Hepatitis B virus-specific human stem cell memory T cells differentiate into cytotoxic T cells and eradicate HBV-infected hepatocytes in mice.

Chronic infection with the hepatitis B virus (HBV) induces progressive hepatic impairment. Achieving complete eradication of the virus remains a formidable challenge. Cytotoxic T lymphocytes, specific to viral antigens, either exhibit a numerical deficiency or succumb to an exhausted state in individuals chronically afflicted with HBV. The comprehension of the genesis and dissemination of stem cell memory T cells (TSCMs) targeting HBV remains inadequately elucidated. We identified TSCMs in subjects with chronic HBV infection and scrutinized their efficacy in a murine model with human hepatocyte transplants, specifically the TK-NOG mice. TSCMs were discerned in all subjects under examination. Introduction of TSCMs into the HBV mouse model precipitated a severe necro-inflammatory response, resulting in the elimination of human hepatocytes. TSCMs may constitute a valuable tool in the pursuit of a remedial therapy for HBV infection.

Deficiency of SCAP inhibits HBV pathogenesis via activation of the interferon signaling pathway.

Hepatitis B virus (HBV) infects the liver and is a major risk factor for liver cirrhosis and hepatocellular carcinoma. Approaches for an effective cure are thwarted by limited knowledge of virus-host interactions. Herein, we identified SCAP as a novel host factor that regulates HBV gene expression. SCAP, sterol regulatory element-binding protein (SREBP) cleavage-activating protein, is an integral membrane protein located in the endoplasmic reticulum. The protein plays a central role in controlling lipid synthesis and uptake by cells. We found that gene silencing of SCAP significantly inhibited HBV replication; furthermore, knockdown of SREBP2 but not SREBP1, the downstream effectors of SCAP, reduced HBs antigen production from HBV infected primary hepatocytes. We also demonstrated that knockdown of SCAP resulted in activation of interferons (IFNs) and IFN stimulated genes (ISGs). Conversely, ectopic expression of SREBP2 in SCAP-deficient cells restored expression of IFNs and ISGs. Importantly, expression of SREBP2 restored HBV production in SCAP knockdown cells, suggesting that SCAP participates in HBV replication through an effect on IFN production via its downstream effector SREBP2. This observation was further confirmed by blocking IFN signaling by an anti-IFN antibody, which restored HBV infection in SCAP-deficient cells. This led to the conclusion that SCAP regulates the IFN pathway through SREBP, thereby affecting the HBV lifecycle. This is the first study to reveal the involvement of SCAP in regulation of HBV infection. These results may facilitate development of new antiviral strategies against HBV.

HBV with precore and basal core promoter mutations exhibits a high replication phenotype and causes ER stress-mediated cell death in humanized liver chimeric mice.

BACKGROUND AND AIMS: Mutations within the precore (PC) and basal core promoter (BCP) regions of the HBV genome are associated with fulminant hepatitis and HBV reactivation. These mutations may enhance viral replication, but little is known about whether they directly induce damage to the liver. We investigated mechanisms of direct cytopathic effects induced by the infection with PC/BCP mutants in the absence of immune response in vitro and in vivo . APPROACH AND RESULTS: Mice with humanized livers and hepatocytes derived from humanized mice were infected with either wild-type or mutant-type PC/BCP HBV, and the HBV replication and human hepatocyte damage were evaluated. HBV proliferated vigorously in mice with PC/BCP-mutant infection, and the severe loss of human hepatocytes with a slight human ALT elevation subsequently occurred only in PC/BCP mutant mice. In PC/BCP mutant infection, the accumulation of HBsAg in humanized livers colocalized with the endoplasmic reticulum, leading to apoptosis through unfolded protein response in HBV-infected hepatocytes. RNA-sequencing revealed the molecular characteristics of the phenotype of PC/BCP mutant infection in a humanized mouse model. Reduced ALT elevation and higher HBV DNA levels in this model are consistent with characteristics of HBV reactivation, indicating that the hepatocyte damage in this model might mimic HBV reactivation followed by hepatocyte damage under immunosuppressive conditions. CONCLUSION: PC and BCP mutations were associated with enhanced viral replication and cell death induced by ER stress using HBV infection models. These mutations might be associated with liver damage in patients with fulminant hepatitis or HBV reactivation.

