Biology subjects

Shimizu, R.

Publications and source records attributed to Shimizu, R..

12 recordsLinked to original sources

bioRxiv2026

Biosynthesis of prenylated flavonoids by two membrane-bound prenyltransferases in glandular trichomes of Macaranga tanarius.

Macaranga tanarius (Euphorbiaceae), a myrmecophilic plant grown in subtropical countries, was identified as a source of Okinawan propolis, a honeybee product, which is glandular trichomes developed on the surface of the fruits. This tissue contains a high amount of characteristic geranylated flavonoids known as nymphaeols (nymphaeol A - C and isonymphaeol B), which exhibit strong antioxidant activity. In this study, we characterized the structure of glandular trichomes of M. tanarius, showing that they are sac-like structures containing drupelet-like aggregates of secretary cells, and a subcuticular cavity was also observed. From a cDNA library, we identified two prenyltransferase (PT) genes involved in biosynthesis of nymphaeols, and a PT gene grouped in a primary metabolic PT. Our biochemical experiments demonstrated that one of the former secondary metabolic PTs (MtPT1) is a B-ring-specific geranyltransferase for eriodictyol to yield nymphaeols B and isonymphaeol B. MtPT1 was unusual for a plant PT, an enzyme that yielded multiple reaction products. The other PT (MtPT3) transferred a geranyl moiety to the A-ring of eriodictyol to form nymphaeol A. Although dimethylallyl diphosphate (DMAPP) was not recognized as its prenyl donor substrate of eriodictyol, giving a B-ring geranylated eriodyctiol as the prenyl acceptor substrate, this enzyme used DMAPP to attach the prenyl moiety to the A-ring and produced nymphaeol C. These data suggest that M. tanarius has evolved its PT function to produce a diverse range of prenylated flavonoids using a limited set of genes.

biochemistry↗

bioRxiv2026

APOBEC3 antagonism fully explains HIV-1 Vif essentiality under interferon and differentiation conditions

HIV-1 virion infectivity factor (Vif) counteracts APOBEC3 (A3) proteins by targeting them for proteasomal degradation, thereby preventing lethal G-to-A mutations and loss of infectivity. However, Vif also degrades additional cellular proteins, raising the possibility that its essential role in infectious virion production may extend beyond A3 antagonism, particularly under inflammatory or differentiation conditions. Whether such conditions reveal additional essential Vif targets remains unresolved. Here, using interferon (IFN)-stimulated or phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 monocytic cells, we directly addressed this question. Although type I IFN and PMA markedly suppressed viral production through Vif-independent mechanisms, {Delta}Vif viruses produced from stimulated parental cells exhibited severely reduced infectivity. In contrast, disruption of A3A-A3G fully restored {Delta}Vif infectivity to wild-type levels under all conditions tested. G-to-A mutations were attributable exclusively to A3 proteins under both IFN and PMA stimulation, whereas IFN-induced, A3-independent blocks to reverse transcription were not antagonized by Vif. Across diverse HIV-1 strains, the requirement for Vif was strictly dependent on A3 family proteins. These findings demonstrate that the essential role of Vif is fully explained by antagonism of A3-mediated restriction and that evolutionary pressure on Vif is driven predominantly by the need to counteract A3-mediated restriction and mutagenesis. ImportanceA3 proteins are potent intrinsic antiviral factors that restrict HIV-1 by inducing lethal mutations and impairing reverse transcription. The viral protein Vif counteracts A3-mediated restriction, yet it also targets additional host proteins, raising questions about whether its essential role extends beyond A3 antagonism under inflammatory conditions. Myeloid cells mount IFN responses and undergo transcriptional reprogramming upon differentiation, environments in which additional Vif functions could emerge. Using an A3A-A3G-null monocytic cell model, we show that even under IFN stimulation or PMA-induced differentiation, the requirement for Vif in maintaining HIV-1 infectivity is due to A3 proteins. In contrast, Vif does not counteract IFN-induced, A3-independent restriction pathways, highlighting functional specificity. These findings establish the A3-Vif axis as the central determinant of Vif dependency and indicate that evolutionary pressure on Vif is driven predominantly by the need to evade A3-mediated restriction. Targeting Vif may therefore expose HIV-1 to intrinsic inactivation, even in inflammatory environments.

