Hepatitis D infection induces IFN-ß-mediated NK cell activation and TRAIL-dependent cytotoxicity.

Background and aims: The co-infection of hepatitis B (HBV) patients with the hepatitis D virus (HDV) causes the most severe form of viral hepatitis and thus drastically worsens the course of the disease. Therapy options for HBV/HDV patients are still limited. Here, we investigated the potential of natural killer (NK) cells that are crucial drivers of the innate immune response against viruses to target HDV-infected hepatocytes. Methods: We established in vitro co-culture models using HDV-infected hepatoma cell lines and human peripheral blood NK cells. We determined NK cell activation by flow cytometry, transcriptome analysis, bead-based cytokine immunoassays, and NK cell-mediated effects on T cells by flow cytometry. We validated the mechanisms using CRISPR/Cas9-mediated gene deletions. Moreover, we assessed the frequencies and phenotype of NK cells in peripheral blood of HBV and HDV superinfected patients. Results: Upon co-culture with HDV-infected hepatic cell lines, NK cells upregulated activation markers, interferon-stimulated genes (ISGs) including the death receptor ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), produced interferon (IFN)-γ and eliminated HDV-infected cells via the TRAIL-TRAIL-R2 axis. We identified IFN-ß released by HDV-infected cells as an important enhancer of NK cell activity. In line with our in vitro data, we observed activation of peripheral blood NK cells from HBV/HDV co-infected, but not HBV mono-infected patients. Conclusion: Our data demonstrate NK cell activation in HDV infection and their potential to eliminate HDV-infected hepatoma cells via the TRAIL/TRAIL-R2 axis which implies a high relevance of NK cells for the design of novel anti-viral therapies.

Medical Advances in Hepatitis D Therapy: Molecular Targets.

An approximate number of 250 million people worldwide are chronically infected with hepatitis B virus, making them susceptible to a coinfection with hepatitis D virus. The superinfection causes the most severe form of a viral hepatitis and thus drastically worsens the course of the disease. Until recently, the only available therapy consisted of interferon-α, only eligible for a minority of patients. In July 2020, the EMA granted Hepcludex conditional marketing authorization throughout the European Union. This first-in-class entry inhibitor offers the promise to prevent the spread in order to gain control and eventually participate in curing hepatitis B and D. Hepcludex is an example of how understanding the viral lifecycle can give rise to new therapy options. Sodium taurocholate co-transporting polypeptide, the virus receptor and the target of Hepcludex, and other targets of hepatitis D therapy currently researched are reviewed in this work. Farnesyltransferase inhibitors such as Lonafarnib, targeting another essential molecule in the HDV life cycle, represent a promising target for hepatitis D therapy. Farnesyltransferase attaches a farnesyl (isoprenyl) group to proteins carrying a C-terminal Ca1a2X (C: cysteine, a: aliphatic amino acid, X: C-terminal amino acid) motif like the large hepatitis D virus antigen. This modification enables the interaction of the HBV/HDV particle and the virus envelope proteins. Lonafarnib, which prevents this envelopment, has been tested in clinical trials. Targeting the lifecycle of the hepatitis B virus needs to be considered in hepatitis D therapy in order to cure a patient from both coexisting infections. Nucleic acid polymers target the hepatitis B lifecycle in a manner that is not yet understood. Understanding the possible targets of the hepatitis D virus therapy is inevitable for the improvement and development of a sufficient therapy that HDV patients are desperately in need of.

Lonafarnib: First Approval.

Lonafarnib (Zokinvy™) is an orally active farnesyltransferase inhibitor developed by Eiger BioPharmaceuticals under license from Merck & Co. for the treatment of hepatitis D virus (HDV) infections, and progeria and progeroid laminopathies. The drug was originally discovered by Merck & Co as an investigational drug in oncology. In progeria, lonafarnib inhibits farnesyltransferase to prevent farnesylation and subsequent accumulation of progerin and progerin-like proteins in the nucleus and cellular cytoskeleton. In November 2020, lonafarnib received its first approval in the USA to reduce the risk of mortality in Hutchinson-Gilford Progeria Syndrome (HGPS) and for the treatment of processing-deficient progeroid laminopathies (with either heterozygous LMNA mutation with progerin-like protein accumulation, or homozygous or compound heterozygous ZMPSTE24 mutations) in patients ≥ 12 months of age with a body surface area (BSA) of ≥ 0.39 m2. Lonafarnib is under regulatory review in the European Union. Clinical development for the treatment of HDV infections is underway in multiple countries. This article summarizes the milestones in the development of lonafarnib leading to this first approval.

