FGF21 upregulation by hepatitis C virus via the eIF2α-ATF4 pathway: implications for interferon signaling suppression and TRIM31-mediated TSC degradation.

Hepatitis C virus (HCV) infection is a major cause of chronic liver diseases and is known to induce endoplasmic reticulum (ER) stress, which alters cellular homeostasis and metabolic processes. While ER stress is implicated in HCV-related diseases, its precise role remains unclear. This study identifies fibroblast growth factor 21 (FGF21) as a key host factor significantly upregulated by HCV infection. Mechanistic analyses reveal that the activation of the FGF21 promoter by HCV is primarily mediated by the transcription factor ATF4, which is upregulated through the phosphorylation of eIF2α induced by ER stress. Additionally, CREBH activation further enhances ATF4 expression, contributing to increased FGF21 levels. TRIB3, upregulated by ATF4, acts as a negative regulator of FGF21 expression. The study also identifies FGF21-dependent upregulation of SOCS2 and TRIM31 in HCV-infected cells. SOCS2 contributes to the suppression of type 1 interferon signaling, aiding viral persistence, while TRIM31 promotes the degradation of the tumor suppressor protein TSC, activating the mTORC1 pathway and potentially promoting liver cell proliferation. These findings suggest that FGF21 upregulation in HCV-infected cells may play a role in both immune response regulation and cell proliferation, contributing to sustained viral infection and disease progression.

Obesity impairs ciliary function and mucociliary clearance in the murine airway epithelium.

Obesity is a risk factor for increased morbidity and mortality in viral respiratory infection. Mucociliary clearance (MCC) in the airway is the primary host defense against viral infections. However, the impact of obesity on MCC is unclear, prompting this study. Using murine tracheal tissue culture and in vitro influenza A virus (IAV) infection models, we analyzed cilia-driven flow and ciliary beat frequency (CBF) in the airway epithelium to evaluate MCC. Short-term IAV infection increased cilia-driven flow and CBF in control mice, but not in high-fat diet-induced obese mice. Basal cilia-driven flow and CBF were also lower in obese mice than in control mice. Mechanistically, the increase of extracellular adenosine triphosphate (ATP) release during IAV infection, which was observed in the control mice, was abolished in the obese mice; however, the addition of ATP increased cilia-driven flow and CBF both in control and obese mice to a similar extent. In addition, RNA sequencing and reverse transcription-polymerase chain reaction revealed the downregulation of several cilia-related genes, including Dnah1, Dnal1, Armc4, and Ttc12 (the dynein-related genes); Ulk4 (the polychaete differentiation gene); Cep164 (the ciliogenesis and intraflagellar transport gene); Rsph4a, Cfap206, and Ppil6 (the radial spoke structure and assembly gene); and Drc3(the nexin-dynein regulatory complex genes) in obese murine tracheal tissues compared with their control levels. In conclusion, our studies demonstrate that obesity attenuates MCC under basal conditions and during IAV infection by downregulating the expression of cilia-related genes and suppressing the release of extracellular ATP, thereby increasing the susceptibility and severity of IAV infection.NEW & NOTEWORTHY Our study shows that obesity impairs airway mucociliary clearance (MCC), an essential physical innate defense mechanism for viral infection. Mechanically, this is likely due to the obesity-induced downregulation of cilia-related genes and attenuation of extracellular ATP release. This study provides novel insights into the mechanisms driving the higher susceptibility and severity of viral respiratory infections in individuals with obesity.

Impact of amino acid substitutions in hepatitis C virus core region on the severe oxidative stress.