microbiology↗

bioRxiv2025

Immunological characterization of peritoneal exudate cells in liver cirrhosis patients

BACKGROUND AND AIMSLiver cirrhosis (LC) is the end stage of liver fibrosis caused by various chronic liver diseases. Patients with LC often develop ascites containing peritoneal exudate cells (PECs). However, those cells have not been fully immunologically characterized. In this study, we clarify immune cell profiles of PECs from patients with LC. APPROACH AND RESULTSPECs were collected from patients with LC or patients receiving continuous ambulatory peritoneal dialysis (CAPD) as a non-cirrhotic control and subjected to single-cell RNA sequencing (scRNA-seq), bulk RNA sequencing (RNA-seq) and flowcytometry analyses. Analysis of scRNA-seq revealed that dendritic cells (DCs) and macrophages were major populations in CAPD patient-derived PECs, while those cells were decreased and T cells were most abundant in LC patient-derived PECs. Notably, FCGR3A-expressing macrophages were dominant over DCs and GATA6-expressing macrophages in LC patient-derived PECs. Bulk RNA-seq analysis further clarified expression of a set of genes was up- or down-regulated along with LC severity. Especially, expression of T cell signature genes was featured by its increase at Child-Pugh class B, but decrease at Child-Pugh class C. Flowcytometry analysis showed increase of T cells, decrease of DCs and macrophages, and increased expression of CD16 and CD163 in CD1cintCD14high cells corresponding to FCGR3A-expressing macrophages in LC patient-derived PECs. CONCLUSIONSIn LC patient-derived PECs, myeloid and T cell populations and their gene expression profiles were fluctuated with severity. Our findings should contribute to further development of diagnosis or therapeutic maneuver for LC.

immunology↗

bioRxiv2023

Virological characteristics of the SARS-CoV-2 BA.2.86 variant

In late 2023, a lineage of SARS-CoV-2 emerged and was named the BA.2.86 variant. BA.2.86 is phylogenetically distinct from other Omicron sublineages identified so far, displaying an accumulation of over 30 amino acid mutations in its spike protein. Here, we performed multiscale investigations to reveal the virological characteristics of the BA.2.86 variant. Our epidemic dynamics modeling suggested that the relative reproduction number of BA.2.86 is significantly higher than that of EG.5.1. Experimental studies showed that four clinically-available antivirals were effective against BA.2.86. Although the fusogenicity of BA.2.86 spike is similar to that of the parental BA.2 spike, the intrinsic pathogenicity of BA.2.86 in hamsters was significantly lower than that of BA.2. Since the growth kinetics of BA.2.86 is significantly lower than that of BA.2 in both in vitro cell cultures and in vivo, it is suggested that the attenuated pathogenicity of BA.2.86 is due to its decreased replication capacity.

microbiology↗

bioRxiv2023

Transmissibility, infectivity, and immune resistance of the SARS-CoV-2 BA.2.86 variant

