Biology subjects

Akaike, Y.

Publications and source records attributed to Akaike, Y..

3 recordsLinked to original sources

bioRxiv2025

Generation of a HiBiT-expressing recombinant rat hepacivirus supporting both in vivo and in vitro infection

The lack of immuno-competent animal models of hepatitis C virus (HCV) infection has been an obstacle to vaccine development and research on immune responses. Hepacivirus ratti (Norway rat hepacivirus-1: NRHV1) is a virus closely related to HCV that specifically infects the liver and induces hepatocellular carcinoma in rats, making it a promising surrogate model for HCV. NRHV1 expressing a reporter gene serves as a powerful tool for analyzing the in vivo dynamics and pathogenicity mechanisms of NRHV1. In this study, we developed a reporter NRHV1 capable of infection and replication in both mice and cultured cells and established a platform for generating diverse reporter viruses. A reporter virus containing the HiBiT gene in the coding region of NS5A domain III was constructed using circular polymerase extension reaction (CPER). Infection with this reporter virus led to HiBiT activity in infected cells and the activity was correlated with the amount of intracellular viral RNA. In addition, this reporter virus established persistent infection in NOD-SCID mice and led to the generation of HiBiT activity in the livers of infected mice. Furthermore, reporter virus recovered from infected mice could infect and generate HiBiT activity in cultured cells. These results demonstrate that the reporter virus can infect hepatocytes in vivo and in vitro. This platform provides a versatile tool for in vitro quantitative antiviral screening and in vivo pathogenicity studies. Author summaryHepatitis C virus (HCV) infects millions of people and causes severe liver diseases, yet vaccine development has been hindered by the lack of practical small-animal models with intact immunity. Hepacivirus ratti (NRHV1), a close relative of HCV that naturally infects rats, is a promising HCV surrogate, but tools for monitoring NRHV1 infection dynamics have been limited. Here, we developed the first reporter NRHV1 by inserting a small luminescent HiBiT tag into a tolerable region of the NS5A protein. Using a CPER-based reverse genetics system, we generated a recombinant virus that efficiently infected cultured cells and established persistent infection in mice while retaining liver tropism. HiBiT activity closely reflected viral RNA levels, enabling the quantitative monitoring of infection and sensitive evaluation of antiviral drugs and neutralizing antibodies. This reporter NRHV1 system provides a versatile platform for studying hepacivirus pathogenesis and antiviral immunity and will support future efforts toward HCV vaccine development.

microbiology↗

bioRxiv2025

Identification of claudin-3 as an entry factor for rat hepacivirus

Approximately 58 million people worldwide are believed to be infected with hepatitis C virus (HCV), a major causative agent of chronic liver diseases. Hepacivirus ratti strain rn-1, which was discovered from Rattus norvegicus (Norway rat) and designated Norway rat hepacivirus 1 (NRHV1), shares similar properties with HCV in terms of genetic homology, target cell tropism, pathogenicity and the immune response. In vivo infection systems for NRHV1 will help overcome the challenges in HCV research for vaccine development. However, the virological characteristics of NRHV1, such as the mechanisms of cell entry, remain largely unexplored, in part owing to a paucity of cell culture systems for NRHV1. Here, we identified the host factors that facilitate NRHV1 entry by profiling the gene expression of two cell lines with different susceptibilities to NRHV1 infection. NRHV1 employs rodent orthologues of HCV entry factors, including scavenger receptor class BI, CD81, and occludin, and utilizes claudin-3 (CLDN3) but not claudin-1. The expression of rat and mouse, but not human, CLDN3 facilitates the entry of NRHV1 into murine cell lines that are insusceptible to NRHV1 infection. The host-specific cell entry of CLDN3 is determined by two amino acid residues, Ile44 and Trp46, in extracellular loop 1. These findings suggest that some hepacivirus species share an HCV-like entry mechanism but show evidence of adaptive evolution of their virus-specific entry factors to host animals.

microbiology↗

bioRxiv2024

HCV infection activates the proteasome via PA28γ acetylation and heptamerization to facilitate the degradation of RNF2, a catalytic component of polycomb repressive complex 1.

We previously reported that hepatitis C virus (HCV) infection or HCV core protein expression induces HOX gene expression by impairing histone H2A monoubiquitination via a proteasome-dependent reduction in the level of RNF2, a key catalytic component of polycomb repressive complex 1 (PRC1) (J. Virol, 2021, 95, e01784-20). In this study, we aimed to investigate the mechanism by which HCV infection accelerates RNF2 degradation. Yeast two-hybrid screening and an immunoprecipitation assay revealed that RNF2 is a PA28{gamma}-binding protein. The proteasome activator PA28{gamma} destabilized the RNF2 protein in a proteasome-dependent manner, since RNF2 degradation was impaired by PA28{gamma} knockout or MG132 treatment. HCV infection or core protein expression reduced the levels of RNF2 and histone H2A K119 monoubiquitination and induced the expression of HOX genes in the presence of PA28{gamma}, while PA28{gamma} knockout reversed these changes. Treatment with a lysine acetyltransferase inhibitor inhibited the acetylation of PA28{gamma} at K195 and the degradation of the RNF2 protein, while treatment with a lysine deacetylase inhibitor accelerated these events in a PA28{gamma}-dependent manner. RNF2 protein degradation was increased by expression of the acetylation mimetic PA28{gamma} mutant but not by expression of the acetylation-defective mutant or the proteasome activation-defective mutant. Furthermore, HCV infection or core protein expression facilitated the interaction between PA28{gamma} and the lysine acetyltransferase CBP/p300 and then accelerated PA28{gamma} acetylation and heptamerization to promote RNF2 degradation. These data suggest that HCV infection accelerates the acetylation-dependent heptamerization of PA28{gamma} to increase the proteasomal targeting of RNF2. IMPORTANCEHCV is a causative agent of HCV-related liver diseases, including hepatic steatosis, cirrhosis and hepatocellular carcinoma. PA28{gamma}, which, in heptameric form, activates the 20S core proteasome for the degradation of PA28{gamma}-binding proteins, is responsible for HCV-related liver diseases. HCV core protein expression or HCV infection accelerates RNF2 degradation, leading to the induction of HOX gene expression via a decrease in the level of H2Aub on HOX gene promoters. However, the mechanism of RNF2 degradation in HCV-infected cells has not been clarified. The data presented in this study suggest that PA28{gamma} acetylation and heptamerization are promoted by HCV infection or by core protein expression to activate the proteasome for the degradation of RNF2 and are responsible for HCV propagation. This study provides novel insights valuable for the development of therapies targeting both HCV propagation and HCV-related diseases.

microbiology↗