Biology subjects

Takemura, M.

Publications and source records attributed to Takemura, M..

7 recordsLinked to original sources

bioRxiv2024

Subnanometer structure of medusavirus capsid during maturation using cryo-electron microscopy

Medusavirus is a giant virus classified into an independent family of Mamonoviridae. Medusavirus-infected amoebae release immature particles in addition to the virions. These particles were suggested to exhibit the maturation process of this virus, but the structure of these capsids during maturation remains unknown. Here, we applied a block-based reconstruction method in cryo-electron microscopy (cryo-EM) single particle analysis to these viral capsids, extending these resolutions to 7-10 [A]. The map revealed a novel network consisted of minor capsid proteins (mCPs), supporting major capsid proteins (MCPs). A predicted molecular model of the MCP fitted into the cryo-EM map clarified the boundaries between the MCP and the underlining mCPs and between the MCP and the outer spikes, identifying the molecular interactions between the MCP and these components. Several structural changes of mCPs were observed beneath the 5-fold vertices of immature particles, depending on the presence or absence of the underlying internal membrane. Furthermore, the lower part of penton proteins on the 5-fold vertices were also lost in the mature virions. These dynamic structural changes of mCPs exhibit important functions in the maturation process of medusavirus. ImportanceStructural changes in giant virus capsids during maturation have not been known. Medusavirus is a unique giant virus, where amoebae infected with the virus release immature particles in addition to mature virions. In this study, immature and mature medusavirus particles were investigated using cryo-electron microscopy, and firstly reported the structural changes in the viral capsid during maturation. In DNA empty particles, the conformation of the minor capsid proteins dynamically changed depending on the presence or absence of the underlying internal membranes. In DNA full particles, the lower part of the penton proteins was lost. These are the first report of the structural changes in the viral capsid during giant virus maturation.

molecular biology↗

bioRxiv2021

Particle morphology of medusavirus inside and outside the cells reveals a new maturation process of giant viruses

Medusavirus, a giant virus, is phylogenetically closer to eukaryotes than the other giant viruses and has been recently classified as an independent species. However, details of its morphology and maturation process in host cells remain unclear. Here, we investigated the particle morphology of medusavirus inside and outside infected cells using conventional transmission electron microscopy (C-TEM) and cryo-electron microscopy (cryo-EM). The C-TEM of amoeba infected with the medusavirus showed four types of particles: empty, DNA-full, and the corresponding intermediates. Time-dependent changes in the proportion and following intracellular localization of these particles suggested a new maturation process for the medusavirus. Empty particles and viral DNAs were produced independently in the cytoplasm and nucleus, respectively, and only empty particles located near the nucleus incorporated the viral DNA into the capsid. All four types of particles were also found outside the cells. The cryo-EM of these particles showed that the intact capsid structure, covered with three different types of spikes, was conserved among all particle types, although with minor size-related differences. The internal membrane exhibited a structural array similar to that of the capsid, interacted closely with the capsid, and displayed open membrane structures in the empty and empty-intermediate particles. This result suggests that the open structures in the internal membrane are used for an exchange of scaffold proteins and viral DNA during the maturation process. This new model of the maturation process of medusavirus provides insight into the structural and behavioral diversity of giant viruses. ImportanceGiant viruses exhibit diverse morphologies and maturation processes. In the present study, medusavirus showed four types of particle morphologies both inside and outside the infected cells, when propagated in the laboratory using amoeba culture. Time-course analysis of the medusavirus particles in the infected cells reveals a new maturation process. Empty viral particles and viral DNAs were produced independently in the cytoplasm and nucleus, and only the empty particles located near the nucleus incorporated the viral DNA. Consequently, many immature particles, along with mature virions, were released from the host cells. Except for showing a small change in size, the capsid structures were well preserved during the maturation process. The empty viral particles and corresponding intermediates showed open membrane structures, which are presumably used for exchanging scaffold proteins and viral DNAs.

microbiology↗

bioRxiv2021

RNA-seq of the medusavirus suggests remodeling of the host nuclear environment at an early infection stage

