Biology subjects

Haga, K.

Publications and source records attributed to Haga, K..

7 recordsLinked to original sources

bioRxiv2026

Near-atomic structures of human astrovirus capsids reveal a subunit-specific maturation mechanism

Human astrovirus (HAstV) is a major cause of acute gastroenteritis, yet the structural basis of capsid maturation and infectivity remains poorly understood. Here, we report near-atomic cryo-electron microscopy structures of HAstV serotype 4 in immature and mature states at 1.79 [A] and 1.73 [A] resolution, respectively. These structures reveal a subunit-specific maturation mechanism in which differential protease accessibility drives selective removal of spike (P2) domains. Structural comparisons show that only A-B dimers form a broad cavity beneath the P2 domains that is permissive for trypsin entry, whereas the corresponding region in C-C dimers is sterically restricted. Proteolytic maturation induces pronounced conformational rearrangements, particularly in subunit B, linking capsid geometry to asymmetric protease susceptibility. Despite these changes, the inner capsid architecture and presumptive RNA-associated densities remain conserved, indicating that genome release occurs at a later stage of infection. Together, our findings establish a mechanistic framework in which capsid geometry governs proteolysis-driven activation.

microbiology↗

bioRxiv2023

Production of Infectious Reporter Murine Norovirus by VP2 trans-Complementation

Human norovirus (HuNoV) causes gastroenteritis, a disease with no effective therapy or vaccine. Murine norovirus (MNV) easily replicates in cell culture and small animals and has often been used as a model to elucidate the structural and functional characteristics of HuNoV. A MNV plasmid-based reverse genetics system was developed to produce modified recombinant virus. In this study, we attempted to construct the recombinant virus by integrating a foreign gene into MNV ORF3 that encodes the minor structural protein VP2. We found that 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, we found that the coding sequence of C-terminal ORF3 was essential for cDNA clones compensated with VP2 to produce infectious particles. Further, the recombinant virus with exogenous reporter genes in place of the dispensable ORF3 coding region was able to propagate 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. IMPORTANCEIn this study, we revealed that some of the coding regions of ORF3 could be replaced by foreign gene and infectious virus could be produced under conditions with VP2 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 virus lifecycle and to the development of other caliciviral vectors for recombinant attenuated live enteric virus vaccines or therapeutics.

microbiology↗

bioRxiv2022

Neonatal Fc receptor is a functional receptor for human astrovirus

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 of FCGRT or B2M, 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.

microbiology↗

bioRxiv2021

Nasal delivery of single-domain antibodies improve symptoms of SARS-CoV-2 infection in an animal model

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the disease COVID-19 can lead to serious symptoms, such as severe pneumonia, in the elderly and those with underlying medical conditions. While vaccines are now available, they do not work for everyone and therapeutic drugs are still needed particularly for treating life-threatening conditions. Here, we showed nasal delivery of a new, unmodified camelid single-domain antibody (VHH), termed K-874A, effectively inhibited SARS-CoV-2 titers in infected lungs of Syrian hamsters without causing weight loss and cytokine induction. In vitro studies demonstrated that K-874A neutralized SARS-CoV-2 in both VeroE6/TMPRSS2 and human lung-derived alveolar organoid cells. Unlike other drug candidates, K-874A blocks viral membrane fusion rather than viral attachment. Cryo-electron microscopy revealed K-874A bound between the receptor binding domain and N-terminal domain of the virus S protein. Further, infected cells treated with K-874A produced fewer virus progeny that were less infective. We propose that direct administration of K-874A to the lung via a nebulizer could be a new treatment for preventing the reinfection of amplified virus in COVID-19 patients. Author summaryVaccines for COVID-19 are now available but therapeutic drugs are still needed to treat life-threatening cases and those who cannot be vaccinated. We discovered a new heavy-chain single-domain antibody that can effectively neutralize the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes COVID-19. Unlike other drug candidates, which prevent the virus from attaching to the receptor on the host cell, this new antibody acts by blocking the virus membrane from fusing with the host cell membrane. We studied the behavior of the new antibody in vitro using VeroE6/TMPRSS2 cells and human lung organoids. When delivered through the nose to infected Syrian hamsters, we found that this antibody could prevent the typical symptoms caused by SARS-CoV-2. Our results are significant because delivering simple drugs directly to infected lungs using a nebulizer could increase the potency of the drugs while reducing the risk of immune reaction that could occur if the drugs escape or are delivered through the blood stream.

microbiology↗

bioRxiv2020

The Phosphorylation Status of NPH3 Affects Photosensory Adaptation During the Phototropic Response

