Biology subjects

Matsui, C.

Publications and source records attributed to Matsui, C..

3 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↗

bioRxiv2025

Hepatitis C virus NS3/4A protease cleaves SPG20, a key regulator of lipid droplet turnover, to promote lipid droplet formation

ABSTRACTSHepatitis C virus (HCV) assembles in close proximity to lipid droplets (LDs), which play important roles in HCV RNA replication. HCV infection often causes the accumulation of large LDs in hepatocytes. However, the molecular mechanism underlying HCV-induced large LD formation is poorly understood. It has been reported that the SPG20/Spartin protein associates with the LD surface and plays a crucial role in LD turnover by recruiting the ubiquitin ligase Itch to promote the ubiquitin-dependent degradation of adipophilin (ADRP), which protects LDs from lipase-mediated degradation. To elucidate the mechanism underlying HCV-induced large LD formation, we investigated the SPG20 proteins role in LD formation in HCV J6/JFH1-infected Huh-7.5 cells. Immunoblot analysis revealed that HCV infection promoted SPG20 protein cleavage. Transfection of increasing amounts of NS3/4A, but not the inactive NS3/4A mutant, resulted in SPG20 cleavage, implicating the NS3/4A protease in this cleavage. Site-directed mutagenesis suggested that the NS3/4A protease cleaves SPG20 at Cys504 and Cys562. The SPG20 protein was co-immunoprecipitated with the LD-attached protein TIP47. Increasing amounts of NS3/4A protease, but not inactive NS3/4A, decreased the co-precipitation of SPG20 with TIP47. The siRNA-mediated knockdown of Itch in Huh-7.5 cells restored ADRP levels, suggesting that Itch mediates ubiquitylation-dependent ADRP degradation. Immunofluorescence staining of HCV-infected cells revealed that ADRP was localized mainly around LDs in HCV-infected cells, whereas cytosolic ADRP was decreased. We propose that the HCV NS3/4A protease specifically cleaves SPG20 and inhibits Itch-mediated ubiquitin-dependent degradation of LD-associated ADRP, thereby promoting the formation of large LDs. IMPORTANCEHCV infection often promotes the formation of large LDs in HCV-infected cells. However, the molecular mechanism underlying large LD formation is poorly understood. LD turnover is regulated by SPG20, Itch E3 ligase, and ADRP. To elucidate the mechanism underlying the formation of large LDs induced by HCV infection, we investigated the roles of SPG20, Itch, and ADRP in large LD formation. The HCV NS3/4A protease specifically cleaves SPG20 and disrupts Itch recruitment to LD-associated ADRP. Therefore, LD-associated ADRP can escape and protects LDs from lipase-mediated degradation, thereby promoting LD growth. We propose that HCV NS3/4A protease-mediated cleavage of SPG20 contributes to a previously uncharacterized mechanism underlying the formation of large LDs in HCV-infected cells. These findings may lead to a better understanding of how the virus forms large LDs in infected cells.

microbiology↗

bioRxiv2025

HCV infection induces ubiquitin-dependent degradation of LATS1, inactivating the Hippo pathway and upregulating transcription of the CYR61 and CTGF genes

Hepatitis C virus (HCV) is often associated with chronic liver diseases and significant alterations in host cellular signaling. However, the molecular mechanisms underlying HCV- related liver pathogenesis remain to be elucidated. The Hippo signaling pathway, a key regulator of cell proliferation and survival, plays a critical role in maintaining liver homeostasis. Here we investigated the role of the Hippo pathway in HCV-related pathogenesis. We demonstrated that HCV infection induces degradation of LATS1, a key regulator of the Hippo pathway. Degradation of LATS1 protein was restored by a proteasomal inhibitor, but not a lysosome inhibitor, indicating that HCV promotes proteasomal degradation of LATS1 protein. HCV-induced degradation of LATS1 protein was suppressed in si-Itch-transfected Huh-7.5 cells. These results suggest that Itch ubiquitin ligase is involved in ubiquitin-dependent degradation of LATS1 protein. Cell fractionation assays and immunofluorescence staining revealed that HCV infection promoted nuclear translocation of YAP1 protein, suggesting that HCV infection suppresses the Hippo pathway. Furthermore, the transcription of YAP1 target genes, CYR61 and CTGF, that are involved in tissue remodeling and proliferation, was upregulated in HCV-infected Huh-7.5 cells and in HCV-infected patients. Taken together, we propose that HCV promotes the ubiquitin-dependent proteasomal degradation of LATS1 protein, leading to suppression of the Hippo pathway, thereby upregulating transcription of CYR61 and CTGF genes. DATA SUMMARYAll data are presented in the main figures. 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. The data that support the findings of this study is available at bioRxiv (https://www.biorxiv.org/). IMPACT STATEMENTWe demonstrate evidence suggesting that HCV infection promotes the Itch-mediated ubiquitin-dependent degradation of LATS1 protein, a key factor for the Hippo pathway. HCV-induced ubiquitin-dependent degradation of LATS1 protein promotes inactivation of the Hippo pathway and nuclear translocation of YAP1 protein, thereby upregulating transcription of CYR61 and CTGF genes. We propose a novel molecular mechanism in which HCV infection promotes degradation of LATS1 protein, leading to inactivation of the Hippo pathway. Understanding HCV-induced inactivation of the Hippo pathway may lead to developing new strategies for preventing or treating HCV-related pathogenesis.

microbiology↗