Nucleo-cytoplasmic large DNA viruses (NCLDVs) undergo a cytoplasmic or nucleo-cytoplasmic cycle, and the latter involves both nuclear and cytoplasmic compartments to proceed viral replication. Medusavirus, a recently isolated NCLDV, has a nucleo-cytoplasmic replication cycle in amoebas during which the host nuclear membrane apparently remains intact, a unique feature among amoeba-infecting giant viruses. The medusavirus genome lacks most transcription genes but encodes a full set of histone genes. To investigate the infection strategy, we performed a time-course RNA-seq experiment. All the viral genes were transcribed and classified into five temporal expression clusters. The immediate early genes (cluster 1, 42 genes) were mostly (83%) of unknown functions, frequently (95%) associated with a palindromic promoter-like motif, and enriched (45%) in putative nuclear-targeting genes. The later genes (clusters 2-5) were assigned to various functional categories. The viral linker histone H1 gene was in cluster 1, whereas the four core histone genes were in cluster 3, suggesting they had distinct roles during the course of the virus infection. The transcriptional profile of the host amoeba, Acanthamoeba castellanii, genes was greatly altered post-infection. Several encystment-related host genes showed increased representation levels at 48 hours post-infection, which is consistent with the previously reported amoeba encystment upon medusavirus infection. Overall, the transcriptional landscape during the course of medusavirus infection suggests that the virus modifies the host nuclear environment immediately after the initiation of infection. - ImportanceMedusavirus is an amoeba-infecting giant virus that was isolated from a hot spring in Japan. It belongs to the proposed family "Medusaviridae" in the phylum Nucleocytoviricota. Unlike other amoeba-infecting giant viruses, medusavirus initiates its DNA replication in the host nucleus without disrupting the nuclear membrane. Our RNA-seq analysis of its infection course uncovered ordered viral gene expression profiles. We identified temporal expression clusters of viral genes and associated putative promoter motifs. The subcellular localization prediction showed a clear spatiotemporal correlation between gene expression timing and localization of the encoded proteins. Notably, the immediate early expression cluster was enriched in genes targeting the nucleus, suggesting the priority of remodeling the host intra-nuclear environment during infection. The transcriptional profile of the amoeba genes was greatly altered post-infection. Notably, the expression of encystment-related genes increased 48 hours post-infection, suggesting that encystment may be an antiviral strategy of amoeba.

microbiology↗

bioRxiv2020

DAJIN-assisted multiplex genotyping to validate the outcomes of CRISPR-Cas-based genome editing

Genome editing can introduce designed mutations into a target genomic site. Recent research has revealed that it can also induce various unintended events such as structural variations, small indels, and substitutions at, and in some cases, away from the target site. These rearrangements may result in confounding phenotypes in biomedical research samples and cause a concern in clinical or agricultural applications. However, current genotyping methods do not allow a comprehensive analysis of diverse mutations for phasing and mosaic variant detection. Here, we developed a genotyping method with an on-target site analysis software named Determine Allele mutations and Judge Intended genotype by Nanopore sequencer (DAJIN) that can automatically identify and classify both intended and unintended diverse mutations, including point mutations, deletions, inversions, and cis double knock-in at single-nucleotide resolution. Our approach with DAJIN can handle approximately 100 samples under different editing conditions in a single run. With its high versatility, scalability, and convenience, DAJIN-assisted multiplex genotyping may become a new standard for validating genome editing outcomes.

genomics↗

bioRxiv2020

Comparative analysis of antigen-specific anti-SARS-CoV-2 antibody isotypes in COVID-19 patients

Serological tests for detection of anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies in blood are expected to identify individuals who have acquired immunity against SARS-CoV-2 and indication of seroprevalence of SARS-CoV-2 infection. Many serological tests have been developed to detect antibodies against SARS-CoV-2. However, these tests have considerable variations in their specificity and sensitivity, and whether they can predict levels of neutralizing activity is yet to be determined. This study aimed to investigate the kinetics and neutralizing activity of various antigen-specific antibody isotypes against SARS-CoV-2 in serum of coronavirus disease 2019 (COVID-19) patients confirmed via polymerase chain reaction test. We developed IgG, IgM and IgA measurement assays for each antigen, including receptor-binding domain (RBD) of spike (S) protein, S1 domain, full length S protein, S trimer and nucleocapsid (N) domain, based on enzyme-linked immunosorbent assay. The assays of the S protein for all isotypes showed high specificity, while the assays for all isotypes against N protein showed lower specificity. The sensitivity of all antigen-specific antibody isotypes depended on the timing of the serum collection and all of them, except for IgM against N protein, reached more than 90% at 15-21 days post-symptom onset. The best correlation with virus neutralizing activity was found for IgG against RBD (RBD-IgG), and levels of RBD-IgG in sera from four severe COVID-19 patients increased concordantly with neutralizing activity. Our results provide valuable information regarding the selection of serological test for seroprevalence and vaccine evaluation studies.