Hepatitis delta: In vitro evaluation of cytotoxicity and cytokines involved in PEG-IFN therapy.

The treatment for hepatitis Delta virus (HDV) still consists of Pegylated interferon (PEG-IFN) combined with inhibitors of Hepatitis B virus (HBV) replication. In some patients may be occur a virological response, which means a negative HDV RNA 6 months after stopping treatment. In this study it was conducted an in vitro approach with the aim to mimic possible immunological events that are observed in patients responding to PEG-IFN therapy. Jurkat cells (human T lymphocyte cell line) were employed alone or co-cultured with THP-1 (human monocytic cell line) and stimulated with controls and HBV Surface Antigen (HBsAg), Small-Delta Antigen (SHDAg), and HBsAg + SHDAg combined. Twenty-four hours stimulation with SHDAg and/or HBSAg led to a toxic profile in a co-culture condition and cell supernatants were collected for cytokines quantification. PEG-IFN was added and cells were incubated for additional 24 h. Co-cultured cells incubated with the association (SHDAg + PEG-IFN) significantly produced levels of IFN-γ, IL-2 and IL-12. On the other hand, the HBsAg alone was able to inhibit the production of IFN-γ, suggesting that this antigen may hinder the treatment exclusively with PEG-IFN.

Bona fide receptor for hepatitis B and D viral infections: Mechanism, research models and molecular drug targets.

Hepatitis B infections have become a serious public health issue globally, and the current first-line antiviral treatment for this disease is not a true cure. Recently, sodium taurocholate cotransporting polypeptide (NTCP), a liver-specific bile acid transporter, was identified as a bona fide receptor for hepatitis B virus (HBV) and its satellite virus, hepatitis delta virus (HDV). Identification of the HBV receptor has led to the development of robust cell cultures and provides a potential target for new treatments. This review summarizes the process by which NTCP was discovered and describes its clinical significance as the receptor for HBV and HDV entry.

The Hepatitis Delta Virus accumulation requires paraspeckle components and affects NEAT1 level and PSP1 localization.

The Hepatitis Delta Virus (HDV) relies mainly on host proteins for its replication. We previously identified that PSF and p54nrb associate with the HDV RNA genome during viral replication. Together with PSP1, these proteins are part of paraspeckles, which are subnuclear bodies nucleated by the long non-coding RNA NEAT1. In this work, we established the requirement for PSF, p54nrb and PSP1 in HDV replication using RNAi-mediated knockdown in HEK-293 cells replicating the HDV RNA genome. We determined that HDV replication induces the delocalization of PSP1 to cytoplasmic foci containing PABP and increases NEAT1 level causing an enlargement of NEAT1 foci. Overall, our data support a role for the main paraspeckles proteins in HDV life cycle and indicate that HDV replication causes a cellular stress and induces both a delocalization of the PSP1 to the cytoplasm and a disruption of paraspeckles.

Drug-drug interaction potential of the HBV and HDV entry inhibitor myrcludex B assessed in vitro.