Hepatitis B Virus (HBV) Upregulates TRAIL-R3 Expression in Hepatocytes Resulting in Escape From Both Cell Apoptosis and Suppression of HBV Replication by TRAIL.

BACKGROUND: Hepatitis B virus (HBV) evades host immunity by regulating intracellular signals. To clarify this immune tolerance mechanism, we performed gene expression analysis using HBV-infected humanized mouse livers. METHODS: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor 3 (TRAIL-R3) was significantly upregulated in livers of HBV-infected human hepatocyte transplanted mice by cDNA microarray and next-generation sequencing. We analyzed the significance of TRAIL-R3 upregulation in HBV infection using human hepatocyte transplanted mice and HepG2 cell lines. RESULTS: TRAIL-R3 induction by HBV infection was verified by in vitro and in vivo HBV replication models, and induction was inhibited by antiviral nucleot(s)ide analogue treatment. TRAIL-R3 transcription was regulated by the TRAIL-R3 promoter at -969 to -479 nucleotides upstream from the transcription start site, and by hepatitis B x (HBx) via activation of nuclear factor-κB (NF-κB) signal. TRAIL not only induced cell apoptosis but also inhibited HBV replication. TRAIL-R3 upregulation could inhibit both TRAIL-dependent apoptosis in HBV-infected hepatocytes and TRAIL-mediated suppression of HBV replication. CONCLUSIONS: These results suggest a mechanism by which HBV persists by escaping host immunity through upregulation of TRAIL-R3. Development of novel drugs to inhibit this escape system might lead to complete HBV elimination from human hepatocytes.

A novel cDNA-uPA/SCID/Rag2-/- /Jak3-/- mouse model for hepatitis virus infection and reconstruction of human immune system.

Although human hepatocyte-transplanted immunodeficient mice support infection with hepatitis viruses, these mice fail to develop viral hepatitis due to the lack of an adaptive immune system. In this study, we generated new immunodeficiency cDNA-urokinase-type plasminogen activator (uPA)/SCID/Rag2-/- /Jak3-/- mice and established a mouse model with both a humanized liver and immune system. Transplantation of human hepatocytes with human leukocyte antigen (HLA)-A24 resulted in establishment of a highly replaced liver in cDNA-uPA/SCID/Rag2-/- /Jak3-/- mice. These mice were successfully infected with hepatitis B virus (HBV) and hepatitis C virus (HCV) for a prolonged period and facilitate analysis of the effect of anti-HCV drugs. Administration of peripheral blood mononuclear cells (PBMCs) obtained from an HLA-A24 donor resulted in establishment of 22.6%-81.3% human CD45-positive mononuclear cell chimerism in liver-infiltrating cells without causing graft-versus-host disease in cDNA-uPA/SCID/Rag2-/- /Jak3-/- mice without human hepatocyte transplantation. When mice were transplanted with human hepatocytes and then administered HLA-A24-positive human PBMCs, an alloimmune response between transplanted human hepatocytes and PBMCs occurred, with production of transplanted hepatocyte-specific anti-HLA antibody. In conclusion, we succeeded in establishing a humanized liver/immune system characterized by an allo-reaction between transplanted human immune cells and human liver using a novel cDNA-uPA/SCID/Rag2-/- /Jak3-/- mouse. This mouse model can be used to generate a chronic hepatitis mouse model with a human immune system with application not only to hepatitis virus virology but also to investigation of the pathology of post-transplantation liver rejection.

Untying relaxed circular DNA of hepatitis B virus by polymerase reaction provides a new option for accurate quantification and visualization of covalently closed circular DNA.

