrovirus (ERV) aids in the cell fusion of placenta in humans. Although the placenta serves the same function in all placental mammals, env-derived genes responsible for trophoblast cell fusion and maternal immune tolerance differ among species and remain largely unidentified in the bovine species. To examine env-derived genes playing a role in the bovine placental development comprehensively, we determined the transcriptomic profiles of bovine conceptuses during three crucial windows of implantation periods using a high-throughput sequencer. The sequence reads were mapped into the bovine genome, in which ERV candidates were annotated using RetroTector (c) (7,624 and 1,542 for ERV-derived and env-derived genes, respectively). The mapped reads showed that approximately 18%(284 genes) of env-derived genes in the genome were expressed during placenta formation, and approximately 4% (63 genes) were detected for all days examined. We verified three env-derived genes that are expressed in trophoblast cells by polymerase chain reaction. Out of these three, the sequence of env-derived gene with the longest open reading frame (named BERV-P env) was found to show high expression levels in trophoblast cell lines and to be similar to those of syncytin-Car1 genes found in dogs and cats, despite their disparate origins. These results suggest that placentation depends on various retrovirus-derived genes that could have replaced endogenous predecessors during evolution.
  • Sayumi Shimode, Rokusuke Yoshikawa, Shigeki Hoshino, Yuki Nakaya, Shoichi Sakaguchi, Takeshi Kobayashi, Takayuki Miyazawa
    Virus genes 45 2 393 - 7 2012年10月 [査読有り][通常論文]
     
    RD-114 virus is a replication-competent feline endogenous retrovirus. RD-114 virus contaminates several feline and canine live attenuated vaccines and the issue of contamination of RD-114 virus in vaccines should be solved. To date, three infectious molecular clones (pSc3c, pCRT1, and pRD-UCL) have been reported. In this study, we sequenced the entire nucleotide sequence of pRD-UCL and compared the nucleotide sequences of the three infectious molecular clones. As a result, these three infectious clones were nearly identical with each other in gag-pol and env coding regions. These data support the notion that the active locus of infectious RD-114 virus is single in the feline genome. The length of long terminal repeat (LTR) of pCRT1 was 47 bp shorter than those of pSc3c and pRD-UCL. The 47-bp sequence named direct repeat A (DR-A) was duplicated in the U3 region in pSc3c and pRD-UCL. Although several potential enhancer binding sites are present in the DR-A, there was no significant difference in promoter activities between the LTRs of pRD-UCL and pCRT1 in two human cell lines. We also analyzed the splicing pattern of the RD-114 virus by reverse transcription-polymerase chain reaction and confirmed that RD-114 virus is a simple retrovirus. The data presented here will provide basic information about RD-114 virus to solve the contamination issue in live attenuated vaccines.
  • Eri Takeda, So Nakagawa, Yuki Nakaya, Atsushi Tanaka, Takayuki Miyazawa, Jiro Yasuda
    PLOS ONE 7 7 e41483  2012年07月 [査読有り][通常論文]
     
    Human BST-2 (hBST-2) has been identified as a cellular antiviral factor that blocks the release of various enveloped viruses. Orthologues of BST-2 have been identified in several species, including human, monkeys, pig, mouse, cat and sheep. All have been reported to possess antiviral activity. Duplication of the BST-2 gene has been observed in sheep and the paralogues are referred to as ovine BST-2A and BST2-B, although only a single gene corresponding to BST-2 has been identified in most species. In this study, we identified three isoforms of bovine BST-2, named bBST-2A1, bBST-2A2 and bBST-2B, in bovine cells treated with type I interferon, but not in untreated cells. Both bBST-2A1 and bBST-2A2 are posttranslationally modified by N-linked glycosylation and a GPI-anchor as well as hBST-2, while bBST-2B has neither of these modifications. Exogenous expression of bBST-2A1 or bBST-2A2 markedly reduced the production of bovine leukemia virus and vesicular stomatitis virus from cells, while the antiviral activity of bBST-2B was much weaker than those of bBST-2A1 and bBST-2A2. Our data suggest that bBST-2A1 and bBST-2A2 function as part of IFN-induced innate immunity against virus infection. On the other hand, bBST-2B may have a different physiological function from bBST-2A1 and bBST-2A2.
  • Nakaya Yuki, Shimode Sayumi, Kobayashi Takeshi, Imakawa Kazuhiko, Miyazawa Takayuki
    Xenotransplantation 19 3 177 - 185 2012年05月 [査読有り][通常論文]
     