BACKGROUND: Myrcludex B is a first-in-class virus entry inhibitor for patients with chronic hepatitis B or B/D infections. In patients it will be coadministered with drugs needed for the disease or comorbidities. We aimed to define the risk of drug-drug interactions by characterizing the influence of myrcludex B on relevant drug transporting and metabolizing enzymes in vitro. METHODS: Inhibition of P-glycoprotein (P-gp; ABCB1), breast cancer resistance protein (BCRP/ABCG2), and the organic anion transporting polypeptides 1B1 and 1B3 (OATP1B1/SLCO1B1 and OATP1B3/SLCO1B3) was measured in cells over-expressing the respective transporter using fluorogenic substrates. Inhibition of cytochrome P450 enzymes (CYPs) was assessed with commercially available kits. mRNA induction of drug transporting and metabolizing enzymes was measured in LS180 cells after 4 days of treatment by quantitative real-time PCR. Pregnane X receptor (PXR) activation was assessed using a reporter-gene assay. RESULTS: Whereas activities of P-gp and BCRP were not influenced by myrcludex B, OATP1B1 and OATP1B3 were specifically inhibited with a 50% inhibitory concentration (IC50) of 0.5 and 8.7 µM, respectively. Myrcludex B weakly inhibited all CYPs tested at concentrations ≥10 µM except CYP2D6, which was not inhibited at concentrations up to 2 µM. Myrcludex B had no influence on mRNA expression of CYP1A1, CYP3A4, UGT1A3, ABCB1, ABCC2 and ABCG2, and on PXR activity. CONCLUSIONS: Our in vitro study suggests that myrcludex B is not at major risk of acting as a perpetrator drug. A potential inhibition of the uptake transporters OATP1B1 and OATP1B3 and a previous clinical finding of potential CYP3A inhibition, requires further evaluation and should be carefully addressed in future trials.

Reduced hepatitis B and D viral entry using clinically applied drugs as novel inhibitors of the bile acid transporter NTCP.

The sodium taurocholate co-transporting polypeptide (NTCP, SLC10A1) is the main hepatic transporter of conjugated bile acids, and the entry receptor for hepatitis B virus (HBV) and hepatitis delta virus (HDV). Myrcludex B, a synthetic peptide mimicking the NTCP-binding domain of HBV, effectively blocks HBV and HDV infection. In addition, Myrcludex B inhibits NTCP-mediated bile acid uptake, suggesting that also other NTCP inhibitors could potentially be a novel treatment of HBV/HDV infection. This study aims to identify clinically-applied compounds intervening with NTCP-mediated bile acid transport and HBV/HDV infection. 1280 FDA/EMA-approved drugs were screened to identify compounds that reduce uptake of taurocholic acid and lower Myrcludex B-binding in U2OS cells stably expressing human NTCP. HBV/HDV viral entry inhibition was studied in HepaRG cells. The four most potent inhibitors of human NTCP were rosiglitazone (IC50 5.1 µM), zafirlukast (IC50 6.5 µM), TRIAC (IC50 6.9 µM), and sulfasalazine (IC50 9.6 µM). Chicago sky blue 6B (IC50 7.1 µM) inhibited both NTCP and ASBT, a distinct though related bile acid transporter. Rosiglitazone, zafirlukast, TRIAC, sulfasalazine, and chicago sky blue 6B reduced HBV/HDV infection in HepaRG cells in a dose-dependent manner. Five out of 1280 clinically approved drugs were identified that inhibit NTCP-mediated bile acid uptake and HBV/HDV infection in vitro.

Both interferon alpha and lambda can reduce all intrahepatic HDV infection markers in HBV/HDV infected humanized mice.

Co-infection with hepatitis B (HBV) and D virus (HDV) is associated with the most severe course of liver disease. Interferon represents the only treatment currently approved. However, knowledge about the impact of interferons on HDV in human hepatocytes is scant. Aim was to assess the effect of pegylated interferon alpha (peg-IFNα) and lambda (peg-IFNλ), compared to the HBV-polymerase inhibitor entecavir (ETV) on all HDV infection markers using human liver chimeric mice and novel HDV strand-specific qRT-PCR and RNA in situ hybridization assays, which enable intrahepatic detection of HDV RNA species. Peg-IFNα and peg-IFNλ reduced HDV viremia (1.4 log and 1.2 log, respectively) and serum HBsAg levels (0.9-log and 0.4-log, respectively). Intrahepatic quantification of genomic and antigenomic HDV RNAs revealed a median ratio of 22:1 in untreated mice, resembling levels determined in HBV/HDV infected patients. Both IFNs greatly reduced intrahepatic levels of genomic and antigenomic HDV RNA, increasing the amounts of HDAg- and antigenomic RNA-negative hepatocytes. ETV-mediated suppression of HBV replication (2.1-log) did not significantly affect HBsAg levels, HDV productivity and/or release. In humanized mice lacking adaptive immunity, IFNs but not ETV suppressed HDV. Viremia decrease reflected the intrahepatic reduction of all HDV markers, including the antigenomic template, suggesting that intracellular HDV clearance is achievable.

