Biology subjects

Shimotohno, K.

Publications and source records attributed to Shimotohno, K..

3 recordsLinked to original sources

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↗

bioRxiv2020

MafF is an anti-viral host factor that suppresses transcription from Hepatitis B Virus and Epstein Barr Virus promoters

Hepatitis B Virus (HBV) is a stealth virus that exhibits only minimal induction of the interferon system that is required for both innate and adaptive immune responses. However, 90% of acutely infected adults can clear the virus, suggesting the presence of additional mechanisms that facilitate viral clearance. Herein, we report that Maf bZIP transcription factor F (MafF) promotes host defense against infection with HBV. Using siRNA library and an HBV/NL reporter virus, we screened to identify anti-HBV host factors. Our data showed that silencing of MafF led to a 6-fold increase in luciferase activity after HBV/NL infection. Overexpression of MafF reduced HBV core promoter transcriptional activity, which was relieved upon mutating the putative MafF binding region. Loss of MafF expression by CRISPR/CAS9 (in HepG2-hNTCP-C4 cells) or siRNA silencing (in primary hepatocytes [PXB]), induced HBV core and HBV pregenomic RNA (pgRNA) levels, respectively, after HBV infection. MafF physically binds to HBV core promoter and competitively inhibits HNF-4 binding to an overlapping sequence in HBV enhancer II sequence (EnhII) as seen by ChIP analysis. MafF expression was induced by IL-1{beta}/TNF- treatment in both HepG2 and PXB cells, in an NF-{kappa}B-dependent manner. Consistently, MafF expression levels were significantly enhanced and positively correlated with the levels of these cytokines in patients with chronic HBV infection, especially in the immune clearance phase. ImportanceHBV is a leading cause of chronic liver diseases, infecting about 250 million people worldwide. HBV has developed strategies to escape interferon-dependent innate immune responses. Hence, the identification of other anti-HBV mechanisms is important for understanding HBV pathogenesis, and developing anti-HBV strategies. MafF was shown to suppress transcription from HBV core promoter, leading to a significant suppression of HBV life cycle. Furthermore, MafF expression was induced in chronic HBV patients and in primary human hepatocytes (PXB). This induction correlated with the levels of inflammatory cytokines (IL-1{beta} and TNF-). These data suggest that the induction of MafF contributes to the hosts antiviral defense by suppressing transcription from selected viral promoters. Our data shed light on a novel role for MafF as anti-HBV host restriction factor.

microbiology↗

bioRxiv2018

High-level expression of STING restricts susceptibility to HBV by mediating type III IFN induction

Hepatitis B virus (HBV) is a hepatotropic DNA virus causing hepatic diseases such as chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. To study HBV, human hepatoma HepG2 cells are currently used as an HBV infectious cell culture model worldwide. HepG2 cells exhibit susceptibility to HBV by exogenously expressing sodium taurocholate cotransporting polypeptide (NTCP). We herein demonstrated that human immortalized hepatocyte NKNT-3 cells exhibited susceptibility to HBV by exogenously expressing NTCP (NKNT-3/NTCP cells). By comparing the cGAS-STING signaling pathway in several NKNT-3/NTCP cell-derived cell clones, we found that STING was highly expressed in cell clones exhibiting resistance but not susceptibility to HBV. High-level expression of STING was implicated in HBV-triggered induction of type III IFN and a pro-inflammatory cytokine, IL-6. In contrast, RNAi-mediated knockdown of STING inhibited type III IFN induction and restored the levels of HBV total transcript in an HBV-infected cell clone exhibiting resistance to HBV. These results suggest that STING regulates susceptibility to HBV by its expression levels.\n\nSTING may thus be a novel target for anti-HBV strategies.

microbiology↗