    Nakaya Y, Shimode S, Kobayashi T, Imakawa K, Miyazawa T. Binding of transcription factor activating protein 2? on the 5'-proximal promoter region of human porcine endogenous retrovirus subgroup A receptor 2/GPR172B. Xenotransplantation 2012; 19: 177185. (c) 2012 John Wiley & Sons A/S. Abstract: Background: Xenotransplantation is one of the solutions for the shortage of organ donors, and pigs have been considered to be the most suitable animal donors. Specific pathogen-free pigs are utilized in the xenotransplantation; however, pigs have infectious gammaretroviruses, named porcine endogenous retroviruses (PERVs) in their genome. Of them, PERV-A and PERV-B can infect human cells in vitro and potentially induce diseases like other gammaretroviruses. The human cellular receptors for PERV-A were identified and named human PERV-A receptor (HuPAR)-1 and HuPAR-2 (also called as GPR172A and GPR172B, respectively). We have recently reported that HuPAR-2 expression was regulated by epigenetic modification and preferentially expressed in placenta. However, the detailed mechanisms of HuPAR-2 expression have not been fully characterized. In this study, we analyzed molecular mechanisms associated with HuPAR-2 transcription through the identification of transcription factors that bind to the promoter region of HuPAR-2. Methods: In situ hybridization was performed to identify the cells expressing HuPAR-2 in placental tissues. Transcriptional activities were measured by dual-luciferase reporter assay using serial deletion mutants of HuPAR-2 5'-flanking region. To identify the transcription factors bound to the promoter region, in silico analysis, electrophoresis mobility shift assay, and chromatin immunoprecipitation assay were conducted. The effect of the transcription factor transcription factor activator protein (TFAP)-2? on the promoter activities was investigated by overexpression of the factor. Results: We identified that HuPAR-2 was specifically expressed in villous trophoblast cells. We also identified that a region spanning from -126 to -32 had proximal promoter activities and TFAP-2? bound to a region spanning from -58 to -35 in vitro and in vivo. The overexpression of TFAP-2? also augmented the proximal promoter activity. Conclusion: We demonstrated that TFAP-2? is one of the transcription factors involved in the HuPAR-2 expression in human villous trophoblast cells. By studying transcriptional factors involved in the expression of HuPAR-2, we may find a clue to control the potential risks caused by PERV-A infection in xenotransplantation.
  • Katsuo Koshi, Yasunori Suzuki, Yuki Nakaya, Kei Imai, Misa Hosoe, Toru Takahashi, Keiichiro Kizaki, Takayuki Miyazawa, Kazuyoshi Hashizume
    REPRODUCTIVE BIOLOGY AND ENDOCRINOLOGY 10 41 2012年05月 [査読有り][通常論文]
     
    Background: Endogenous retrovirus (ERV) envelope (env) genes are involved in the differentiation of trophoblastic cells in humans and mice. However, there is limited information about their roles in ruminant trophoblastic cells. Thus, we attempted to explore the possible roles of ERV elements in the binucleation of bovine trophoblastic cells using in vitro bovine trophoblastic (BT) cell lines. Methods: In this study, blastocysts and elongated embryos were obtained from Japanese Black cows, and endometrial and fetal membrane tissues were collected from day 17 to 37 of gestation. The gene expression levels of four ERV elements, bERVE (bovine endogenous retrovirus envelope element-like transcript) -A, bERVE-B, BERV (bovine endogenous retrovirus) -K1 env, and BERV-K2 env, were analyzed in the fetal and endometrial tissue and cultured BT cell lines using quantitative RT-PCR. On-Matrigel gel and on-collagen gel culturing were used to induce binucleate cell (BNC) formation in the BT cell lines. How the culture conditions affected the expression of BNC-specific genes and ERV elements was examined by quantitative RT-PCR and immunocytochemistry. Results: bERVE-A, bERVE-B, BERV-K1 env, and BERV-K2 env were expressed in almost all BT cell lines; however, only bERVE-A and BERV-K1 env were detected in trophoblastic tissues during the peri-implantation period. In the on-Matrigel cultures, the expression levels of BNC-specific genes and molecules were enhanced in the BT cells. The expression levels of bERVE-A and BERV-K1 env were also increased in the BT cells during on-Matrigel culturing. The BT cell expression levels of these ERV elements were consistent with those of BNC-specific genes during on-Matrigel culturing (P<0.01). Conclusions: These results suggest that bERVE-A and BERV-K1 env are involved in the expression of BNC-specific genes and the progression of bovine trophoblastic cell binucleation, as their expression levels increased during periods of increased BNC-specific molecule expression, which is strongly suggestive of the development of BNC from mononucleate trophoblastic cells. The on-Matrigel culture system is a convenient in vitro tool for studying bovine trophoblastic cell lineages.
  • Yuki Nakaya, Shigeki Hoshino, Jiro Yasuda, Takayuki Miyazawa
    JOURNAL OF GENERAL VIROLOGY 92 Pt4 940 - 944 2011年04月 [査読有り][通常論文]
     