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease, leading to liver steatosis, fibrosis, and hepatocellular carcinoma (HCC). Despite the accumulation of clinical data showing the impact of amino acid substitutions at positions 70 (R70Q/H) and/or 91 (L91M) in the HCV core protein in progressive liver diseases, including HCC, the underlying mechanisms have not been elucidated. We analyzed 72 liver biopsy specimens from patients with chronic HCV genotype 1b (HCV-1b) infection prior to antiviral treatment. Levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG) and nuclear factor erythroid 2-related factor 2 (NRF2) in the nucleus were quantified using liver tissue immunohistochemistry. The effects of amino acid substitutions in the HCV core region on hepatocellular oxidative stress were investigated using wild-type or double-mutant (R70Q/H+L91M) HCV-1b core transfection and stable expression in human hepatoma HuH-7 cells. Overall, 24, 19, 11, and 18 patients had the wild-type, R70Q/H, L91M, and R70Q/H+L91M genotypes, respectively, in the HCV core. A significantly higher accumulation of hepatocellular 8-OHdG and a lower NRF2/8-OHdG ratio were observed in patients with R70Q/H+L91M than in those with the wild-type disease. Increased levels of intracellular superoxide and hydrogen peroxide in the cytoplasm and mitochondria, mRNA expression of enzymes generating oxidative stress, and nuclear expression of nicotinamide adenine dinucleotide phosphate oxidase 4 were augmented in cells treated with R70Q+L91M. HCV core proteins harboring either or both substitutions of R70Q/H or L91M enhanced hepatocellular oxidative stress in vivo and in vitro. These amino acid substitutions may affect HCC development by enhancing hepatic oxidative stress in patients with chronic HCV-1b infection.

Orally administered Streptococcus thermophilus YIT 2001 is a vehicle for the delivery of glutathione, a reactive reduced thiol, to the intestine.

AIMS: We aimed to analyze the behavior of cellular glutathione of Streptococcus thermophilus strain YIT 2001 (ST-1) in the gastrointestinal environment to understand how orally administered glutathione in ST-1 cells is delivered stably to the intestine in a reactive form, which is essential for its systemic bioavailability against lipid peroxidation. METHODS AND RESULTS: Intracellular glutathione was labeled with L-cysteine-containing stable isotopes. ST-1 cells from fresh culture or lyophilized powder were treated with simulated gastric and intestinal juices for 60 min each. The release of intracellular glutathione in digestive juices was quantified via LC-MS/MS. Most of the cellular glutathione was retained in the gastric environment and released in response to exposure to the gastrointestinal environment. During digestion, the membrane permeability of propidium iodide increased significantly, especially when cells were exposed to cholate, without change in the cell wall state. CONCLUSIONS: ST-1 cells act as vehicles to protect intracellular reactive components, such as glutathione, from digestive stress, and release them in the upper intestine owing to the disruption of membrane integrity induced by bile acid.

SRPKIN-1 as an inhibitor against hepatitis B virus blocking the viral particle formation and the early step of the viral infection.

New antiviral agents are needed for the treatment of hepatitis B virus (HBV) infection because currently available drugs do not completely eradicate chronic HBV in patients. Phosphorylation dynamics of the HBV core protein (HBc) regulate several processes in the HBV life cycle, including nucleocapsid formation, cell trafficking, and virus uncoating after entry. In this study, the SRPK inhibitors SPHINX31, SRPIN340, and SRPKIN-1 showed concentration-dependent anti-HBV activity. Detailed analysis of the effects of SRPKIN-1, which exhibited the strongest inhibitory activity, on the HBV replication process showed that it inhibits the formation of infectious particles by inhibiting pregenomic RNA packaging into capsids and nucleocapsid envelopment. Mass spectrometry analysis combined with cell-free translation system experiments revealed that hyperphosphorylation of the C-terminal domain of HBc is inhibited by SRPKIN-1. Further, SRPKIN-1 exhibited concentration-dependent inhibition of HBV infection not only in HepG2-hNTCP-C4 cells but also in fresh human hepatocytes (PXB cells) and in the single-round infection system. Treatment with SRPKIN-1 at the time of infection reduced the nuclease sensitivity of HBV DNA in the nuclear fraction. These results suggest that SRPKIN-1 has the potential to not only inhibit the HBV particle formation process but also impair the early stages of viral infection.

Role of hepcidin upregulation and proteolytic cleavage of ferroportin 1 in hepatitis C virus-induced iron accumulation.