In September 2023, the SARS-CoV-2 XBB descendants, such as XBB.1.5 and EG.5.1 (originally XBB.1.9.2.5.1), are predominantly circulating worldwide. Unexpectedly, however, a lineage distinct from XBB was identified and named BA.2.86 on August 14, 2023. Notably, BA.2.86 bears more than 30 mutations in the spike (S) protein when compared to XBB and the parental BA.2, and many of them are assumed to be associated with immune evasion. Although the number of reported cases is low (68 sequences have been reported as of 7 September 2023), BA.2.86 has been detected in several continents (Europe, North America and Africa), suggesting that this variant may be spreading silently worldwide. On 17 August 2023, the WHO designated BA.2.86 as a variant under monitoring. Here we show evidence suggesting that BA.2.86 potentially has greater fitness than current circulating XBB variants including EG.5.1. The pseudovirus assay showed that the infectivity of BA.2.86 was significantly lower than that of B.1.1 and EG.5.1, suggesting that the increased fitness of BA.2.86 is not due to the increased infectivity. We then performed a neutralization assay using XBB breakthrough infection sera to address whether BA.2.86 evades the antiviral effect of the humoral immunity induced by XBB subvariants. The 50% neutralization titer of XBB BTI sera against BA.2.86 was significantly (1.6-fold) lower than those against EG.5.1. The sera obtained from individuals vaccinated with 3rd-dose monovalent, 4th-dose monovalent, BA.1 bivalent, and BA.5 bivalent mRNA vaccines exhibited very little or no antiviral effects against BA.2.86. Moreover, the three monoclonal antibodies (Bebtelovimab, Sotrovimab and Cilgavimab), which worked against the parental BA.2, did not exhibit antiviral effects against BA.2.86. These results suggest that BA.2.86 is one of the most highly immune evasive variants ever.

microbiology↗

bioRxiv2023

Virological characteristics of the SARS-CoV-2 XBB.1.5 variant

Circulation of SARS-CoV-2 Omicron XBB has resulted in the emergence of XBB.1.5, a new Variant of Interest. Our phylogenetic analysis suggests that XBB.1.5 evolved from XBB.1 by acquiring the F486P spike (S) mutation, subsequent to the acquisition of a nonsense mutation in ORF8. Neutralization assays showed similar abilities of immune escape between XBB.1.5 and XBB.1. We determined the structural basis for the interaction between human ACE2 and the S protein of XBB.1.5, showing similar overall structures between the S proteins of XBB.1 and XBB.1.5. The intrinsic pathogenicity of XBB.1.5 in hamsters is lower than that of XBB.1. Importantly, we found that the ORF8 nonsense mutation of XBB.1.5 resulted in impairment of MHC expression. In vivo experiments using recombinant viruses revealed that the XBB.1.5 mutations are involved with reduced virulence of XBB.1.5. Together, these data suggest that the mutations in ORF8 and S could enhance spreading of XBB.1.5 in humans.

microbiology↗

bioRxiv2023

APOBEC3 degradation is the primary function of HIV-1 Vif for virus replication in the myeloid cell line THP-1

HIV-1 must overcome multiple innate antiviral mechanisms to replicate in CD4+ T lymphocytes and macrophages. Previous studies have demonstrated that the APOBEC3 (A3) family of proteins (at least A3D, A3F, A3G, and stable A3H haplotypes) contribute to HIV-1 restriction in CD4+ T lymphocytes. Virus-encoded virion infectivity factor (Vif) counteracts this antiviral activity by degrading A3 enzymes allowing HIV-1 replication in infected cells. In addition to A3 proteins, Vif also targets other cellular proteins in CD4+ T lymphocytes, including PPP2R5 proteins. However, whether Vif primarily degrades only A3 proteins or has additional essential targets during viral replication is currently unknown. Herein, we describe the development and characterization of A3F-, A3F/A3G-, and A3A-to-A3G-null THP-1 cells. In comparison to Vif-proficient HIV-1, Vif-deficient viruses have substantially reduced infectivity in parental and A3F-null THP-1 cells, and a more modest decrease in infectivity in A3F/A3G-null cells. Remarkably, disruption of A3A-A3G protein expression completely restores the infectivity of Vif-deficient viruses in THP-1 cells. These results indicate that the primary function of Vif during HIV-1 replication in THP-1 cells is the targeting and degradation of A3 enzymes. ImportanceHIV-1 Vif neutralizes the HIV-1 restriction activity of A3 proteins. However, it is currently unclear whether Vif has additional essential cellular targets. To address this question, we disrupted A3A to A3G genes in the THP-1 myeloid cell line using CRISPR and compared the infectivity of wildtype HIV-1 and Vif mutants with the selective A3 neutralization activities. Our results demonstrate that the infectivity of Vif-deficient HIV-1 and the other Vif mutants is fully restored by ablating the expression of cellular A3A to A3G proteins. These results indicate that A3 proteins are the only essential target of Vif that is required for HIV-1 replication in THP-1 cells.