The hepatitis delta virus: Replication and pathogenesis.

Hepatitis delta virus (HDV) is a defective virus and a satellite of the hepatitis B virus (HBV). Its RNA genome is unique among animal viruses, but it shares common features with some plant viroids, including a replication mechanism that uses a host RNA polymerase. In infected cells, HDV genome replication and formation of a nucleocapsid-like ribonucleoprotein (RNP) are independent of HBV. But the RNP cannot exit, and therefore propagate, in the absence of HBV, as the latter supplies the propagation mechanism, from coating the HDV RNP with the HBV envelope proteins for cell egress to delivery of the HDV virions to the human hepatocyte target. HDV is therefore an obligate satellite of HBV; it infects humans either concomitantly with HBV or after HBV infection. HDV affects an estimated 15 to 20 million individuals worldwide, and the clinical significance of HDV infection is more severe forms of viral hepatitis--acute or chronic--, and a higher risk of developing cirrhosis and hepatocellular carcinoma in comparison to HBV monoinfection. This review covers molecular aspects of HDV replication cycle, including its interaction with the helper HBV and the pathogenesis of infection in humans.

[HBV COVALENTLY CLOSED CIRCULAR DNA AS A MARKER OF PREVALENCE OF OCCULT HEPATITIS B IN PATIENTS WITH HBV, HDVAND HCV INFECTION IN UZBEKISTAN].

AIM: Evaluate significance of covalently closed circular DNA of hepatitis B virus as a marker for detection of occult viral hepatitis B in Uzbekistan population with hepatitis of various genesis. MATERIALS AND METHODS: Blood plasma and liver biopsy from 39 patients with different severity levels of liver fibrosis and cirrhosis served as study material. HBV covalently closed circular DNA detection was carried out according to Pollicino T et al. (2004). RESULTS: Covalently closed circu- lar DNA of hepatitis B virus was detected in 82% of samples, including in 54.5% of patients with chronic viral hepatitis C (CVHC) and in 100% of patients with hepatitis of unknown etiology. Quantitative evaluation of content of covalently closed circular DNA of hepatitis B virus in liver tissue in patients with CVHB has shown an average of 2.5 copies of HBV genome as ccc DNA per cell, in patients with CVHB + D an average of 0.7 copies/cell, in patients with co-infection by HCV and HBV - 0.5 copies/cell, in patients with CVHC an average of 0.12 copies/cell, and in patients with cryptogenic hepatitis - 0.2 copies/cell. CONCLUSION: Detection of HBV DNA is a complex problem for effective laboratory diagnostics of hepatitis. Detection of HBV ccc DNA as a marker of occult hepatitis B in patients with CVHC and patients with hepatitis of unclear etiol- ogy is an. important factor for diagnostics, selection of adequate therapy, prognosis of disease outcome and prevention of development of severe liver diseases.

Hepatitis D Virus Infection of Mice Expressing Human Sodium Taurocholate Co-transporting Polypeptide.

Hepatitis D virus (HDV) is the smallest virus known to infect human. About 15 million people worldwide are infected by HDV among those 240 million infected by its helper hepatitis B virus (HBV). Viral hepatitis D is considered as one of the most severe forms of human viral hepatitis. No specific antivirals are currently available to treat HDV infection and antivirals against HBV do not ameliorate hepatitis D. Liver sodium taurocholate co-transporting polypeptide (NTCP) was recently identified as a common entry receptor for HDV and HBV in cell cultures. Here we show HDV can infect mice expressing human NTCP (hNTCP-Tg). Antibodies against critical regions of HBV envelope proteins blocked HDV infection in the hNTCP-Tg mice. The infection was acute yet HDV genome replication occurred efficiently, evident by the presence of antigenome RNA and edited RNA species specifying large delta antigen in the livers of infected mice. The resolution of HDV infection appears not dependent on adaptive immune response, but might be facilitated by innate immunity. Liver RNA-seq analyses of HDV infected hNTCP-Tg and type I interferon receptor 1 (IFNα/ßR1) null hNTCP-Tg mice indicated that in addition to induction of type I IFN response, HDV infection was also associated with up-regulation of novel cellular genes that may modulate HDV infection. Our work has thus proved the concept that NTCP is a functional receptor for HDV infection in vivo and established a convenient small animal model for investigation of HDV pathogenesis and evaluation of antiviral therapeutics against the early steps of infection for this important human pathogen.