Hepatitis B virus (HBV) is a small hepatotropic DNA virus that replicates via an RNA intermediate. After entry, the virus capsid carries relaxed circular DNA (rcDNA) into the nucleus where the viral genome is converted into covalently closed circular DNA (cccDNA), which serves as the template for all viral transcripts. To monitor cccDNA levels, preprocessing methods to eliminate rcDNA have emerged for quantitative PCR, although Southern blotting is still the only method to discriminate cccDNA from other DNA intermediates. In this study, we have established a robust method for untying mature rcDNA into double stranded linear DNA using specific polymerases. Untying rcDNA provides not only an alternative method for cccDNA quantification but also a sensitive method for visualizing cccDNA. We combined this method with plasmid-safe DNase and T5 exonuclease preprocessing and revealed that accurate quantification requires cccDNA digestion by a restriction enzyme because heat stability of cccDNA increases after T5 exonuclease treatment. In digital PCR using duplex TaqMan probes, fewer than 1000 copies of cccDNA were successfully visualized as double positive spots that were distinct from single positives derived from untied rcDNA. This method was further applied to the infection model of primary hepatocytes treated with nucleoside analogues and a core protein allosteric modulator to monitor cccDNA levels. Relative quantification of cccDNA by human genome copy demonstrated the possibility of precise evaluation of cccDNA level per nucleus. These results clearly indicate that the sequential reaction from untying rcDNA is useful to investigate cccDNA fates in a small fraction of nuclei.

Construction of an anti-hepatitis B virus preS1 antibody and usefulness of preS1 measurement for chronic hepatitis B patients: Anti-HBV PreS1 antibody.

OBJECTIVES: The preS1 region plays an essential role in hepatitis B virus (HBV) infection. We construct an antibody that binds to preS1 and a measurement system for serum preS1 in chronic HBV-infected patients. METHODS: Hybridoma clones that produce anti-preS1 antibodies were obtained by the iliac lymph node method. Epitope mapping was conducted, and an enzyme-linked immunosorbent assay (ELISA)-based method was developed. Using this ELISA system, serum preS1 levels were measured in 200 chronic HBV-infected patients. RESULTS: Eight types of hybridomas were obtained, of which antibody 3-55 using amino acids 38-47 as the epitope showed high binding affinity to preS1. Serum preS1 levels measured by ELISA using 3-55 antibody were correlated with HBsAg, HBcrAg and HBV DNA levels. Among HBeAg-negative patients without antiviral therapeutic objective (HBV DNA <3.3 log IU/mL or alanine aminotransferase ≤30 U/L), preS1 was significantly higher in subjects who had progressed to the point of requiring antiviral therapy compared to subjects who had maintained their status for the preceding three years (p<0.01). CONCLUSIONS: We constructed an antibody against preS1 and an ELISA system capable of measuring serum preS1 levels. PreS1 may serve as a novel tool to predict the need for antiviral therapy in HBeAg-negative HBV-infected patients.

Serum PreS1 and HBsAg ratio reflects liver fibrosis and predicts the development of hepatocellular carcinoma in chronic hepatitis B patients.

While the preS1 region of the large hepatitis B surface protein plays an essential role in hepatitis B virus (HBV) infection, the effect of preS1 on liver fibrosis and hepatocarcinogenesis in chronic hepatitis B (CHB) patients is not well known. In this study, we measured serum preS1 levels by chemiluminescent immunoassay technology in 690 CHB patients and evaluated the correlation between serum preS1 levels and HBV, liver function markers and liver inflammation, fibrosis assessed by histological findings. Predictive factors for hepatocellular carcinoma (HCC) development in patients who had no previous history of HCC at the time of preS1 level measurement were also analysed. Median hepatitis B surface antigen (HBsAg) and preS1 levels were 3.08 log IU/mL and 98 ng/mL, respectively. PreS1 values were significantly correlated with serum HBsAg (p <0.001), hepatitis B core-related antigen (HBcrAg) (p <0.001) and HBV DNA levels (p <0.01). PreS1 values were also significantly correlated with serum alanine aminotransferase levels (p <0.001) and were significantly higher in patients who had higher grading of liver inflammatory activity (p <0.05). HBsAg level was correlated, but preS1/HBsAg ratio reflected liver fibrosis staging more directly than HBsAg alone. Multivariate analysis identified age ≥53 years (hazard ratio [HR], 18.360 for <53 years; p = 0.021) and preS1/HBsAg ratio ≥0.12 (HR, 6.205 for <0.12; p = 0.040) as significant and independent factors for HCC development in CHB patients. The preS1/HBsAg ratio directly reflects liver fibrosis, and the ratio might be a predictive marker for HCC development in CHB patients.