    Pigs are thought to be the most suitable donor animal for xenotransplantation. However, pigs harbour potentially hazardous infectious agents, termed porcine endogenous retroviruses (PERVs), in their genome. In this study, we generated a mAb against PERV-B surface (SU) envelope protein (Env), designated KRT1. KRT1 binding was detected by an indirect immunofluorescence assay and flow cytometric analysis on cells infected with PERV-B. KRT1 neutralized PERV-B pseudotype virus and specifically recognized PERV-B SU Env, but not PERV-A SU Env by immunoblotting analysis. The peptide-ELISA revealed that KRT1 recognized a linear peptide sequence (ALEPPHNLPVP) residing in a proline-rich region that is one of the subdomains of SU Env. In conclusion, the KRT1 antibody will serve as a useful tool for the study of PERV-B and, more importantly, it may provide new protective strategies against PERV-B infection in xenotransplantation.
  • Kenji Baba, Yuki Nakaya, Takayuki Shojima, Yoshikage Muroi, Keiichiro Kizaki, Kazuyoshi Hashizume, Kazuhiko Imakawa, Takayuki Miyazawa
    JOURNAL OF VIROLOGY 85 3 1237 - 1245 2011年02月 [査読有り][通常論文]
     
    Sequences of retroviral origin occupy approximately 10% of mammalian genomes. Various infectious endogenous retroviruses (ERVs) and functional retroviral elements have been reported for several mammals but not cattle. Here, we identified two proviruses, designated bovine endogenous retrovirus K1 (BERV-K1) and BERV-K2, containing full-length envelope (env) genes in the bovine genome. Phylogenetic analysis revealed that they belong to the genus Betaretrovirus. By reverse transcription (RT)-PCR, both BERV-K1 and -K2 env mRNAs were detected in the placenta and cultured bovine trophoblast cells. Real-time RT-PCR analysis using RNAs isolated from various bovine tissues revealed that BERV-K1 env mRNA was preferentially expressed in the placenta. Moreover, we also found the expression of doubly spliced transcripts, named the REBK1 and REBK2 genes. Both the REBK1 and REBK2 proteins have motifs for a putative nuclear localization signal and a nuclear export signal. REBK1 and REBK2 fused with green fluorescent proteins were localized mainly in the nuclei when they were expressed in bovine and porcine cells. In the env and 3' long terminal repeats of BERV-K1 and -K2, we found regulatory elements responsible for the splicing and transport of viral RNAs and/or translation of the env genes. Although we have not identified the expressed Env proteins in bovine tissues, these data suggest that both BERV-K1 and BERV-K2 express Env proteins and that these proteins may have physiological functions in vivo.
  • Yuki Nakaya, Takayuki Shojima, Jiro Yasuda, Kazuhiko Imakawa, Takayuki Miyazawa
    MICROBES AND INFECTION 13 1 49 - 57 2011年01月 [査読有り][通常論文]
     
