Biology subjects

Ryo, A.

Publications and source records attributed to Ryo, A..

4 recordsLinked to original sources

bioRxiv2026

E3 ubiquitin ligase HUWE1 mediates K6-linked polyubiquitylation and stabilization of Nrf2 in an HBx-dependent manner, thereby inhibit ing hepatitis B virus replication

Hepatitis B virus (HBV) infection remains a major global health burden, and HBV X protein (HBx) plays a central role in modulating host pathways that influence viral replication. We previously reported that the oxidative stress sensor Kelch-like ECH-associated protein 1 (Keap1) recognizes HBx to activate the NF-E2-related factor 2 (Nrf2) signaling pathway to suppress HBV replication. Although canonical K48-linked ubiquitylation is known to control Nrf2 turnover, the contribution of non-canonical ubiquitin linkages to Nrf2 regulation during HBV infection remains unclear. Here, we investigated the role of HECT, UBA, and WWE domain-containing E3 ubiquitin ligase 1 (HUWE1) in the regulation of Nrf2 in the context of HBV replication. Cell-based ubiquitylation assays demonstrated that HUWE1 knockdown reduced HBx-mediated K6-linked polyubiquitylation of Nrf2, while overexpression of wild-type HUWE1, but not the catalytically inactive HUWE1(C4341A) mutant, enhanced it. Coimmunoprecipitation and proximity ligation assays demonstrated that HUWE1 interacts with HBx in the cytoplasm and binds Nrf2 only in the presence of HBx, suggesting that HBx promotes the interaction between HUWE1 and Nrf2. Cycloheximide chase assays demonstrated that HUWE1 knockdown destabilized Nrf2 in HBx-expressing cells. Furthermore, depletion or pharmacological inhibition of HUWE1 increased intracellular HBV RNA and pgRNA levels as well as extracellular HBV DNA and HBsAg levels in HBV-infected cells. Collectively, these results support a model in which HUWE1 mediates HBx-dependent K6-linked polyubiquitylation and stabilization of Nrf2 to restrict HBV replication. This study expands current understanding of non-canonical ubiquitin signaling in HBV-host interactions. DATA SUMMARYAll data are presented in the main figures. The data that support the findings of this study is available at bioRxiv (https://doi.org/10.64898/2026.04.20.719611). Raw sequencing data, microscopy images, materials, and sequence information are available upon request. Correspondence and requests for materials should be addressed to Professor Ikuo Shoji. IMPACT STATEMENTHepatitis B virus (HBV) chronically infects approximately 254 million people worldwide, yet host mechanisms that restrict viral replication remain incompletely understood. The Keap1/ Nrf2 signaling pathway is a central defense against oxidative stress. Under basal conditions, Nrf2 is targeted for degradation via Keap1/Cullin3-mediated K48-linked polyubiquitylation. Here, we provide evidence that the E3 ubiquitin ligase HUWE1 contributes to HBx-dependent K6-linked polyubiquitylation and stabilization of Nrf2. Our findings support a model in which non-canonical ubiquitin signaling helps shape the HBV-host interactions and contributes to suppression of viral replication. This study extends current understanding of the ubiquitin code in HBV infection and highlights HUWE1 as a candidate component of an anti-HBV regulatory pathway.

microbiology↗

bioRxiv2024

Neutralizing antibody evasion of SARS-CoV-2 JN.1 derivatives KP.3, KP.3.1.1, LB.1, and XEC

The emergence of SARS-CoV-2 variants poses ongoing challenges to vaccine efficacy. We evaluated neutralizing antibody responses against JN.1 and its derivatives (KP.3, KP.3.1.1, LB.1, and XEC) in healthcare workers who received seven doses of BNT162b2, including XBB.1.5 monovalent vaccine. In COVID-19-naive individuals, KP.3.1.1 and LB.1 showed substantial immune escape, while previously infected individuals maintained neutralization activity against all variants. We also demonstrated that JN.1-based immunization induces robust cross-neutralizing activity against emerging variants. A single amino acid deletion at position 31 in the spike protein significantly impacted immune evasion. These findings support the potential effectiveness of JN.1-based vaccines while highlighting the need for continued surveillance and vaccine optimization.

immunology↗

bioRxiv2024

Rapid neutralizing assay for circulating H5N1 influenza virus in dairy cows

A rapid and safe neutralization assay is required for emerging highly pathogenic avian influenza viruses, including the H5N1 subtype, which was recently found in cows. Herein, we report a novel neutralization assay using HiBiT-tagged virus-like particles (hiVLPs). Our hiVLP-based neutralization test demonstrated a higher quantitative value and shorter assay time than conventional methods. We used this assay to evaluate whether the neutralizing antibodies induced by the candidate vaccine virus (NIID-002) were cross-reactive with cow-derived H5N1. Our results suggest that the circulating H5N1 virus in cows shares antigenic characteristics with NIID-002, providing significant implications for the development and preparation of vaccines.

microbiology↗

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↗