Biology subjects

Fukano, K.

Publications and source records attributed to Fukano, K..

3 recordsLinked to original sources

bioRxiv2025

RXR agonist S169 inhibits HBV/HDV entry in vitro by disrupting KIF4-dependent NTCP trafficking.

Chronic hepatitis B (HBV) and hepatitis D (HDV) viral infections pose serious global health challenges, and the lack of curative therapies calls for the development of new antiviral approaches. Recently, we have identified the Kinesin Family Member 4 (KIF4) as a crucial regulator of the surface transport for NTCP (HBV/HDV entry receptor). Our research has shown that the pan-RXR agonist, Bexarotene, suppresses KIF4 expression and inhibits HBV/HDV infections. However, the clinical use of Bexarotene is limited by poor physicochemical and pharmacokinetic (PK) properties and significant side effects. Here we investigated chemically diverse RXR modulators with tuned activity, and identified the RXR agonist S169 as a novel HBV/HDV entry inhibitor. S169 effectively blocks HBV and HDV infections in primary human hepatocyte (PXB) cultures without evident cytotoxicity. S169s suppressive effect on HBV infection was confirmed by reduction of secreted HBsAg and HBeAg, as well as inhibition of HBV cccDNA, pregenomic RNA, and intracellular HBV DNA. Mechanistically, S169 suppressed FOXM1-mediated KIF4 expression, leading to decreased NTCP surface levels and a marked decrease in HBV preS1 peptide binding to the hepatocyte cell surface. S169 is reported to exhibit enhanced oral bioavailability, favorable PK parameters, lower toxicity compared to Bexarotene. Interestingly, S169 induced a modest inhibition of NTCP-mediated bile acid transport up to 10 M, which remained unchanged at higher concentrations. Furthermore, S169 significantly impeded HBV spread in HBV infected long-time cultured PHHs (PXB). We speculate that S169 can be a promising seed for the development of novel oral HBV and HDV antiviral therapeutics.

microbiology↗

bioRxiv2021

The kinesin KIF4 mediates HBV/HDV entry through regulation of surface NTCP localization and can be targeted by RXR agonists in vitro.

Intracellular transport via microtubule-based dynein and kinesin family motors plays a key role in viral reproduction and transmission. We show here that Kinesin Family Member 4 (KIF4) plays an important role in HBV/HDV infection. We intended to explore host factors impacting the HBV life cycle that can be therapeutically addressed using siRNA library transfection and HBV/NLuc (HBV/NL) reporter virus infection in HepG2-hNTCP C4 cells. KIF4 silencing resulted in a 3-fold reduction in luciferase activity following HBV/NL infection and suppressed both wild-type HBV and HDV infection. Transient KIF4 depletion reduced surface and raised intracellular NTCP (HBV/HDV entry receptor) levels, according to both cellular fractionation and immunofluorescence analysis (IF). Overexpression of wild-type KIF4 but not ATPase-null KIF4 regains the surface localization of NTCP in these cells. Furthermore, immunofluorescence (IF) revealed KIF4 and NTCP colocalization across microtubule filaments, and a co-immunoprecipitation study showed that KIF4 physically binds to NTCP. KIF4 expression is regulated by FOXM1. Interestingly, we discovered that RXR agonists (Bexarotene, and Alitretinoin) down-regulated KIF4 expression via FOXM1-mediated suppression, resulting in a substantial decrease in HBV-Pre-S1 protein attachment to HepG2-hNTCP cell surface and subsequent suppression of HBV infection in HepG2-hNTCP and primary human hepatocytes (PXB) (Bexarotene, IC50 1.89 {+/-} 0.98 M). Overall, our findings show that human KIF4 is a critical regulator of NTCP surface transport and localization, which is required for NTCP to function as a receptor for HBV/HDV entry. Furthermore, small molecules that suppress or alleviate KIF4 expression would be potential antiviral candidates that target HBV and HDV entry phases. Author SummaryUnderstanding HBV/HDV entry machinery and the mechanism by which NTCP (HBV/HDV entry receptor) surface expression is regulated is crucial to develop antiviral entry inhibitors. We found that NTCP surface transport is mainly controlled by the motor kinesin KIF4. Surprisingly, KIF4 was negatively regulated by RXR receptors through FOXM1-mediated suppression This study not only mechanistically correlated the role of RXR receptors in regulating HBV/HDV entry but also suggested a novel approach to develop therapeutic rexinoids for preventing HBV and/or HDV infections in important clinical situations, such as in patients undergoing liver transplantation or those who are at a high risk of HBV infection and unresponsive to HBV vaccination.

microbiology↗

bioRxiv2018

A Single Adaptive Mutation in Sodium Taurocholate Cotransporting Polypeptide Induced by Hepadnaviruses Determines Virus Species-specificity

Hepatitis B virus (HBV) and its hepadnavirus relatives infect a wide range of vertebrates from fish to human. Hepadnaviruses and their hosts have a long history of acquiring adaptive mutations. However, there are no reports providing direct molecular evidence for such a coevolutionary \"arms race\" between hepadnaviruses and their hosts. Here, we present evidence suggesting the adaptive evolution of the sodium taurocholate cotransporting polypeptide (NTCP), an HBV receptor, has been influenced by virus infection. Evolutionary analysis of the NTCP-encoding genes from 20 mammals showed that most NTCP residues are highly conserved among species, exhibiting evolution under negative selection (dN/dS < 1); this observation implies that the evolution of NTCP is restricted by maintaining its original protein function. However, 0.7 % of NTCP amino acid (aa) residues exhibit rapid evolution under positive selection (dN/dS > 1). Notably, a substitution at aa 158, a positively selected residue, converting the human NTCP to a monkey-type sequence abrogated the capacity to support HBV infection; conversely, a substitution at this residue converting the monkey Ntcp to the human sequence was sufficient to confer HBV susceptibility. Together, these observations suggested that positive selection at aa 158 was induced by virus infection. Moreover, the aa 158 sequence determined attachment of the HBV envelope protein to host cell, demonstrating the mechanism whereby HBV infection would create positive selection at this residue in NTCP. In summary, we provide the first evidence in agreement with the function of hepadnavirus as a driver for inducing an adaptive mutation in host receptor.\n\nImportanceHepatitis B virus (HBV) and its hepadnavirus relatives infect a wide range of vertebrates, with a long infectious history (hundreds of millions of years). Such a long history generally allows adaptive mutations in hosts to escape from infection, while simultaneously allowing adaptive mutations in viruses to overcome host barriers. However, there is no published molecular evidence for such a coevolutionary \"arms race\" between hepadnaviruses and hosts. In the present study, we performed coevolutionary phylogenetic analysis between hepadnaviruses and the sodium taurocholate cotransporting polypeptide (NTCP), an HBV receptor, combined with virological experimental assays for investigating the biological significance of NTCP sequence variation. Our data provide the first molecular evidences supporting that HBV-related hepadnaviruses drive adaptive evolution in the NTCP sequence, including a mechanistic explanation of how NTCP mutations determine host viral susceptibility. Our novel insights enhance our understanding of how hepadnaviruses evolved with their hosts, permitting the acquisition of strong species-specificity.

evolutionary biology↗