    Porcine endogenous retroviruses (PERVs) have been considered one of the major risks of xenotransplantation from pigs to humans. PERV-A efficiently utilizes human PERV-A receptor 2 (HuPAR-2)/GPR172B to infect human cells; however, there has been no study on the regulation mechanisms of HuPAR-2/GPR172B expression. In this study, we examined the expression of HuPAR-2/GPR172B from the standpoint of epigenetic regulation and discussed the risks of PERV-A infection in xenotransplantation. Quantitative real-time RT-PCR revealed that HuPAR-2 mRNA was preferentially expressed in placental tissue, whereas it was highly suppressed in BeWo cells (a human choriocarcinoma cell line) and HEK293 cells. A CpG island containing the HuPAR-2 transcription starting site was identified by in silico analysis. The DNA methylation ratio (the relative quantity of methylcytosine to total cytosine) and histone modification (H3K9me3) levels in the CpG island measured by bisulfite genomic sequencing and ChIP assay, respectively, were inversely correlated with the mRNA levels. Both HuPAR-2 mRNA and HuPAR-2 protein were up-regulated in HEK293 cells by inhibiting DNA methylation and histone deacetylation. Additionally, promoter/enhancer activities within the CpG island were suppressed by in vitro DNA methylation. Our results demonstrated that epigenetic modification regulates HuPAR-2 expression. (C) 2010 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
  • Yuki Nakaya, Takayuki Shojima, Jiro Yasuda, Takayuki Miyazawa
    JOURNAL OF VETERINARY MEDICAL SCIENCE 72 1 67 - 71 2010年01月 [査読有り][通常論文]
     
    In xenotransplantation from pigs to humans, a bio-artificial endocrine pancreas (Bio-AEP), in which pancreatic endocrine cells are encapsulated within a semipermeable membrane of 100 nm pore size, has been developed. We evaluated the permeability of porcine endogenous retroviruses (PERVs) through membrane filters using a pseudotype virus (LacZ(PERV-A)) containing a viral core derived from murine leukemia virus and ail envelope (Env) from PERV Subgroup A. Contrary to our expectations, LacZ(PERV-A) lost its infectivity by filtration through a 200 nm membrane filter. This unusual phenotype was not observed in pseudotype Viruses harboring Envs from other gammaretroviruses. The infectivity of LacZ(PERV-A) was significantly decreased by repeated freeze/thaw treatment, indicating that LacZ(PERV-A) was physically labile. In addition, LacZ(PERV-A) may be agglutinated because copy numbers of viral RNA after filtration were significantly reduced by filtration through the 200 nm membrane. This phenotype is advantageous to develop a safe Bio-AEP blocking PI-RV infection.
  • Yuki Nakaya, Takayuki Shojima, Shigeki Hoshino, Takayuki Miyazawa
    JOURNAL OF VETERINARY MEDICAL SCIENCE 72 1 117 - 121 2010年01月 [査読有り][通常論文]
     
    T-lymphotropic feline leukemia virus (FeLV-T) induces immunodeficiency in cats. FeLV-T is fusion-detective and requires a cofactor, termed FeLIX, for infection. FeLIX is a truncated envelope glycoprotein of an endogenous FeLV and mediates infection by binding a phosphate transporter Pit-1. In this Study, we established a feline sarcoma-positive leukemia-negative cell line expressing FeLIX, named QN/FeLIX cells. Upon infection, FeLV-T induced prominent foci with syncytia in QN/FeLIX cells and could be titrated by the focus assay. In addition, we established a FeLIX-expressing feline Fibroblast cell line, named AH/FeLIX cells. FeLV-T productively infected AH/FeLIX cells and induced severe CPE with syncytia. QN/FeLIX and AH/FeLIX cells will be useful for the study of FeLIX-dependent mutants in FeLV-infected cats.
  • Yuki Nakaya, Keiichiro Kizaki, Toru Takahashi, Osman V. Patel, Kazuiyoshi Hashizume
    BMC MOLECULAR BIOLOGY 10 19 2009年03月 [査読有り][通常論文]
     