Hepatitis C virus (HCV) is a pathogen characterized not only by its persistent infection leading to the development of cirrhosis and hepatocellular carcinoma (HCC), but also by metabolic disorders such as lipid and iron dysregulation. Elevated iron load is commonly observed in the livers of patients with chronic hepatitis C, and hepatic iron overload is a highly profibrogenic and carcinogenic factor that increases the risk of HCC. However, the underlying mechanisms of elevated iron accumulation in HCV-infected livers remain to be fully elucidated. Here, we observed iron accumulation in cells and liver tissues under HCV infection and in mice expressing viral proteins from recombinant adenoviruses. We established two molecular mechanisms that contribute to increased iron load in cells caused by HCV infection. One is the transcriptional induction of hepcidin, the key hormone for modulating iron homeostasis. The transcription factor cAMP-responsive element-binding protein hepatocyte specific (CREBH), which was activated by HCV infection, not only directly recognizes the hepcidin promoter but also induces bone morphogenetic protein 6 (BMP6) expression, resulting in an activated BMP-SMAD pathway that enhances hepcidin promoter activity. The other is post-translational regulation of the iron-exporting membrane protein ferroportin 1 (FPN1), which is cleaved between residues Cys284 and Ala285 in the intracytoplasmic loop region of the central portion mediated by HCV NS3-4A serine protease. We propose that host transcriptional activation triggered by endoplasmic reticulum stress and FPN1 cleavage by viral protease work in concert to impair iron efflux, leading to iron accumulation in HCV-infected cells.

Potent bystander effect and tumor tropism in suicide gene therapy using stem cells from human exfoliated deciduous teeth.

Herpes simplex virus thymidine kinase (HSVTK)/ganciclovir (GCV) suicide gene therapy has a long history of treating malignant gliomas. Recently, stem cells from human exfoliated deciduous teeth (SHED), which are collected from deciduous teeth and have excellent harvestability, ethical aspects, and self-renewal, have been attracting attention mainly in the field of gene therapy. In the present study, we assessed SHED as a novel cellular vehicle for suicide gene therapy in malignant gliomas, as we have previously demonstrated with various cell types. SHED was transduced with the HSVTK gene (SHEDTK). In vitro experiments showed a significant bystander effect between SHEDTK and glioma cell lines in coculture. Furthermore, apoptotic changes caused by caspase 3/7 activation were simultaneously observed in SHEDTK and glioma cells. Mice implanted with a mixture of U87 and SHEDTK and treated with intraperitoneal GCV survived for longer than 100 days. Additionally, tumors in treatment model mice were significantly reduced in size during the treatment period. SHEDTK implanted at the contralateral hemisphere migrated toward the tumor crossing the corpus callosum. These results suggested that SHEDTK-based suicide gene therapy has potent tumor tropism and a bystander-killing effect, potentially offering a new promising therapeutic modality for malignant gliomas.

Efficacy of HSV-TK/GCV system suicide gene therapy using SHED expressing modified HSV-TK against lung cancer brain metastases.

Lung cancer is one of the most common cancers, and the number of patients with intracranial metastases is increasing. Previously, we developed an enzyme prodrug suicide gene therapy based on the herpes simplex virus thymidine kinase (HSV-TK)/ganciclovir (GCV) system using various mesenchymal stem cells to induce apoptosis in malignant gliomas through bystander killing effects. Here, we describe stem cells from human exfoliated deciduous teeth (SHED) as gene vehicles of the TK/GCV system against a brain metastasis model of non-small cell lung cancer (NSCLC). We introduced the A168H mutant TK (TKA168H) into SHED to establish the therapeutic cells because of the latent toxicity of wild type. SHED expressing TKA168H (SHED-TK) exhibited chemotaxis to the conditioned medium of NSCLC and migrated toward implanted NSCLC in vivo. SHED-TK demonstrated a strong bystander effect in vitro and in vivo and completely eradicated H1299 NSCLC in the brain. SHED-TK cells implanted intratumorally followed by GCV administration significantly suppressed the growth of H1299 and improved survival time. These results indicate that the TKA168H variant is suitable for establishing therapeutic cells and that intratumoral injection of SHED-TK followed by GCV administration may be a useful strategy for therapeutic approaches.