Hepatitis D virus infection, replication and cross-talk with the hepatitis B virus.

Viral hepatitis remains a worldwide public health problem. The hepatitis D virus (HDV) must either coinfect or superinfect with the hepatitis B virus (HBV). HDV contains a small RNA genome (approximately 1.7 kb) with a single open reading frame (ORF) and requires HBV supplying surface antigens (HBsAgs) to assemble a new HDV virion. During HDV replication, two isoforms of a delta antigen, a small delta antigen (SDAg) and a large delta antigen (LDAg), are produced from the same ORF of the HDV genome. The SDAg is required for HDV replication, whereas the interaction of LDAg with HBsAgs is crucial for packaging of HDV RNA. Various clinical outcomes of HBV/HDV dual infection have been reported, but the molecular interaction between HBV and HDV is poorly understood, especially regarding how HBV and HDV compete with HBsAgs for assembling virions. In this paper, we review the role of endoplasmic reticulum stress induced by HBsAgs and the molecular pathway involved in their promotion of LDAg nuclear export. Because the nuclear sublocalization and export of LDAg is regulated by posttranslational modifications (PTMs), including acetylation, phosphorylation, and isoprenylation, we also summarize the relationship among HBsAg-induced endoplasmic reticulum stress signaling, LDAg PTMs, and nuclear export mechanisms in this review.

Modification of the hepatitis B virus envelope protein glycosylation pattern interferes with secretion of viral particles, infectivity, and susceptibility to neutralizing antibodies.

UNLABELLED: The envelope proteins of hepatitis B virus (HBV) bear an N-linked glycosylation site at N146 within the immunodominant a-determinant in the antigenic loop (AGL) region. This glycosylation site is never fully functional, leading to a nearly 1/1 ratio of glycosylated/nonglycosylated isoforms in the viral envelope. Here we investigated the requirement for a precise positioning of N-linked glycan at amino acid 146 and the functions associated with the glycosylated and nonglycosylated isoforms. We observed that the removal of the N146 glycosylation site by mutagenesis was permissive to envelope protein synthesis and stability and to secretion of subviral particles (SVPs) and hepatitis delta virus (HDV) virions, but it was detrimental to HBV virion production. Several positions in the AGL could substitute for position 146 as the glycosylation acceptor site. At position 146, neither a glycan chain nor asparagine was absolutely required for infectivity, but there was a preference for a polar residue. Envelope proteins bearing 5 AGL glycosylation sites became hyperglycosylated, leading to an increased capacity for SVP secretion at the expense of HBV and HDV virion secretion. Infectivity-compatible N-glycosylation sites could be inserted at 3 positions (positions 115, 129, and 136), but when all three positions were glycosylated, the hyperglycosylated mutant was substantially attenuated at viral entry, while it acquired resistance to neutralizing antibodies. Taken together, these findings suggest that the nonglycosylated N146 is essential for infectivity, while the glycosylated form, in addition to its importance for HBV virion secretion, is instrumental in shielding the a-determinant from neutralizing antibodies. IMPORTANCE: At the surface of HBV particles, the immunodominant a-determinant is the main target of neutralizing antibodies and an essential determinant of infectivity. It contains an N-glycosylation site at position 146, which is functional on only half of the envelope proteins. Our data suggest that the coexistence of nonglycosylated and glycosylated N146 at the surface of HBV reflects the dual function of this determinant in infectivity and immune escape. Hence, a modification of the HBV glycosylation pattern affects not only virion assembly and infectivity but also immune escape.

Viral entry of hepatitis B and D viruses and bile salts transportation share common molecular determinants on sodium taurocholate cotransporting polypeptide.