    Background: Bovine trophoblast binucleate cells (BNC) express a plethora of molecules including bovine placental lactogen (bPL, gene name is bCSH1) and bovine prolactin-related protein-1 (bPRP1). BCSH1 and bPRP1 are members of the growth hormone (GH)/prolactin (PRL) gene family, which are expressed simultaneously in BNC and are central to placentation and the progression of pregnancy in cattle. However, there is a paucity of information on the transcriptional regulatory mechanisms of both the bCSH1 and bPRP1 genes. Recent studies, however, have demonstrated that the expression of a number of genes is controlled by the methylation status of their promoter region. In the present study, we examined the cell-type-specific epigenetic alterations of the 5'-flanking region of the bCSH1 and bPRP1 genes to gain an insight into their regulatory mechanisms. Results: Analysis of 5-aza-2'-deoxycytidine treatment demonstrated that bCSH1 expression is moderately induced in fibroblast cultures but enhanced in BT-1 cells. Sodium bisulfite based sequencing revealed that bCSH1 is hypomethylated in the cotyledonary tissue but not in the fetal skin, and this pattern was not altered with the progression of pregnancy. On the other hand, the methylation status of bPRP1 was similar between the cotyledon and fetal skin. The bPRP1 gene was exclusively hypermethylated in a bovine trophoblast cell-derived BT-1 cell-line. While the activity of bCSH1 was similar in both BT-1 and bovine fibroblast cells, that of bPRP1 was specific to BT-1. Treatment with a demethylating agent and luciferase assays provided in vitro evidence of the positive regulation of bCSH1 but not bPRP1. Conclusion: This is the first report to identify the differential regulatory mechanisms of the bCSH1 and bPRP1 genes and indicates that bCSH1 might potentially be the only transcript that is subject to DNA methyltransferase regulation. The data indicates the possibility of novel kinetics of induction of the synchronously expressed BNC-specific bCSH1 and bPRP1 transcripts, which may aid the understanding of the intricate regulation and specific role(s) of these important molecules in bovine placentogenesis and the progression of pregnancy.

MISC

  • 安浦 雅人, 仲屋 友喜, 藤巻 真 「センサ・マイクロマシンと応用システム」シンポジウム論文集 電気学会センサ・マイクロマシン部門 [編] 35 4p 2018年10月 [査読無し][通常論文]
  • 仲屋 友喜, 宮沢 孝幸 生物科学 67 (1) 28 -37 2015年12月 [査読無し][通常論文]
  • 唄花子, 櫻井敏博, 青木悦成, 中川草, 仲屋友喜, 宮沢孝幸, 出田篤司, 青柳敬人, 五篠堀孝, 今川和彦 日本畜産学会大会講演要旨 116th 97 -97 2013年03月 [査読無し][通常論文]
  • 下出紗弓, 吉川禄助, 仲屋友喜, 中岡里江, 小林剛, 宮沢孝幸 日本獣医学会学術集会講演要旨集 155th 231 2013年03月 [査読無し][通常論文]
  • 宮沢孝幸, 星野重樹, 仲屋友喜, 吉川禄助, 下出紗弓, 坂口翔一, 中岡里江 日本野生動物医学会大会・講演要旨集 19th 64 2013年 [査読無し][通常論文]
  • 仲屋友喜, 越勝男, 中川草, 木崎景一郎, 小林剛, 橋爪一善, 宮沢孝幸 日本ウイルス学会学術集会プログラム・抄録集 60th 291 2012年10月 [査読無し][通常論文]
  • 仲屋友喜, 越勝男, 中川草, 木崎景一郎, 小林剛, 橋爪一善, 宮沢孝幸 日本獣医学会学術集会講演要旨集 154th 241 -241 2012年08月 [査読無し][通常論文]
  • 下出紗弓, 吉川禄助, 星野重樹, 仲屋友喜, 小林剛, 宮沢孝幸 日本分子生物学会年会プログラム・要旨集(Web) 35th 4P-0116 (WEB ONLY) 2012年 [査読無し][通常論文]
  • Katsuo Koshi, Yasunori Suzuki, Yuki Nakaya, Kei Imai, Toru Takahashi, Keiichiro Kizaki, Takayuki Miyazawa, Kazuyoshi Hashizume BIOLOGY OF REPRODUCTION 85 2011年07月 [査読無し][通常論文]
  • 仲屋友喜, 越勝男, 木崎景一郎, 馬場健司, 今川和彦, 橋爪一善, 宮沢孝幸 日本獣医学会学術集会講演要旨集 151st 78 2011年03月 [査読無し][通常論文]
  • 越 勝男, 鈴木 康規, 仲屋 友喜, 木崎 景一郎, 宮沢 孝幸, 橋爪 一善 日本繁殖生物学会 講演要旨集 104 (0) 228 -228 2011年 [査読無し][通常論文]
     