Virus-like particles with FLAG-tagged envelope protein as a tetravalent dengue vaccine candidate.

The global incidence of dengue, which is caused by dengue virus (DENV) infection, has grown dramatically in recent decades and secondary infection with heterologous serotype of the virus may cause severe symptoms. Efficacious dengue vaccines should be able to provide long-lasting immunity against all four DENV serotypes simultaneously. In this study, we constructed a novel vaccine platform based on tetravalent dengue virus-like particles (DENV-LPs) in which envelope (E) protein carried a FLAG tag sequence at the position located not only in the exterior loop on the protruding domain but outside of dimerization interface of the protein. We demonstrated an effective strategy to produce the DENV-LPs by transient transfection with expression plasmids for pre-membrane and E proteins of DENV-1 to DENV-4 in mammalian cells and to concentrate and purify them with one-step affinity chromatography. Characteristic features of VLPs such as particle size, shape and density were comparable to flavivirus-like particles reported. The neutralizing activity against all four DENV serotypes was successfully induced by immunization with the purified tetravalent VLPs in mice. Simple, one-step purification systems for VLP vaccine platforms using epitope-tagging strategy should be advantageous for vaccine development not only for dengue but for emerging pandemics in the future.

MafF Is an Antiviral Host Factor That Suppresses Transcription from Hepatitis B Virus Core Promoter.

Hepatitis B virus (HBV) is a stealth virus that exhibits only minimal induction of the interferon system, which 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. Here, we report that Maf bZIP transcription factor F (MafF) promotes host defense against infection with HBV. Using a small interfering RNA (siRNA) library and an HBV/NanoLuc (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 mutation of the putative MafF binding region. Loss of MafF expression through CRISPR/Cas9 editing (in HepG2-hNTCP-C4 cells) or siRNA silencing (in primary hepatocytes [PXB cells]) induced HBV core RNA and HBV pregenomic RNA (pgRNA) levels, respectively, after HBV infection. MafF physically binds to the HBV core promoter and competitively inhibits HNF-4α binding to an overlapping sequence in the HBV enhancer II sequence (EnhII), as seen by chromatin immunoprecipitation (ChIP) analysis. MafF expression was induced by interleukin-1ß (IL-1ß) or tumor necrosis factor alpha (TNF-α) treatment in both HepG2 and PXB cells, in an NF-κB-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. IMPORTANCE HBV 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. Therefore, 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 the HBV core promoter, leading to significant suppression of the HBV life cycle. Furthermore, MafF expression was induced in chronic HBV patients and in primary human hepatocytes (PXB cells). This induction correlated with the levels of inflammatory cytokines (IL-1ß and TNF-α). These data suggest that the induction of MafF contributes to the host's antiviral defense by suppressing transcription from selected viral promoters. Our data shed light on a novel role for MafF as an anti-HBV host restriction factor.

G ATA2 mediates the negative regulation of the prepro-thyrotropin-releasing hormone gene by liganded T3 receptor ß2 in the rat hypothalamic paraventricular nucleus.