Novel drug resistance-associated substitutions against pibrentasvir emerged in genotype 1b hepatitis C virus-infected human hepatocyte transplanted mice.

Combination therapy with glecaprevir and pibrentasvir (PIB) has high efficacy for patients with hepatitis C virus (HCV) infection except among those who experienced NS5A-P32 deletion (del) mutation during prior DAA treatment failure. However, some patients fail to achieve SVR through combination treatment even in the absence of NS5A-P32del. We analyzed emergence of NS5A resistance-associated substitutions (RASs) against PIB using HCV-infected mice. Male human hepatocyte transplanted mice were infected with genotype 1b wild-type HCV. Mice were treated with PIB, resulting in a transient decrease in serum HCV RNA levels but followed by relapse during the treatment. Direct sequence analysis showed emergences of various mutations in the NS5A region, including L31V/P32del, L31F/P32del/Y93H, NS5A-P29del/Y85C, and NS5A-F37Y. PIB was less effective in mice with NS5A-F37Y mutations compared to mice with wild-type HCV. NS5A-F37Y showed 5.4-fold resistance to PIB relative to wild-type based on analysis using HCV subgenomic replicon systems. The present in vivo and in vitro studies identified NS5A-F37Y as a novel RAS against PIB and showed the possibility of emergence of various NS5A RASs including P29del, P32del and F37Y following PIB treatment. These mutations might emerge and lead to failure to respond to DAA therapies including PIB-based regimens in chronic hepatitis C patients.

Abatacept treatment for patients with severe acute hepatitis caused by hepatitis B virus infection-Pilot study.

Although glucocorticoids have been used for immunosuppression of patients with primary hepatitis B virus (HBV) infection-induced severe hepatitis, the treatment is associated with a high frequency of adverse events. We conducted a pilot study for evaluating the efficacy and safety of abatacept, a cytotoxic T lymphocyte antigen-4 immunoglobulin (CTLA4), for acute hepatitis B. Five patients with severe acute hepatitis B (prothrombin activity ≤ 60%) were treated for immunosuppression by abatacept. Four patients received abatacept concurrently with methylprednisolone, and another patient was treated with abatacept alone. Rapid decrease in serum alanine aminotransferase levels, increase in prothrombin activity and improvement of general condition were obtained in four out of five patients. The patient with the most severe hepatitis underwent liver transplantation due to exacerbation of hepatitis in spite of treatment with both abatacept and methylprednisolone. None of the patients developed significant adverse events associated with the use of abatacept. Hepatitis B surface antigen (HBsAg) became negative in all five patients. The effect of abatacept and methylprednisolone for severe hepatitis B was compared using a mouse model. Rapid reduction in mouse serum HBV DNA and human albumin levels and elevation of serum interferon-gamma and granzyme A levels were observed in HBV-infected human hepatocyte-transplanted immunodeficient mice that were administered human peripheral blood mononuclear cells. These hepatocyte injuries were inhibited to a greater extent by abatacept compared to methylprednisolone. Abatacept might be an effective therapy alternative to methylprednisolone to reduce acute massive liver damage for patients with severe acute hepatitis caused by HBV infection.

In vitro analysis of hepatic stellate cell activation influenced by transmembrane 6 superfamily 2 polymorphism.

Non­alcoholic steatohepatitis (NASH) may progress via liver fibrosis along with hepatic stellate cell (HSC) activation. A single nucleotide polymorphism (SNP; rs58542926) located in transmembrane 6 superfamily 2 (TM6SF2) has been reported to be significantly associated with fibrosis in patients with NASH, but the precise mechanism is still unknown. The present study aimed to explore the role of TM6SF2 in HSC activation in vitro. Plasmids producing TM6SF2 wild-type (WT) and mutant type (MT) containing E167K amino acid substitution were constructed, and the activation of LX­2 cells was analyzed by overexpressing or knocking down TM6SF2 under transforming growth factor ß1 (TGFß) treatment. Intracellular α­smooth muscle actin (αSMA) expression in LX­2 cells was significantly repressed by TM6SF2­WT overexpression and increased by TM6SF2 knockdown. Following treatment with TGFß, αSMA expression was restored in TM6SF2­WT overexpressed LX­2 cells and was enhanced in TM6SF2 knocked­down LX­2 cells. Comparing αSMA expression under TM6SF2­WT or ­MT overexpression, expression of αSMA in TM6SF2­MT overexpressed cells was higher than that in TM6SF2­WT cells and was further enhanced by TGFß treatment. The present study demonstrated that intracellular αSMA expression in HCS was negatively regulated by TM6SF2 while the E167K substitution released this negative regulation and led to enhanced HSC activation by TGFß. These results suggest that the SNP in TM6SF2 may relate to sensitivity of HSC activation.