    【目的】内在性レトロウイルス関連遺伝子は、ヒト、マウスにおいて胎盤、栄養膜細胞の分化に必要であるとの報告がある。我々はこれまでに、ウシ胎盤、栄養膜細胞に発現する内在性レトロウイルス関連遺伝子が存在することを明らかにした。しかしウシにおいて、栄養膜細胞の分化過程に、内在性レトロウイルス関連遺伝子が関連するかどうかは不明である。本研究では、ウシ培養栄養膜細胞を用い、二核細胞分化誘導時に内在性レトロウイルス関連遺伝子がどのような発現変化をみせるのか明らかにすることを目的とした。
    【方法】我々が樹立したウシ培養栄養膜細胞(BT-B、BT-C、BT-K)を用い、コラーゲンゲル上培養、DHSを用いた血清飢餓培養、マウスEHS腫瘍由来基底膜様物質であるマトリゲルを用いたゲル上培養を8日間行うことで二核細胞を誘導した。またこれらの細胞を材料に定量的PCR法を行い、各細胞系列における二核細胞特異的遺伝子 (bCSH1bPRP-1bPAG-1)、IFNT並びに内在性レトロウイルス関連遺伝子(BERV-K1 env、BERV-K2 envbERVE-AP1x)のRNA発現変化を検討した。
    【結果】マトリゲル上で培養したBT-B、BT-Cでは、二核細胞特異的遺伝子の発現上昇が生じたが、BT-Kでは発現の変化は確認できなかった。コラーゲンゲル上で培養した場合、いずれの細胞系でもIFNTの発現上昇が認められた。このことから、BT-B、BT-Cのマトリゲル上培養において二核細胞への分化誘導が生じていると考えられた。また内在性レトロウイルス関連遺伝子の発現変化として、BT-B、BT-Cのマトリゲル上培養においてBERV-K1 envbERVE-Aの発現上昇が、血清飢餓培養でbERVE-Aの発現上昇がそれぞれ認められた。一方、BERV-K2 envP1xについては培養条件による発現変化は確認されなかった。これらの結果より、二核細胞への分化もしくは、二核細胞の形質に内在性レトロウイルス関連遺伝子が関与することが示唆された。
  • 仲屋 友喜, 越 勝男, 馬場 健司, 木崎 景一郎, 小林 剛, 今川 和彦, 橋爪 一善, 宮沢 孝幸 日本繁殖生物学会 講演要旨集 104 (0) 5 -5 2011年 [査読無し][通常論文]
     
    【目的】祖先動物の生殖細胞に感染し、子孫にゲノムの一部として遺伝したレトロウイルスは内在性レトロウイルス(ERV)と定義される。多くのERVは変異や欠失によりそのオープンリーディングフレーム(ORF)を失っているが、一部のERVにはORFが保存され、宿主の生理機能を担うものが存在する。ヒトやマウスでは、胎盤形成時に必須である栄養膜細胞同士の融合をERVのエンベロープ(遺伝子;env,タンパク;Env)が担うと考えられている。ウシ胎盤においては、栄養膜二核細胞(BNC)と子宮内膜細胞(EMC)との融合細胞の存在が報告されているが、その形成に関わる機構は明らかになっていない。本研究では、最近我々が同定したウシ胎盤で発現する、ウシERV(BERV)-K1およびBERV-K2 envの融合細胞形成への関与を明らかにすべく実験を行った。
    【方法】FLAGタグ付BERV-K Env発現プラスミドを構築し、Cos-7細胞に導入後、イムノブロット(IB)により細胞溶解液中における両Envの検出を試みた。各Env発現Cos-7細胞を、初代培養ウシEMCと共培養し、細胞融合依存的ルシフェラーゼアッセイにより両Envの細胞融合活性を調べた。さらに、妊娠約30、90、150、230日齢のウシ胎盤組織における、両envmRNAならびにBERV-K1 Envの発現を、in situハイブリダイゼーション(ISH)ならびに免疫組織化学染色(IHC)により調べた。
    【結果と考察】IBの結果、BERV-K1およびBERV-K2 Envともにその発現を確認できたことから、両envはEnv産生能を有することが明らかとなった。さらに、BERV-K1 Envの細胞融合活性は、陽性対照と同様に高い値を示したのに対し、BERV-K2 Envの値は低かった。ISHの結果、ウシ胎盤ではBERV-K1 envのみ検出でき、その発現はBNCに特異的であった。そこで、BERV-K1 Envに対する抗体を作製し、IHCを行ったところ、ISHの結果と同様に、BERV-K1 EnvはBNC特異的に発現していることが明らかとなった。以上の結果より、BNCとEMCとの細胞融合過程に、BERV-K1 Envが中心的な役割を担うことが強く示唆された。
  • Hilling Karen, Hanel Rita, 仲屋 友喜 J-vet 23 (12) 21 -33,96 2010年12月 [査読無し][通常論文]
  • 仲屋友喜, 越勝男, 木崎景一郎, 馬場健司, 今川和彦, 橋爪一善, 宮沢孝幸 日本ウイルス学会学術集会プログラム・抄録集 58th 266 2010年10月 [査読無し][通常論文]
  • 仲屋友喜, 星野重樹, 庄嶋貴之, 安田二朗, 宮沢孝幸 日本獣医学会学術集会講演要旨集 150th 2010年
  • 吉川禄助, 仲屋友喜, 馬場健司, 宮沢孝幸 日本ウイルス学会学術集会プログラム・抄録集 57th 419 2009年10月 [査読無し][通常論文]
  • 吉川禄助, 庄嶋貴之, 馬場健司, 仲屋友喜, 宮沢孝幸 日本獣医学会学術集会講演要旨集 147th 248 2009年03月 [査読無し][通常論文]
  • 吉川禄助, 馬場健司, 仲屋友喜, 宮沢孝幸 日本獣医学会学術集会講演要旨集 148th 207 2009年 [査読無し][通常論文]
  • Kazuyoshi Hashizume, Yuki Nakaya, Keiichiro Kizaki, Toru Takahashi BIOLOGY OF REPRODUCTION 295 -296 2008年 [査読無し][通常論文]
  • Keiichiro Kizaki, Kanako Maeda, Yuki Nakaya, Yoshio Yamamoto, Kazuyoshi Hashizume BIOLOGY OF REPRODUCTION 104 -105 2007年 [査読無し][通常論文]