Thyroid hormone (T3) inhibits thyrotropin-releasing hormone (TRH) synthesis in the hypothalamic paraventricular nucleus (PVN). Although the T3 receptor (TR) ß2 is known to mediate the negative regulation of the prepro-TRH gene, its molecular mechanism remains unknown. Our previous studies on the T3-dependent negative regulation of the thyrotropin ß subunit (TSHß) gene suggest that there is a tethering mechanism, whereby liganded TRß2 interferes with the function of the transcription factor, GATA2, a critical activator of the TSHß gene. Interestingly, the transcription factors Sim1 and Arnt2, the determinants of PVN differentiation in the hypothalamus, are reported to induce expression of TRß2 and GATA2 in cultured neuronal cells. Here, we confirmed the expression of the GATA2 protein in the TRH neuron of the rat PVN using immunohistochemistry with an anti-GATA2 antibody. According to an experimental study from transgenic mice, a region of the rat prepro-TRH promoter from nt. -547 to nt. +84 was able to mediate its expression in the PVN. We constructed a chloramphenicol acetyltransferase (CAT) reporter gene containing this promoter sequence (rTRH(547)-CAT) and showed that GATA2 activated the promoter in monkey kidney-derived CV1 cells. Deletion and mutation analyses identified a functional GATA-responsive element (GATA-RE) between nt. -357 and nt. -352. When TRß2 was co-expressed, T3 reduced GATA2-dependent promoter activity to approximately 30%. Unexpectedly, T3-dependent negative regulation was maintained after mutation of the reported negative T3-responsive element, site 4. T3 also inhibited the GATA2-dependent transcription enhanced by cAMP agonist, 8-bromo-cAMP. A rat thyroid medullary carcinoma cell line, CA77, is known to express the preproTRH mRNA. Using a chromatin immunoprecipitation assay with this cell line where GATA2 expression plasmid was transfected, we observed the recognition of the GATA-RE by GATA2. We also confirmed GATA2 binding using gel shift assay with the probe for the GATA-RE. In CA77 cells, the activity of rTRH(547)-CAT was potentiated by overexpression of GATA2, and it was inhibited in a T3-dependent manner. These results suggest that GATA2 transactivates the rat prepro-TRH gene and that liganded TRß2 interferes with this activation via a tethering mechanism as in the case of the TSHß gene.

Influenza A virus enhances ciliary activity and mucociliary clearance via TLR3 in airway epithelium.

BACKGROUND: Viral respiratory tract infections, such as influenza A virus (IAV), are common and life-threatening illnesses worldwide. The mechanisms by which viruses are removed from the respiratory tract are indispensable for airway host defense. Mucociliary clearance is an airway defense mechanism that removes pathogens from the respiratory tract. The coordination and modulation of the ciliary beating of airway epithelial cells play key roles in maintaining effective mucociliary clearance. However, the impact of respiratory virus infection on ciliary activity and mucociliary clearance remains unclear. METHODS: Tracheal samples were taken from wild-type (WT) and Toll-like receptor 3 (TLR3)-knockout (KO) mice. Transient organ culture of murine trachea was performed in the presence or absence of IAV, polyI:C, a synthetic TLR3 ligand, and/or reagents. Subsequently, cilia-driven flow and ciliary motility were analyzed. To evaluate cilia-driven flow, red fluorescent beads were loaded into culture media and movements of the beads onto the tracheal surface were observed using a fluorescence microscope. To evaluate ciliary motility, cilia tips were labeled with Indian ink diluted with culture medium. The motility of ink-labeled cilia tips was recorded by high-speed cameras. RESULTS: Short-term IAV infection significantly increased cilia-driven flow and ciliary beat frequency (CBF) compared with the control level in WT culture. Whereas IAV infection did not elicit any increases of cilia-driven flow and CBF in TLR3-KO culture, indicating that TLR3 was essential to elicit an increase of cilia-driven flow and CBF in response to IAV infection. TLR3 activation by polyI:C readily induced adenosine triphosphate (ATP) release from the trachea and increases of cilia-driven flow and CBF in WT culture, but not in TLR3-KO culture. Moreover, blockade of purinergic P2 receptors (P2Rs) signaling using P2R antagonist, suramin, suppressed polyI:C-mediated increases of cilia-driven flow and CBF, indicating that TLR3-mediated ciliary activation depended on released extracellular ATP and the autocrine ATP-P2R loop. CONCLUSIONS: IAV infection readily increases ciliary activity and cilia-driven flow via TLR3 activation in the airway epithelium, thereby hastening mucociliary clearance and "sweeping" viruses from the airway as an initial host defense response. Mechanically, extracellular ATP release in response to TLR3 activation promotes ciliary activity through autocrine ATP-P2R loop.

Prevalence and viral loads of polyomaviruses BKPyV, JCPyV, MCPyV, TSPyV and NJPyV and hepatitis viruses HBV, HCV and HEV in HIV-infected patients in China.