microbiology↗

bioRxiv2022

Virological characteristics of the novel SARS-CoV-2 Omicron variants including BA.2.12.1, BA.4 and BA.5

After the global spread of SARS-CoV-2 Omicron BA.2 lineage, some BA.2-related variants that acquire mutations in the L452 residue of spike protein, such as BA.2.9.1 and BA.2.13 (L452M), BA.2.12.1 (L452Q), and BA.2.11, BA.4 and BA.5 (L452R), emerged in multiple countries. Our statistical analysis showed that the effective reproduction numbers of these L452R/M/Q-bearing BA.2-related Omicron variants are greater than that of the original BA.2. Neutralization experiments revealed that the immunity induced by BA.1 and BA.2 infections is less effective against BA.4/5. Cell culture experiments showed that BA.2.12.1 and BA.4/5 replicate more efficiently in human alveolar epithelial cells than BA.2, and particularly, BA.4/5 is more fusogenic than BA.2. Furthermore, infection experiments using hamsters indicated that BA.4/5 is more pathogenic than BA.2. Altogether, our multiscale investigations suggest that the risk of L452R/M/Q-bearing BA.2-related Omicron variants, particularly BA.4 and BA.5, to global health is potentially greater than that of original BA.2. HighlightsO_LISpike L452R/Q/M mutations increase the effective reproduction number of BA.2 C_LIO_LIBA.4/5 is resistant to the immunity induced by BA.1 and BA.2 infections C_LIO_LIBA.2.12.1 and BA.4/5 more efficiently spread in human lung cells than BA.2 C_LIO_LIBA.4/5 is more pathogenic than BA.2 in hamsters C_LI

microbiology↗

bioRxiv2022

SARS-CoV-2 spike S375F mutation characterizes the Omicron BA.1 variant

Recent studies have revealed the unique virological characteristics of Omicron, the newest SARS-CoV-2 variant of concern, such as pronounced resistance to vaccine-induced neutralizing antibodies, less efficient cleavage of the spike protein, and poor fusogenicity. However, it remains unclear which mutation(s) in the spike protein determine the virological characteristics of Omicron. Here, we show that the representative characteristics of the Omicron spike are determined by its receptor-binding domain. Interestingly, the molecular phylogenetic analysis revealed that the acquisition of the spike S375F mutation was closely associated with the explosive spread of Omicron in the human population. We further elucidate that the F375 residue forms an interprotomer pi-pi interaction with the H505 residue in another protomer in the spike trimer, which confers the attenuated spike cleavage efficiency and fusogenicity of Omicron. Our data shed light on the evolutionary events underlying Omicron emergence at the molecular level. HighlightsO_LIOmicron spike receptor binding domain determines virological characteristics C_LIO_LISpike S375F mutation results in the poor spike cleavage and fusogenicity in Omicron C_LIO_LIAcquisition of the spike S375F mutation triggered the explosive spread of Omicron C_LIO_LIF375-H505-mediated {pi}-{pi} interaction in the spike determines the phenotype of Omicron C_LI

microbiology↗

bioRxiv2022

Virological characteristics of SARS-CoV-2 BA.2 variant

Soon after the emergence and global spread of a new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron lineage, BA.1 (ref1, 2), another Omicron lineage, BA.2, has initiated outcompeting BA.1. Statistical analysis shows that the effective reproduction number of BA.2 is 1.4-fold higher than that of BA.1. Neutralisation experiments show that the vaccine-induced humoral immunity fails to function against BA.2 like BA.1, and notably, the antigenicity of BA.2 is different from BA.1. Cell culture experiments show that BA.2 is more replicative in human nasal epithelial cells and more fusogenic than BA.1. Furthermore, infection experiments using hamsters show that BA.2 is more pathogenic than BA.1. Our multiscale investigations suggest that the risk of BA.2 for global health is potentially higher than that of BA.1.

