Biology subjects

Anzai, I.

Publications and source records attributed to Anzai, I..

5 recordsLinked to original sources

bioRxiv2025

A divergent betacoronavirus with a functional furin cleavage site in South American bats

Bats are natural reservoirs for a wide range of RNA viruses. Members of the genus Betacoronavirus, including Severe Acute Respiratory Syndrome virus 2 (SARS-CoV-2) and Middle East Respiratory Syndrome virus (MERS-CoV), have attracted particular attention due to their recent zoonotic emergence. However, much of the known diversity of betacoronaviruses is based on data from Asia, Africa, and the Middle East, with limited genomic information available from the Americas. Herein, we report the complete genome of a novel bat betacoronavirus identified from a Pteronotus parnellii bat sampled in Brazil. Phylogenetic analysis revealed that this virus is sufficiently distinct from the five recognized Betacoronavirus subgenera to represent a new subgenus. Of note, the spike protein of this novel bat coronavirus possesses a functional furin cleavage site at the S1/S2 junction with a unique amino acid sequence motif (RDAR) that differs from that found in SARS-CoV-2 (RRAR) by only one amino acid. Comparative structural analysis identified other betacoronaviruses in bats with furin cleavage sites at the S1/S2 junction, suggesting that this region is a structurally permissive "hotspot" for cleavage site incorporation. Our study provides a broader understanding of the phylogenetic and functional diversity of bat coronaviruses as well as their zoonotic potential.

microbiology↗

bioRxiv2021

Epoxidized graphene grid for high-throughput high-resolution cryoEM structural analysis

Many specimens suffer from low particle density and/or preferred orientation in cryoEM specimen grid preparation, making data collection and structure determination time consuming. We developed an epoxidized graphene grid (EG-grid) that effectively immobilizes protein particles by applying an oxidation reaction using photoactivated ClO2* and further chemical modification. The particle density and orientation distribution are both dramatically improved, having enabled us to reconstruct the density map of GroEL and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), at 1.99 and 2.16 [A] resolution from only 504 and 241 micrographs, respectively. A low concentration sample solution of 0.1 mg ml-1 was sufficient to reconstruct a 3.10 [A] resolution density map of SARS-CoV-2 spike protein from 1,163 micrographs. The density maps of V1-ATPase, {beta}-galactosidase, and apoferritin were also reconstructed at 3.03, 1.81, and 1.29 [A] resolution, respectively. These results indicate that the EG-grid will be a powerful tool for high-throughput cryoEM data collection to accelerate high-resolution structural analysis of biological macromolecules.

molecular biology↗

bioRxiv2021

Nanobodies recognizing conserved hidden clefts of all SARS-CoV-2 spike variants

We are in the midst of the historic coronavirus infectious disease 2019 (COVID-19) pandemic caused by severe respiratory syndrome coronavirus 2 (SARS-CoV-2). Although countless efforts to control the pandemic have been attempted--most successfully, vaccination1-3--imbalances in accessibility to vaccines, medicines, and diagnostics among countries, regions, and populations have been problematic. Camelid variable regions of heavy chain-only antibodies (VHHs or nanobodies)4 have unique modalities: they are smaller, more stable, easier to customize, and, importantly, less expensive to produce than conventional antibodies5, 6. We present the sequences of nine alpaca nanobodies that detect the spike proteins of four SARS-CoV-2 variants of concern (VOCs)--namely, the alpha, beta, gamma, and delta variants. We show that they can quantify or detect spike variants via ELISA and lateral flow, kinetic, flow cytometric, microscopy, and Western blotting assays7. The panel of nanobodies broadly neutralized viral infection by pseudotyped SARS-CoV-2 VOCs. Structural analyses showed that a P86 clone targeted epitopes that were conserved yet unclassified on the receptor-binding domain (RBD) and located inside the N-terminal domain (NTD). Human antibodies have hardly accessed both regions; consequently, the clone buries hidden crevasses of SARS-CoV-2 spike proteins undetected by conventional antibodies and maintains activity against spike proteins carrying escape mutations.

molecular biology↗

bioRxiv2021

Identification of a Pathway for Electron Uptake in Shewanella oneidensis

Extracellular electron transfer (EET) could enable electron uptake into microbial metabolism for the synthesis of complex, energy dense organic molecules from CO2 and renewable electricity1-6 . EET could do this with an efficiency comparable to H2 -oxidation7,8 but without the need for a volatile intermediate and the problems it causes for scale up9. However, naturally occurring electroactive organisms suffer from a number of technical drawbacks. Correcting these will require extensive knowledge of the genetics and mechanisms of electron uptake. To date, studies of electron uptake in electroactive microbes have focused on shared molecular machinery also used for anaerobic mineral reduction, like the Mtr EET complex in the electroactive microbe Shewanella oneidensis MR-110-14. However, this shared machinery cannot explain all features of electron uptake, hindering efforts to engineer electron uptake. To address this, we screened gene disruption mutants for 3,667 genes, representing {approx} 99% of all non-essential genes, from the S. oneidensis whole genome knockout collection using a redox dye oxidation assay as a proxy for electron uptake. Confirmation of electron uptake using electrochemical testing allowed us to identify five genes from S. oneidensis that are indispensable for electron uptake from a cathode. Knockout of each gene eliminates extracellular electron uptake, yet in 4 of the 5 cases produces no significant defect in electron donation to an anode, highlighting a distinct role for these loci in electron uptake vs. donation. This result highlights an electronic connection between aerobic and anaerobic electron transport chains that allow electrons from the reversible EET machinery to be coupled to different respiratory processes in S. oneidensis. Furthermore, we find homologs to these genes across many different genera suggesting that electron uptake by EET coupled to respiration could be a widespread phenomenon. These gene discoveries provide a foundation for studying this phenotype in exotic metal-oxidizing autotrophic microbes. Additionally, we anticipate that the characterization of these genes will allow for the genetic improvement of electron uptake in S. oneidensis; and genetically engineering electron uptake into a highly tractable host like E. coli to complement recent advances in synthetic CO2 fixation15.

systems biology↗

bioRxiv2020

Establishment of a reverse genetics system for SARS-CoV-2 using circular polymerase extension reaction

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been identified as the causative agent of coronavirus disease 2019 (COVID-19). While the development of specific treatments and a vaccine is urgently needed, functional analyses of SARS-CoV-2 have been limited by the lack of convenient mutagenesis methods. In this study, we established a PCR-based, bacterium-free method to generate SARS-CoV-2 infectious clones. Recombinant SARS-CoV-2 could be rescued at high titer with high accuracy after assembling 10 SARS-CoV-2 cDNA fragments by circular polymerase extension reaction (CPER) and transfection of the resulting circular genome into susceptible cells. Notably, the construction of infectious clones for reporter viruses and mutant viruses could be completed in two simple steps: introduction of reporter genes or mutations into the desirable DNA fragments (~5,000 base pairs) by PCR and assembly of the DNA fragments by CPER. We hope that our reverse genetics system will contribute to the further understanding of SARS-CoV-2.

microbiology↗