Human polyomaviruses (PyVs) and hepatitis viruses are often more prevalent or persistent in human immunodeficiency virus (HIV)-infected persons and the associated diseases are more abundant than in immunocompetent individuals. Here, we evaluated seroreactivities and viral loads of human PyVs and hepatitis viruses in HIV/AIDS patients and the general population in China in the combination antiretroviral therapy (cART) era. A total of 810 HIV-1-infected patients and age- and sex-matched HIV-negative individuals were enrolled to assess seroprevalence of PyVs BKPyV, JCPyV, MCPyV, TSPyV, and NJPyV and hepatitis viruses HBV, HCV, and HEV. 583 (72%) patients received cART, and among them, 31.2% had undetectable HIV RNA. While no significant difference was observed in prevalence of anti-PyV antibodies between HIV-positive and -negative groups, serum DNA positivity and DNA copy level of MCPyV were higher in the HIV-positive group. Among HIV-infected patients, BKPyV DNA positivity was significantly higher in patients with CD4 + cell counts < 200 cells/mm3 compared to those with CD4 + cell counts > 500 cells/mm3, suggesting possible reactivation caused by HIV-induced immune suppression. Higher HBV and HCV seropositivities but not HEV seropositivity were also observed in the HIV-positive group. Further correlation analyses demonstrated that HBV and HEV are potential risk factors for increased prevalence of PyV infection.

Partial silencing of fucosyltransferase 8 gene expression inhibits proliferation of Ishikawa cells, a cell line of endometrial cancer.

Endometrial cancer is the most common gynecologic malignancy and is associated with increased morbidity each year, including young people. However, its mechanisms of proliferation and progression are not fully elucidated. It is well known that abnormal glycosylation is involved in oncogenesis, and fucosylation is one of the most important types of glycosylation. In particular, fucosyltransferase 8 (FUT8) is the only FUT responsible for α1, 6-linked fucosylation (core fucosylation), and it is involved in various physiological as well as pathophysiological processes, including cancer biology. Therefore, we aimed to identify the expression of FUT8 in endometrial endometrioid carcinoma and investigate the effect of the partial silencing of the FUT8 gene on the cell proliferation of Ishikawa cells, an epithelial-like endometrial cancer cell line. Quantitative real-time PCR analysis showed that FUT8 gene expression was significantly elevated in the endometrial endometrioid carcinoma, compared to the normal endometrium. The immunostaining of FUT8 and Ulex europaeus Agglutinin 1 (UEA-1), a kind of lectin family specifically binding to fucose, was detected endometrial endometrioid carcinoma. The proliferation assay showed FUT8 partial knockdown by transfection of siRNA significantly suppressed the proliferation of Ishikawa cells, concomitant with the upregulation in the gene expressions associated with the interesting pathways associated with de-ubiquitination, aspirin trigger, mesenchymal-epithelial transition (MET) et al. It was suggested that the core fucosylation brought about by FUT8 might be involved in the proliferation of endometrial endometrioid carcinoma cells.

Characterization of the self-assembly of New Jersey polyomavirus VP1 into virus-like particles and the virus seroprevalence in Japan.

New Jersey polyomavirus (NJPyV) was discovered in 2014 in a pancreatic transplant recipient's vascular endothelial cells. Here, in the recombinant baculovirus system, VP1 protein of NJPyV expressed in insect cells was processed. The protein self-assembled into virus-like particles (NJPyV-LPs) in a cell-type-dependent manner, and the particles were then released into the culture media. Spherical ~50-nm-dia. NJPyV-LPs of uniform size with morphology resembling that of the native particles of polyomaviruses were purified from the fraction at 1.33 g/cm3 in supernatants of VP1-expressing Sf9 cells. We investigated the antigenic properties of purified NJPyV-LPs and performed a VLP-based enzyme immunoassay to determine the age-specific prevalence of NJPyV infection in a general Japanese population aged 1-70 years. The overall seropositivity rate of anti-NJPyV antibodies was only 1.8%. This might be explained by the low circulation of NJPyV in Japan. This is the first report of a large-scale serological survey of NJPyV in Asia (n = 1,050).

Cell Type Diversity in Hepatitis B Virus RNA Splicing and Its Regulation.