産業財産権

  • 特願2020-054331:PCRでインフルエンザウイルスの感染性を評価する方法  2020年03月25日
    仲屋友喜, 福田隆史, 藤巻真  国立研究開発法人産業技術総合研究所
  • 特開2019-211453:標的物質検出方法  2019年12月12日
    仲屋友喜, 藤巻真  国立研究開発法人産業技術総合研究所

受賞

  • 2013年04月 独立行政法人日本学術振興会(学振) 日本学術振興会特別研究員(SPD)
     
    受賞者: 仲屋 友喜
  • 2011年10月 World Congress of Reproductive Biology Centre for Reproductive Genomics Poster Award
     
    受賞者: 仲屋 友喜
  • 2011年03月 日本獣医学会 第151回日本獣医学会学術集会大会会長賞
     
    受賞者: 仲屋 友喜
  • 2010年04月 独立行政法人日本学術振興会(学振) 日本学術振興会特別研究員(DC2)
     
    受賞者: 仲屋 友喜
  • 2008年03月 岩手大学 岩手大学獣医学科優秀卒業論文研究賞
     
    受賞者: 仲屋 友喜

共同研究・競争的資金等の研究課題

  • 内在性レトロウイルス由来RNA核外輸送タンパク質の生理学的機能の解明
    日本学術振興会:科学研究費助成事業 基盤研究(C)(一般)
    研究期間 : 2021年04月 -2024年03月 
    代表者 : 仲屋友喜
  • 磁気微粒子マトリクスを用いたデジタルウイルス検出法の研究開発
    日本医療研究開発機構:ウイルス等感染症対策技術開発事業(基礎研究支援)
    研究期間 : 2020年09月 -2021年03月
  • ウシ内在性レトロウイルスK1由来エンベロープの受容体分子の探索
    日本学術振興会:科学研究費助成事業 若手研究
    研究期間 : 2019年04月 -2021年03月 
    代表者 : 仲屋 友喜
     
    本研究では、ウシやスイギュウなどの胎盤で発現する内在性レトロウイルス由来エンベロープタンパク質であるFematrin-1のリガンド分子、すなわち結合受容体を探索することを目的としている。当該分子のスクリーニングにはゲノムワイドハプロイドスクリーニング法を用いる計画である。その中で、水疱性口炎ウイルス(VSV)にFematrin-1を外包させたシュードタイプウイルスを作製する必要がある。VSVは農林水産省が定める届出伝染病に該当するため、それに準じた研究施設が必要である。このため、今年度は本研究代表者が所属する機関において、VSVを導入するための手続きに従事した。その他、解析に必要な装置や消耗品などを購入し、研究環境の整備も合わせて行った。
  • 内在化に伴うレトロウイルスの適応と不活化
    日本学術振興会:科学研究費助成事業 特別研究員奨励費
    研究期間 : 2013年04月 -2016年03月 
    代表者 : 仲屋 友喜
     