Signal Activation of Hepatitis B Virus-Related Hepatocarcinogenesis by Up-regulation of SUV39h1.

BACKGROUND: Hepatitis B virus (HBV) X (HBx) protein is associated with hepatocellular carcinogenesis via the induction of malignant transformation and mitochondrial dysfunction. However, the association between HBx and histone methyltransferase in carcinogenesis has not been fully clarified. In the current study, we analyzed the association between HBx and the histone methyltransferase suppressor of variegation 3-9 homolog 1 (SUV39h1) using HBV replication models. METHODS: We constructed several HBx and SUV39h1 expression plasmids and analyzed the association between HBx and SUV39h1 with respect to HBV replication and hepatocarcinogenesis. RESULTS: SUV39h1 up-regulation was observed in HBV-infected humanized mouse livers and clinical HBV-related hepatocellular carcinoma tissues, indicating that SUV39h1 expression might be regulated by HBV infection. Through in vitro analysis, we determined that the coactivator domain of HBx interacts with the PSET (PostSET) and SET (Su(var)3-9, Enhancer-of-zeste, Trithorax) domains of SUV39h1. The expression levels of 4 genes, activating transcription factor 6, α-fetoprotein, growth arrest and DNA damage-inducible 45a, and dual-specificity phosphatase 1, known to induce carcinogenesis via HBx expression, were up-regulated by HBx and further up-regulated in the presence of both HBx and SUV39h1. Furthermore, histone methyltransferase activity, the main function of SUV39h1, was enhanced in the presence of HBx. CONCLUSIONS: We demonstrated that SUV39h1 and HBx enhance each other's activity, leading to HBx-mediated hepatocarcinogenesis. We propose that regulation of this interaction could help suppress development of hepatocellular carcinoma.

Circulating cytokines and angiogenic factors based signature associated with the relative dose intensity during treatment in patients with advanced hepatocellular carcinoma receiving lenvatinib.

BACKGROUND: Although lenvatinib was recently approved for treatment of advanced unresectable hepatocellular carcinoma (HCC) based on the phase III REFLECT trial, no biomarkers for management of lenvatinib treatment have been established. The aim of this study is to identify predictive biomarkers for the management of lenvatinib treatment in advanced HCC patients. METHODS: A total of 41 patients with advanced HCC were enrolled in this retrospective study. Serum levels of 22 circulating cytokines and angiogenic factors (CAFs) were measured by multiplex Luminex assay. Profiles of CAFs, clinical chemistry/hematology parameters, and clinical background were evaluated to explore biomarkers associated with clinical outcomes. RESULTS: Relative dose intensity (RDI) decreased significantly between weeks 1-2 and 3-4 (p < 0.001), and RDI during weeks 3-4 was a prominent indicator of progression-free survival (PFS). A signature based on baseline serum levels of nine CAFs associated with low RDI was identified. In a multivariate Cox regression analysis, patients with a favorable 9-CAFs signature [hazard ratio (HR) 0.42, 95% confidence interval (CI) 0.18-0.96, p = 0.040] had lower risk, and Child-Pugh grade B (HR 1.6, 95% CI 1.1-8.3, p = 0.026) and presence of macrovascular invasion (MVI; HR 2.9, 95% CI 1.0-8.3, p = 0.045) had higher risk of shorter PFS. CONCLUSION: This study demonstrates that RDI is an important predictive factor for longer PFS during lenvatinib treatment. In this hypothesis-generating exploratory analysis, we report that a CAF-signature associated with adverse events and RDI could predict PFS, which might contribute to improved management of lenvatinib treatment in HCC patients.

Publisher Correction: Regulation of the Hepatitis B virus replication and gene expression by the multi-functional protein TARDBP.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Comparative study on the replication of HCV1b genome between wild-type and cell culture-adaptive mutant in regard to sensitivities against anti-HCV drugs.