Although RNA splicing of hepatitis B virus (HBV) is a commonly observed in livers of hepatitis B patients as well as in the cultured cells replicating the viral genome, its biological significance in the HBV life cycle and the detailed regulatory mechanisms are still largely unclear. In this study, we found cell-type dependency of HBV splicing of the 3.5 kb pregenomic RNA, which is efficiently spliced in human hepatoma cells but not in cells derived from human hepatic stellate, mouse hepatoma and human non-hepatic cells. It may be likely that RNA splicing is one of the determinants of host range restriction of HBV. Given the finding indicating the difference in cell-type dependency of the splicing efficiency between HBV and simian virus 40, we carried out intron-swapping experiments. The results suggest the presence of putative exonic splicing enhancer that possibly works in the cell-type dependent fashion. Together with further mutational analyses, a novel 50-nt intronic splicing silencer, whose secondary structure is well conserved among the HBV strains, was identified. It appears that this intronic silencer functions effectively independent of cell backgrounds.

Peroxiredoxin 1, a Novel HBx-Interacting Protein, Interacts with Exosome Component 5 and Negatively Regulates Hepatitis B Virus (HBV) Propagation through Degradation of HBV RNA.

Hepatitis B virus (HBV) infection is a major risk factor for the development of chronic liver diseases, including cirrhosis and hepatocellular carcinoma (HCC). A growing body of evidence suggests that HBV X protein (HBx) plays a crucial role in viral replication and HCC development. Here, we identified peroxiredoxin 1 (Prdx1), a cellular hydrogen peroxide scavenger, as a novel HBx-interacting protein. Coimmunoprecipitation analysis coupled with site-directed mutagenesis revealed that the region from amino acids 17 to 20 of the HBx, particularly HBx Cys17, is responsible for the interaction with Prdx1. Knockdown of Prdx1 by siRNA significantly increased the levels of intracellular HBV RNA, HBV antigens, and extracellular HBV DNA, whereas knockdown of Prdx1 did not increase the activities of HBV core, enhancer I (Enh1)/X, preS1, and preS2/S promoters. Kinetic analysis of HBV RNA showed that knockdown of Prdx1 inhibited HBV RNA decay, suggesting that Prdx1 reduces HBV RNA levels posttranscriptionally. The RNA coimmunoprecipitation assay revealed that Prdx1 interacted with HBV RNA. The exosome component 5 (Exosc5), a member of the RNA exosome complexes, was coimmunoprecipitated with Prdx1, suggesting its role in regulation of HBV RNA stability. Taken together, these results suggest that Prdx1 and Exosc5 play crucial roles in host defense mechanisms against HBV infection.IMPORTANCE Hepatitis B virus (HBV) infection is a major global health problem. HBx plays important roles in HBV replication and viral carcinogenesis through its interaction with host factors. In this study, we identified Prdx1 as a novel HBx-binding protein. We provide evidence suggesting that Prdx1 promotes HBV RNA decay through interaction with HBV RNA and Exosc5, leading to downregulation of HBV RNA. These results suggest that Prdx1 negatively regulates HBV propagation. Our findings may shed new light on the roles of Prdx1 and Exosc5 in host defense mechanisms in HBV infection.

Involvement of PUF60 in Transcriptional and Post-transcriptional Regulation of Hepatitis B Virus Pregenomic RNA Expression.

Here we identified PUF60, a splicing factor and a U2 small nuclear ribonucleoprotein auxiliary factor, as a versatile regulator of transcriptional and post-transcriptional steps in expression of hepatitis B virus (HBV) 3.5 kb, precore plus pregenomic RNA. We demonstrate that PUF60 is involved in: 1) up-regulation of core promoter activity through its interaction with transcription factor TCF7L2, 2) promotion of 3.5 kb RNA degradation and 3) suppression of 3.5 kb RNA splicing. When the 1.24-fold HBV genome was introduced into cells with the PUF60-expression plasmid, the 3.5 kb RNA level was higher at days 1-2 post-transfection but declined thereafter in PUF60-expressing cells compared to viral replication control cells. Deletion analyses showed that the second and first RNA recognition motifs (RRMs) within PUF60 are responsible for core promoter activation and RNA degradation, respectively. Expression of PUF60 mutant deleting the first RRM led to higher HBV production. To our knowledge, this is the first to identify a host factor involved in not only positively regulating viral gene expression but also negative regulation of the same viral life cycle. Functional linkage between transcriptional and post-transcriptional controls during viral replication might be involved in mechanisms for intracellular antiviral defense and viral persistence.