microbiology↗

bioRxiv2021

SARS-CoV-2 spike P681R mutation enhances and accelerates viral fusion

During the current SARS-CoV-2 pandemic, a variety of mutations have been accumulated in the viral genome, and currently, four variants of concerns (VOCs) are considered as the hazardous SARS-CoV-2 variants to the human society1. The newly emerging VOC, the B.1.617.2/Delta variant, closely associates with a huge COVID-19 surge in India in Spring 20212. However, its virological property remains unclear. Here, we show that the B.1.617.2/Delta variant is highly fusogenic, and notably, more pathogenic than prototypic SARS-CoV-2 in infected hamsters. The P681R mutation in the spike protein, which is highly conserved in this lineage, facilitates the spike protein cleavage and enhances viral fusogenicity. Moreover, we demonstrate that the P681R-bearing virus exhibits higher pathogenicity than the parental virus. Our data suggest that the P681R mutation is a hallmark that characterizes the virological phenotype of the B.1.617.2/Delta variant and is closely associated with enhanced pathogenicity.

microbiology↗

bioRxiv2019

A non-mosaic humanized mouse model of Down syndrome, trisomy of the long arm of human chromosome 21 in mouse chromosome background

Down syndrome (DS) is a complex human condition, and animal models trisomic for human chromosome 21 (HSA21) genes or orthologs provide insights into better understanding and treating DS. However, HSA21 orthologs are distributed into three mouse chromosomes, preventing us from generating mouse models trisomy of a complete set of HSA21 orthologs. The only existing humanized mouse DS model, Tc1, carries a HSA21 with over 20% of protein coding genes (PCGs) disrupted. More importantly, due to the human centromere, Tc1 is mosaic (a mix of euploid and trisomic cells), which makes every mouse unique and compromises interpretation of results. Here, we used mouse artificial chromosome (MAC) technology to "clone" the 34 MB long arm of HSA21 (HSA21q). Through multiple steps of microcell-mediated chromosome transfer we created a new humanized DS mouse model, Tc(HSA21q;MAC)1Yakaz ("TcMAC21"). Constitutive EGFP expression from the transchromosome and fluorescent in situ hybridization validate that TcMAC21, containing a hybrid chromosome of HSA21q and mouse centromere, is not mosaic. Whole genome sequencing shows that TcMAC21 contains a nearly complete copy of HSA21q with 93% of intact PCGs, while RNA-seq and additional mRNA/protein expression analyses confirm that PCGs are transcribed and regulated. A battery of tests show that TcMAC21 recapitulates many DS phenotypes including morphological anomalies in heart, craniofacial skeleton and brain, pathologies at molecular and cellular level, and impairments in learning, memory and synaptic plasticity. TcMAC21 is the most complete mouse model of DS extant and has potential for supporting a wide range of basic and preclinical research. Significance StatementIn the last 25 years, mouse models of trisomy 21 have supported research into Down syndrome, from defining the basis for developmental effects up to support for clinical trials. However, existing models have significant shortfalls, especially for preclinical studies. These deficiencies include incomplete or inappropriate representation of trisomic genes, absence of an extra chromosome, and mosaicism. Using cutting edge technologies we produced a mouse artificial chromosome containing the entire 34Mb long arm of human chromosome 21 and, with assisted reproductive technologies, established it in the germ line of mice. This trisomic mouse manifests developmental and functional features of Down syndrome, including hippocampal-based learning and memory deficits. This is the most complete model of Down syndrome produced to date.

genetics↗