The replicon system, which mimics viral genome replication in culture cells, has been widely used to analyze the genome replication of the hepatitis C virus (HCV). However, most HCV genomes used in the system include adaptive mutations (AMs) that are vital for replication in culture cells despite the nonexistence of such mutations in the genome of wild-type (WT) HCV in patients. In order to study the genome replications of WT HCV, new HCV subgenomic replicon (SGR) systems were established using Huh-7.5-derived cells producing Sec14-like protein 2 constitutively and SGR of KT9 (one of the HCV genotype 1b clones) with WT genome (SGR KT9WT) in this study. The replication efficiency and sensitivities of SGR KT9WT to anti-HCV drugs in the cloned cells permanently bearing replicon RNA, HS55-4 cells, were similar to those of reports using SGR, including AM. The SGR transient transfection system using SGR KT9WT and SGR KT9AM encoding secreted Nano-luciferase and HS55-4C cells established by the elimination of SGR KT9 RNA from HS55-4 cells, however, showed that the replication efficiency of SGR KT9WT was much lower than that of SGR KT9AM under a same condition. Furthermore, the sensitivities of SGR KT9WT to almost all tested anti-HCV reagents, except the inhibitor of miR-122, a cellular factor important for HCV replication, were quite low compared with SGR KT9AM. These results suggested that the new replicon systems might not only provide information about precise responses against new anti-HCV drugs but also reveal novel molecular mechanisms supporting negligent proliferation of HCV.

Ribavirin induces hepatitis C virus genome mutations in chronic hepatitis patients who failed to respond to prior daclatasvir plus asunaprevir therapy.

Ribavirin (RBV) induces nucleotide (nt) substitutions in hepatitis C virus (HCV) genome nonstructural (NS) regions. Although emergence of drug resistance-associated variants is associated with direct-acting antiviral treatment failure, the effect of RBV on genome substitutions in such patients is unknown. Genotype 1b HCV subgenomic replicon cells were treated with RBV for 120 hours. Six patients with chronic genotype 1b with HCV-infected patients who failed to respond to prior daclatasvir plus asunaprevir (DCV/ASV) therapy were treated with 12 weeks of sofosbuvir and ledipasvir plus RBV after 4 weeks of RBV monotherapy. RBV-induced genome mutations in the HCV NS region (nt3493-9301) in replicon cells and in patients during 4 weeks of RBV monotherapy were analyzed by deep sequencing. RBV-associated G-to-A and C-to-U transitions increased in a dose-dependent manner in HCV replicon cells after the RBV treatment. In patients with prior DCV/ASV treatment failures, the median serum HCV RNA level was 6.25 ± 0.31 log IU/mL at the start of RBV therapy and decreased significantly to 5.95 ± 0.4 log IU/mL (P = .03) after 4 weeks of RBV monotherapy. Although predominant HCV genome substitutions rates were similar between nontreatment and RBV-treatment periods (0.042 and 0.031 per base pair, respectively; P = .248), the frequencies of G-to-A and C-to-U transitions significantly increased after RBV monotherapy. These transitions were enriched, particularly within the HCV NS3 region in all patients. RBV treatment induces G-to-A and C-to-U transitions in the HCV genome even in chronic patients with hepatitis C with prior DCV/ASV treatment failures.

Regulation of the Hepatitis B virus replication and gene expression by the multi-functional protein TARDBP.

Hepatitis B virus (HBV) infects the liver and is a key risk factor for hepatocellular carcinoma. Identification of host factors that support viral replication is important to understand mechanisms of viral replication and to develop new therapeutic strategies. We identified TARDBP as a host factor that regulates HBV. Silencing or knocking out the protein in HBV infected cells severely impaired the production of viral replicative intermediates, mRNAs, proteins, and virions, whereas ectopic expression of TARDBP rescued production of these products. Mechanistically, we found that the protein binds to the HBV core promoter, as shown by chromatin precipitation as well as mutagenesis and protein-DNA interaction assays. Using LC-MS/MS analysis, we also found that TARDBP binds to a number of other proteins known to support the HBV life cycle, including NPM1, PARP1, Hsp90, HNRNPC, SFPQ, PTBP1, HNRNPK, and PUF60. Interestingly, given its key role as a regulator of RNA splicing, we found that TARDBP has an inhibitory role on pregenomic RNA splicing, which might help the virus to export its non-canonical RNAs from the nucleus without being subjected to unwanted splicing, even though mRNA nuclear export is normally closely tied to RNA splicing. Taken together, our results demonstrate that TARDBP is involved in multiple steps of HBV replication via binding to both HBV DNA and RNA. The protein's broad interactome suggests that TARDBP may function as part of a RNA-binding scaffold involved in HBV replication and that the interaction between these proteins might be a target for development of anti-HBV drugs.