Development of hepatoma-derived, bidirectional oval-like cells as a model to study host interactions with hepatitis C virus during differentiation.

Directed differentiation of human stem cells including induced pluripotent stem cells into hepatic cells potentially leads to acquired susceptibility to hepatitis C virus (HCV) infection. However, cellular determinants that change their expression during cell reprogramming or hepatic differentiation and are pivotal for supporting the HCV life cycle remain unclear. In this study, by introducing a set of reprogramming factors, we established HuH-7-derived oval-like cell lines, Hdo-17 and -23, which possess features of bipotential liver precursors. Upon induction of hepatocyte differentiation, expression of mature hepatocyte markers and hepatoblast markers in cells increased and decreased, respectively. In contrast, in response to cholangiocytic differentiation induction, gene expression of epithelium markers increased and cells formed round cysts with a central luminal space. Hdo cells lost their susceptibility to HCV infection and viral RNA replication. Hepatic differentiation of Hdo cells potentially led to recovery of permissiveness to HCV RNA replication. Gene expression profiling showed that most host-cell factors known to be involved in the HCV life cycle, except CD81, are expressed in Hdo cells comparable to HuH-7 cells. HCV pseudoparticle infectivity was significantly but partially recovered by ectopic expression of CD81, suggesting possible involvement of additional unidentified factors in HCV entry. In addition, we identified miR200a-3p, which is highly expressed in Hdo cells and stem cells but poorly expressed in differentiated cells and mature hepatocytes, as a novel negative regulator of HCV replication. In conclusion, our results showed that epigenetic reprogramming of human hepatoma cells potentially changes their permissivity to HCV.

Critical role of CREBH-mediated induction of transforming growth factor ß2 by hepatitis C virus infection in fibrogenic responses in hepatic stellate cells.

Mechanisms of hepatic fibrogenesis induced by hepatitis C virus (HCV), one of the leading causes of liver fibrosis, are not fully understood. We studied transcriptional up-regulation of transforming growth factor ß (TGF-ß), especially TGF-ß2, which is mediated by activation of liver-enriched transcription factor cAMP-responsive element-binding protein, hepatocyte specific (CREBH) triggered by HCV infection and its functional significance for induction of profibrogenic phenotypes by interaction of HCV-infected cells with hepatic stellate cells (HSCs). Compared to TGF-ß1, expression of TGF-ß2 mRNA was induced faster and to a higher level upon HCV infection. Serum TGF-ß2 levels in hepatitis C patients were higher compared to those in healthy individuals and were positively correlated with hepatic fibrosis stages F0-F2. TGF-ß2 promoter activity was decreased and increased, respectively, by silencing and overexpression of CREBH. CREBH recognition sites were identified in the TGF-ß2 promoter. CREBH binding to the promoter and its increase in cells expressing HCV Core-NS2 were shown by gel mobility shift and chromatin immunoprecipitation, respectively. The active form of CREBH was detectable in HCV-infected chimeric mice with human livers and cells expressing HCV proteins. Involvement of CREBH in HCV-induced fibrogenic response was further demonstrated in the CREBH null-mutant mouse model. Fibrogenic phenotypes were assessed using co-cultures of HCV-infected cells and HSCs. Expressions of fibrogenic factors and TGF-ß1 increasing in the co-cultures was prevented by TGF-ß2- or CREBH silencing. CONCLUSION: CREBH was identified as a key positive regulator of TGF-ß2 transcription in HCV-infected cells. TGF-ß2 released from infected cells potentially contributes to cross-induction of TGF-ß in an autocrine manner through its own signaling pathway, leading to an increase in fibrogenic responses in adjacent HSCs. (Hepatology 2017;66:1430-1443).