    平成27年度は平成26年度から継続して海外渡航制度を利用し、米国University of Pennsylvaniaにおいて、内在性レトロウイルスと自己免疫疾患に関する研究を行った。いくつかの自己免疫疾患は、内在性レトロウイルスに由来する細胞質DNA、すなわち自己由来DNAが細胞質に存在するDNAセンサー分子によってが認識され、I型インターフェロン反応を引き起こすことに起因すると考えられている。しかしながら、それらセンサー分子に関する詳細は明らかになっていない。本研究ではセンサー分子の同定、それらによるインターフェロン反応発生のメカニズムを明らかにすることとした。 平成26年度までに、マウスのマクロファージ細胞株NR9456においてsiRNAによるセンサーのスクリーニングを行ったところ、Trex1とAim2が免疫応答の抑制に必要なことが明らかとなった。今年度はさらに解析を進め、Aim2様受容体の一つであるIFI205が免疫応答の活性化に必要なことを見出した。これらは初代培養マクロファージにおいても同様の結果であった。共免疫沈降法やProximity Ligation Assayなどを用いて分子間の相互作用を調べたところ、Aim2とIFI205が結合すること、両分子は当該免疫応答を調節する分子であるStingに結合すること、両分子のStingへの結合は拮抗し合うことを見出した。DNAプルダウンアッセイなどを行った結果、Aim2とIFI205はともにマクロファージ細胞質内に局在し、細胞質DNAにすることも明らかにした。 以上の結果より、マウスにおける内在性レトロウイルス由来DNAに起因する自己免疫応答は、Aim2(抑制作用)とIFI205(活性作用)の2つのセンサー分子によって調整されていることが考えられた。これらの分子のホモログはヒトに存在するため、同様のメカニズムが考えられる。
  • 胎盤構築過程におけるウシ内在性レトロウイルスの貢献
    日本学術振興会:科学研究費助成事業 特別研究員奨励費
    研究期間 : 2010年 -2011年 
    代表者 : 仲屋 友喜
     
    本研究の目的は、ウシ胎盤で機能する内在性レトロウイルスを同定することである。昨年度までに、代表者らが同定したウシ内在性レトロウイルスK1(BERV-K1)のエンベロープ(Env)が胎盤の栄養膜二核細胞で特異的に発現していること、ならびにBERV-K1 Envがin vitroの解析で、ウシ子宮内膜細胞に対して高い細胞融合活性を示すことを明らかとした。このことから、BERV-K1 Envはウシの胎盤に特徴的な組織像である三核細胞の形成に中心的な役割を担っていると考えられた。 本年度の計画は、ウシ子宮内膜細胞上で発現しているBERV-K1 Envの受容体を同定することとしたが、まだ特定には至っていない。しかしながら、BERV-K1 Envがウシ子宮内膜細胞に結合することが確認できたことから、当該組織の細胞がBERV-K1 Envの受容体を発現していることが強く示唆された。 さらに詳細な解析を行った結果、BERV-K1はウシFAT2遺伝子のイントロン18に挿入されていることが明らかとなった。そこで、様々な動物のFAT2遺伝子におけるBERV-K1の有無をゲノムPCRによって解析したところ、ウシ科の中でもバリ牛、スイギュウ、シタツンガなどウシ亜科に属する動物のみがBERV-K1を有していることが明らかとなった。また、これらのBERV-K1は家畜ウシと同様にEnvのみが保存され、それらは高い細胞融合活性を示したことから、BERV-K1 Envはウシ亜科動物の進化の過程で積極的に保存され、これらの胎盤の発達に大きく寄与してきたものと考えられる。 ウシFAT2遺伝子に関する解析を行ったところ、BERV-K1と同様に胎盤で特異的に発現していたことから、BERV-K1はFAT2遺伝子の発現機構を利用することで胎盤特異的に発現するようになったことが示唆された。 代表者は、以上の研究成果を「The 2nd World Congress on Reproductive Biology」で発表し、「Centre for Reproductive Genomics Poster Award 2011」を受賞した。

その他

  • 2016年07月 - 2016年07月  2016年度 卓越研究員事業 卓越研究員候補者 
    雇用調整不良により辞退


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