UNLABELLED: The liver bile acids transporter sodium taurocholate cotransporting polypeptide (NTCP) is responsible for the majority of sodium-dependent bile salts uptake by hepatocytes. NTCP also functions as a cellular receptor for viral entry of hepatitis B virus (HBV) and hepatitis D virus (HDV) through a specific interaction between NTCP and the pre-S1 domain of HBV large envelope protein. However, it remains unknown if these two functions of NTCP are independent or if they interfere with each other. Here we show that binding of the pre-S1 domain to human NTCP blocks taurocholate uptake by the receptor; conversely, some bile acid substrates of NTCP inhibit HBV and HDV entry. Mutations of NTCP residues critical for bile salts binding severely impair viral infection by HDV and HBV; to a lesser extent, the residues important for sodium binding also inhibit viral infection. The mutation S267F, corresponding to a single nucleotide polymorphism (SNP) found in about 9% of the East Asian population, renders NTCP without either taurocholate transporting activity or the ability to support HBV or HDV infection in cell culture. These results demonstrate that molecular determinants critical for HBV and HDV entry overlap with that for bile salts uptake by NTCP, indicating that viral infection may interfere with the normal function of NTCP, and bile acids and their derivatives hold the potential for further development into antiviral drugs. IMPORTANCE: Human hepatitis B virus (HBV) and its satellite virus, hepatitis D virus (HDV), are important human pathogens. Available therapeutics against HBV are limited, and there is no drug that is clinically available for HDV infection. A liver bile acids transporter (sodium taurocholate cotransporting polypeptide [NTCP]) critical for maintaining homeostasis of bile acids serves as a functional receptor for HBV and HDV. We report here that the NTCP-binding lipopeptide that originates from the first 47 amino acids of the pre-S1 domain of the HBV L protein blocks taurocholate transport. Some bile salts dose dependently inhibit HBV and HDV infection mediated by NTCP; molecular determinants of NTCP critical for HBV and HDV entry overlap with that for bile acids transport. This work advances our understanding of NTCP-mediated HBV and HDV infection in relation to NTCP's physiological function. Our results also suggest that bile acids or their derivatives hold potential for development into novel drugs against HBV and HDV infection.

High prevalence and significance of hepatitis D virus infection among treatment-naïve HBsAg-positive patients in Northern Vietnam.

BACKGROUND: Hepatitis D virus (HDV) infection is considered to cause more severe hepatitis than hepatitis B virus (HBV) monoinfection. With more than 9.5 million HBV-infected people, Vietnam will face an enormous health burden. The prevalence of HDV in Vietnamese HBsAg-positive patients is speculative. Therefore, we assessed the prevalence of HDV in Vietnamese patients, determined the HDV-genotype distribution and compared the findings with the clinical outcome. METHODS: 266 sera of well-characterized HBsAg-positive patients in Northern Vietnam were analysed for the presence of HDV using newly developed HDV-specific RT-PCRs. Sequencing and phylogenetic analysis were performed for HDV-genotyping. RESULTS: The HDV-genome prevalence observed in the Vietnamese HBsAg-positive patients was high with 15.4% while patients with acute hepatitis showed 43.3%. Phylogenetic analysis demonstrated a predominance of HDV-genotype 1 clustering in an Asian clade while HDV-genotype 2 could be also detected. The serum aminotransferase levels (AST, ALT) as well as total and direct bilirubin were significantly elevated in HDV-positive individuals (p<0.05). HDV loads were mainly low (<300 to 4.108 HDV-copies/ml). Of note, higher HDV loads were mainly found in HBV-genotype mix samples in contrast to single HBV-infections. In HBV/HDV-coinfections, HBV loads were significantly higher in HBV-genotype C in comparison to HBV-genotype A samples (p<0.05). CONCLUSION: HDV prevalence is high in Vietnamese individuals, especially in patients with acute hepatitis B. HDV replication activity showed a HBV-genotype dependency and could be associated with elevated liver parameters. Besides serological assays molecular tests are recommended for diagnosis of HDV. Finally, the high prevalence of HBV and HDV prompts the urgent need for HBV-vaccination coverage.

Proteomic changes in HEK-293 cells induced by hepatitis delta virus replication.