Efficacy of glecaprevir and pibrentasvir treatment for genotype 1b hepatitis C virus drug resistance-associated variants in humanized mice.

Combination therapy with glecaprevir (GLE) and pibrentasvir (PIB) has high efficacy for pan-genotypic hepatitis C virus (HCV)-infected patients. However, the efficacy for patients who acquired potent NS5A inhibitor resistance-associated variants (RAVs) as a result of failure to respond to previous direct-acting antiviral (DAA) therapies is unclear. We investigated the efficacy of GLE/PIB treatment for genotype 1b HCV strains containing RAVs using subgenomic replicon systems and human hepatocyte transplanted mice. Mice were injected with serum samples obtained from a DAA-naïve patient or daclatasvir plus asunaprevir (DCV/ASV) treatment failures including NS5A-L31M/Y93H, -P58S/A92K or -P32 deletion (P32del) RAVs, then treated with GLE/PIB. HCV was eliminated by GLE/PIB treatment in mice with wild-type and NS5A-L31M/Y93H but relapsed in mice with NS5A-P58S/A92K, followed by emergence of additional NS5A mutations after cessation of the treatment. In NS5A-P32del-infected mice, serum HCV RNA remained positive during the GLE/PIB treatment. NS5A-P58S/A92K showed 1.5-fold resistance to PIB relative to wild-type based on analysis using HCV subgenomic replicon systems. When mice were administered various proportions of HCV wild-type and P32del strains and treated with GLE/PIB, serum HCV RNA remained positive in mice with high frequencies of P32del. In these mice, the P32del was undetectable by deep sequencing before GLE/PIB treatment, but P32del strains relapsed after cessation of the GLE/PIB treatment. GLE/PIB is effective for wild-type and NS5A-L31M/Y93H HCV strains, but the effect seems to be low for P58S/A92K and NS5A-P32del RAVs. Although NS5A-P32del was not detected, the mutation may be present at low frequency in DCV/ASV treatment failures.

Comparison of intracellular responses between HBV genotype A and C infection in human hepatocyte chimeric mice.

BACKGROUND AND AIMS: The clinical course and responsiveness to antiviral treatments differs among hepatitis B virus (HBV) genotypes. However, the cause of these differences is unclear. In the present study, we compared mRNA expression profiles in human hepatocyte chimeric mice infected with HBV genotypes A and C. METHODS: Fifteen chimeric mice were prepared and divided into the following three groups: uninfected control mice, HBV genotype A-infected mice, and HBV genotype C-infected mice. Human hepatocytes were collected from these mouse livers and gene expression analyses were performed using next-generation RNA sequencing. RESULTS: Although similar pathways were influenced by HBV infection, including inflammation mediated by chemokine and cytokine signaling, p53, and integrin signaling pathways, expression levels of up-regulated genes by HBV genotype A or C infection were quite different. In HBV genotype A-infected hepatocytes, 172 genes, including KRT23 and C10orf54, were significantly more highly expressed than in HBV genotype C-infected cells, whereas 10 genes, including SPX and IER3, were expressed at significantly lower levels. Genes associated with the p53 pathway and the inflammation mediated by chemokine and cytokine signaling pathway were more highly expressed in cells with HBV genotype A infection, whereas genes associated with CCKR signaling map and oxidative stress response were more highly expressed in cells with HBV genotype C infection. CONCLUSION: Several differences in gene expression with respect to HBV genotype A and C infection were detected in human hepatocytes. These differences might be associated with genotypic difference in the clinical course or responsiveness to treatment.