Photosensory adaptation, which can be classified as sensor or effector adaptation, optimizes the light sensing of living organisms by tuning their sensitivity to changing light conditions. During the phototropic response in Arabidopsis (Arabidopsis thaliana), the light-dependent expression controls of blue-light photoreceptor phototropin1 (phot1) and its modulator ROOT PHOTOTROPISM2 (RPT2) are known as the molecular mechanisms underlying sensor adaptation. However, little is known about effector adaption in plant phototropism. Here we show that control of the phosphorylation status of NONPHOTOTROPIC HYPOCOTYL3 (NPH3) leads to effector adaptation in hypocotyl phototropism. We identified seven phosphorylation sites of NPH3 proteins in the etiolated seedlings of Arabidopsis and generated unphosphorable and phosphomimetic NPH3 proteins on those sites. Unphosphorable NPH3 showed a shortening of its subcellular localization in the cytosol and caused an inability to adapt to very low fluence rates of blue light ([~]10-5 {micro}mol m-2 s-1) during the phototropic response. In contrast, the phosphomimetic NPH3 proteins had a lengthened subcellular localization in the cytosol and could not lead to the adaptation for blue light at fluence rates of 10-3 {micro}mol m-2 s-1 or more. Our results suggest that the activation levels of phot1 and the corresponding phosphorylation levels of NPH3 determine the rate of plasma membrane-cytosol shuttling of NPH3, which moderately maintains the active state of phot1 signaling across a broad range of blue-light intensities and contributes to the photosensory adaptation of phot1 signaling during the phototropic response in hypocotyls. One sentence summaryThe phosphorylation status of NON-PHOTOTROPIC HYPOCOTYL3 proteins affects their subcellular localization and the photosensory adaptation of phot1 signaling.

plant biology↗

bioRxiv2019

Dynamic rotation of the protruding domain enhances the infectivity of norovirus

Norovirus is the major cause of epidemic nonbacterial gastroenteritis worldwide. Lack of structural information on infection and replication mechanisms hampers the development of effective vaccines and remedies. Here, using cryo-electron microscopy, we show that the capsid structure of murine noroviruses changes in response to aqueous conditions. By twisting the flexible hinge connecting two domains, the protruding (P) domain reversibly rises off the shell (S) domain in solutions of higher pH, but rests on the S domain in solutions of lower pH. Metal ions help to stabilize the latter conformation in this process. Furthermore, in the resting conformation, the cellular receptor CD300lf is readily accessible, and thus infection efficiency is significantly enhanced. Two P domain conformations were also found in the human norovirus GII.3 capsid. These results provide new insights into the infection mechanisms of the non-envelope viruses that function in dramatic environmental changes such as the digestive tract. Significance StatementThe capsid structures of caliciviruses have been reported to be classified into two different types, according to species and genotype. One is the rising P domain type as shown in human norovirus GII.10 and rabbit hemorrhagic disease virus, where the P domain rises from the S domain surface. The other is the resting P domain type as shown in human norovirus GI.1, sapovirus and San Miguel sea lion virus, where the P domain rests upon the S domain. Here, we demonstrate that the P domain of the murine norovirus infectious particles changes reversibly between the rising and resting P domain types in response to aqueous conditions. Our findings provide new insights into the mechanisms of viral infection of caliciviruses.

pathology↗

bioRxiv2019

Membrane Protrusion Formation Mediated by Rho/ROCK Signalling and Modulation of Chloride Flux

Membrane protrusion is an important structural property associated with various cellular functions. The pentaspan membrane protein Prominin-1 (Prom1/CD133) is known to be localised to the protrusions and plays a pivotal role in migration and the determination of cellular morphology; however, the underlying mechanisms have been elusive. Here, we demonstrate that Prom1 is sufficient to trigger membrane protrusion formation. Overexpression of Prom1 in the RPE-1 cells triggers multiple long cholesterol-enriched protrusions, independently from actin and tubulin polymerisation. For this protrusion formation, the five amino acid stretch located at the carboxyl cytosolic region is essential. Moreover, the small GTPase Rho and its effector kinase ROCK are essential for this protrusion formation, and the intersection point of active Rho and Prom1 is where the protrusion formation initiates. Importantly, Prom1 causes the chloride ion efflux induced by calcium ion uptake, and protrusion formation is closely associated with the chloride efflux activity. Altogether, this study has elucidated that Prom1 plays critical roles for the membrane morphology and chloride ion flux.

cell biology↗