Hepatitis delta virus (HDV) infection greatly increases the risk of hepatocellular carcinoma in hepatitis B virus chronically infected patients. HDV is highly dependent on host factors for accomplishment of the replication cycle. However, these factors are largely unknown and the mechanisms involved in the pathogenicity of the virus still remain elusive. Here, we made use of the HEK-293 cell line, which was engineered in order to mimic HDV replication. Five different proteomes were analyzed and compared using a MS-based quantitative proteomics approach by (18)O/(16)O stable isotope labeling. About 3000 proteins were quantified and 89 found to be differentially expressed as a consequence HDV RNA replication. The down-regulation of p53 , HSPE, and ELAV as well as the up-regulation of Transportin 1 , EIF3D, and Cofilin 1 were validated by Western blot. A systems biology approach was additionally used to analyze altered pathways and networks. The G2/M DNA damage checkpoint and pyruvate metabolism were among the most affected pathways, and Cancer was the most likely disease associated to HDV replication. Western blot analysis allowed identifying 14-3-3 σ interactor as down-regulated protein acting in the G2/M cell cycle control checkpoint. This evidence supports deregulation of G2/M checkpoint as a possible mechanism involved in the promotion of HDV associated hepatocellular carcinoma. BIOLOGICAL SIGNIFICANCE: This manuscript provides a description of changes observed in the cellular proteome that arise as result of expression of the hepatitis delta virus (HDV) antigen as well as virus genome replication. Using a systems biology approach cancer was found to be the most probable disease associated with HDV replication. Additionally, results show that HDV alters the regulation of G2/M cell cycle control checkpoint. Taken together, our data provide new insights into probable mechanisms associated with the increased incidence of hepatocellular carcinoma observed in HDV infected patients.

Distribution of copper, iron, and zinc in biological samples (scalp hair, serum, blood, and urine) of Pakistani viral hepatitis (A-E) patients and controls.

The aim of the present study was to compare the level of copper (Cu), iron (Fe) and zinc (Zn) in biological samples (serum, blood, urine, and scalp hair) of patients suffering from different viral hepatitis (A, B, C, D, and E; n = 521) of both gender age ranged 31-45 years. For comparative study, 255 age-matched control subjects, of both genders residing in the same city were selected as referents. The elements in the biological samples were analyzed by flame atomic absorption spectrophotometry, prior to microwave-assisted acid digestion. The validity and accuracy of the methodology was checked by using certified reference materials (CRMs) and with those values obtained by conventional wet acid digestion method on same CRMs. The results of this study showed that the mean values of Cu and Fe were higher in blood, sera, and scalp hair samples of hepatitis patients, while Zn level was found to be lower than age-matched control subjects. The urinary levels of these elements were found to be higher in the hepatitis patients than in the age-matched healthy controls (p < 0.05). These results are consistent with literature-reported data, confirming that the deficiency of zinc and hepatic iron and copper overload can directly cause lipid peroxidation and eventually hepatic damage.

Large hepatitis delta antigen modulates transforming growth factor-beta signaling cascades: implication of hepatitis delta virus-induced liver fibrosis.

BACKGROUND & AIMS: Transforming growth factor-beta (TGF-beta) has been implicated in the pathogenesis of liver disease. TGF-beta is involved in liver regeneration and in the fibrotic and cirrhotic transformation with hepatitis viral infection. Hepatitis delta virus (HDV) infection causes fulminant hepatitis and liver cirrhosis. To elucidate the molecular mechanism of HDV pathogenesis, we examined the effects of HDV-encoded-only protein, the small hepatitis delta antigen (SHDAg), and the large hepatitis delta antigen (LHDAg), on TGF-beta- and c-Jun-induced signaling cascades. METHODS: The effects of either SHDAg or LHDAg on TGF-beta- and c-Jun-induced signaling cascades in Huh7 and Cos7 cells were investigated by luciferase reporter gene assay, immunoprecipitation assay, electrophoretic mobility shift assay, Western blot analysis, and confocal microscopy analysis. RESULTS: The LHDAg, but not the SHDAg, potentiated TGF-beta- and c-Jun-induced signal activation, and the isoprenylation of LHDAg played a major role in signaling cascades. LHDAg synergistically activated hepatitis B virus X protein-mediated TGF-beta and AP-1 signaling cascades. In addition, LHDAg enhanced the protein expression level of TGF-beta-induced plasminogen activator inhibitor-1. CONCLUSIONS: LHDAg may induce liver fibrosis through the regulation of TGF-beta-induced signal transductions. This regulation of TGF-beta-mediated signaling is accomplished by the isoprenylation of LHDAg, which is a novel mechanism involved in HDV pathogenesis.