immunology↗

bioRxiv2019

Streptococcus pneumoniae evades host cell phagocytosis and limits host mortality through its cell wall anchoring protein PfbA

Streptococcus pneumoniae is a Gram-positive bacterium belonging to the oral streptococcus species, mitis group. This pathogen is a leading cause of community-acquired pneumonia, which often evades host immunity and causes systemic diseases, such as sepsis and meningitis. Previously, we reported that PfbA is a {beta}-helical cell surface protein contributing to pneumococcal adhesion to and invasion of human epithelial cells in addition to its survival in blood. In the present study, we investigated the role of PfbA in pneumococcal pathogenesis. Phylogenetic analysis indicated that the pfbA gene is specific to S. pneumoniae within the mitis group. Our in vitro assays showed that PfbA inhibits neutrophil phagocytosis, leading to pneumococcal survival. We found that PfbA activates NF-{kappa}B through TLR2, but not TLR4. In addition, TLR2/4 inhibitor peptide treatment of neutrophils enhanced the survival of the S. pneumoniae {Delta}pfbA strain as compared to a control peptide treatment, whereas the treatment did not affect survival of a wild-type strain. In a mouse pneumonia model, the host mortality and level of TNF- in bronchoalveolar lavage fluid were comparable between wild-type and {Delta}pfbA-infected mice, while deletion of pfbA increased the bacterial burden in bronchoalveolar lavage fluid. In a mouse sepsis model, the {Delta}pfbA strain demonstrated significantly increased host mortality and TNF- levels in plasma, but showed reduced bacterial burden in lung and liver. These results indicate that PfbA may contribute to the success of S. pneumoniae species by inhibiting host cell phagocytosis, excess inflammation, and mortality.\n\nImportanceStreptococcus pneumoniae is often isolated from the nasopharynx of healthy children, but the bacterium is also a leading cause of pneumonia, meningitis, and sepsis. In this study, we focused on the role of a cell wall anchoring protein, PfbA, in the pathogenesis of S. pneumoniae-related disease. We found that PfbA is a pneumococcus-specific anti-phagocytic factor that functions as a TLR2 ligand, indicating that PfbA may represent a pneumococcal-specific therapeutic target. However, a mouse pneumonia model revealed that PfbA deficiency reduced the bacterial burden, but did not decrease host mortality. Furthermore, in a mouse sepsis model, PfbA deficiency increased host mortality. These results suggest that S. pneumoniae optimizes reproduction by regulating host mortality through PfbA; therefore, PfbA inhibition would not be an effective strategy for combatting pneumococcal infection. Our findings underscore the challenges involved in drug development for a bacterium harboring both commensal and pathogenic states.

microbiology↗

bioRxiv2018

Chondroitin sulfate proteoglycan Windpipe modulates Hedgehog signaling in Drosophila

Proteoglycans, a class of carbohydrate-modified proteins, often modulate growth factor signaling on the cell surface. However, the molecular mechanism by which proteoglycans regulate signal transduction is largely unknown. In this study, using a recently-developed glycoproteomic method, we found that Windpipe (Wdp) is a novel chondroitin sulfate proteoglycan (CSPG) in Drosophila. Wdp is a single-pass transmembrane protein with leucin-rich repeat (LRR) motifs and bears three CS sugar chain attachment sites in the extracellular domain. Here we show that Wdp modulates the Hedgehog (Hh) pathway. Overexpression of wdp inhibits Hh signaling in the wing disc, which is dependent on its CS chains and the LRR motifs. Conversely, loss of wdp leads to the upregulation of Hh signaling. Furthermore, knockdown of wdp increase the cell surface accumulation of Smoothened (Smo), suggesting that Wdp inhibits Hh signaling by regulating Smo stability. Our study demonstrates a novel role of CSPG in regulating Hh signaling.

cell biology↗