A novel target of hepatitis B virus mutations: splicing of surface RNA.
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Hepatitis B↗
Biomedical subjects
Hubert E Blum
Publications and source records attributed to Hubert E Blum.
APOBEC-mediated interference with hepadnavirus production.
APOBEC3G is a cellular cytidine deaminase displaying broad antiretroviral activity. Recently, it was shown that APOBEC3G can also suppress hepatitis B virus (HBV) production in human hepatoma cells. In the present study, we characterized the mechanisms of APOBEC-mediated antiviral activity against HBV and related hepadnaviruses. We show that human APOBEC3G blocks HBV production in mammalian and nonmammalian cells and is active against duck HBV as well. Early steps of viral morphogenesis, including RNA and protein synthesis, binding of pregenomic RNA to core protein, and self-assembly of viral core protein, were unaffected. However, APOBEC3G rendered HBV core protein-associated full-length pregenomic RNA nuclease-sensitive. Ongoing reverse-transcription in capsids that had escaped the block in morphogenesis was not significantly inhibited. The antiviral effect was not modulated by abrogating or enhancing expression of the accessory HBV X protein, suggesting that HBV X protein does not represent a functional homologue to the HIV vif protein. Furthermore, human APOBEC3F but not rat APOBEC1 inhibited HBV DNA production. Viral RNA and low-level DNA produced in the presence of APOBEC3F or rat APOBEC1 occasionally displayed mutations, but the majority of clones were wild-type. In conclusion, APOBEC3G and APOBEC3F but not rat APOBEC1 can downregulate the production of replication-competent hepadnaviral nucleocapsids. In contrast to HIV and other retroviruses, however, APOBEC3G/3F-mediated editing of nucleic acids does not seem to represent an effective innate defense mechanism for hepadnaviruses.
APOBEC-1 Deaminase↗
Intrahepatic CD8+ T-cell failure during chronic hepatitis C virus infection.
The precise mechanisms responsible for the failure of intrahepatic hepatitis C virus (HCV)-specific CD8+ T cells to control the virus during persistent infection have not been fully defined. We therefore studied the CD8+ T-cell response in 27 HLA-A2-positive patients using four previously well-defined HLA-A2-restricted HCV epitopes. The corresponding HCV sequences were determined in several patients and compared with the intrahepatic HCV-specific CD8+ T-cell response. The results of the study indicate: (1) intrahepatic HCV-specific CD8+ T cells are present in the majority of patients with chronic HCV infection and overlap significantly with the response present in the peripheral blood. (2) A large fraction of intrahepatic HCV-specific CD8+ T cells are impaired in their ability to secrete interferon gamma (IFN-gamma). This dysfunction is specific for HCV-specific CD8+ T cells, since intrahepatic Flu-specific CD8+ T cells readily secrete this cytokine. (3) T-cell selection of epitope variants may have occurred in some patients. However, it is not an inevitable consequence of a functional virus-specific CD8+ T-cell response, since several patients with IFN-gamma-producing CD8+ T-cell responses harbored HCV sequences identical or cross-reactive with the prototype sequence. (4) The failure of intrahepatic virus-specific CD8+ T cells to sufficiently control the virus occurs despite the presence of virus-specific CD4+ T cells at the site of disease. In conclusion, different mechanisms contribute to the failure of intrahepatic CD8+ T cells to eliminate HCV infection, despite their persistence and accumulation in the liver.
Adult↗
Treatment of hepatocellular carcinoma.
Hepatocellular carcinoma (HCC) is one of the most common malignant tumours worldwide. The major etiologies and risk factors for HCC development are well defined and some of the multiple steps involved in hepatocarcinogenesis have been elucidated in recent years. Despite these scientific advances and the implementation of measures for early HCC detection in patients at risk, patient survival has not improved during the last three decades. This is due in part to the advanced stage of the disease at the time of clinical presentation, in part due to the limited therapeutic options. These fall into four main categories: (1) surgical interventions, including tumour resection and liver transplantation, (2) percutaneous interventions, including ethanol injection and radiofrequency thermal ablation, (3) transarterial interventions, including embolisation and chemoembolisation and (4) drugs as well as gene and immune therapies. These therapeutic strategies have been evaluated in part in randomised controlled clinical trials that are the basis for therapeutic recommendations. While surgery and percutaneous as well as transarterial interventions are effective in patients with limited disease (1-3 lesions, < 5 cm in diameter) and compensated underlying liver disease (cirrhosis Child A), at the time of diagnosis more than 80% patients present with multicentric HCC and advanced liver disease or comorbidities that restrict the therapeutic measures to best supportive care. In order to reduce morbidity and mortality from HCC, therefore, early diagnosis and the development of novel systemic therapies for advanced disease, including drugs, gene and immune therapies as well as primary HCC prevention are of paramount importance. Further, secondary HCC prevention after successful therapeutic interventions needs to be improved in order to make an impact on the survival of patients with HCC. New technologies, including gene expression profiling and proteomic analyses, should further elucidate the molecular events underlying HCC development and identify novel diagnostic markers as well as therapeutic and preventive targets.
Carcinoma, Hepatocellular↗
Chronic infection with hepatitis B viruses and antiviral drug evaluation in uPA mice after liver repopulation with tupaia hepatocytes.
BACKGROUND/AIMS: Transplantation of primary human hepatocytes and establishment of hepatitis B virus (HBV) infection in immunodeficient urokinase plasminogen activator (uPA) transgenic mice was shown. However, the availability of usable primary human hepatocytes is very limited. Therefore, alternative and more accessible sources of hepatocytes permissive for HBV infection are highly desirable. Here we investigated the potential of primary hepatocytes from the tree shrew Tupaia belangeri that were shown to be susceptible to HBV infection. METHODS: Freshly isolated or cryopreserved primary tupaia hepatocytes were transplantated via intrasplenic injection into immunodeficient uPA/RAG-2 mice. Engrafted mice were then infected with HBV and woolly monkey (WM)-HBV positive sera. RESULTS: Extensive proliferation of xenografted cells was demonstrated by the stable production of tupaia alpha1-antitrypsin in serum and liver of transplanted mice. Quantitative PCR assays demonstrated the presence of circulating viral particles as well as intracellular viral DNA, including covalently closed circular (ccc) DNA, in transplanted mice. Viral infection could be serially passaged in mice. Furthermore, viral replication was strongly inhibited by treating mice with adefovir dipivoxil. CONCLUSIONS: uPA mice repopulated with tupaia hepatocytes represent a useful and more accessible model for HBV infection studies, including the evaluation of antiviral therapy and cccDNA.
Adenine↗
Liver cancer.
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Carcinoma, Hepatocellular↗
Pathogenesis of hepatocellular carcinoma.
Hepatocellular carcinoma (HCC) is one of the most common malignant tumours worldwide. The major aetiologies and risk factors for the development of HCC are well defined and some of the multiple steps involved in hepatocarcinogenesis have been elucidated in recent years. However, no clear picture of how and in what sequence these factors interact at the molecular level has emerged yet. Malignant transformation of hepatocytes may occur as a consequence of various aetiologies, such as chronic viral hepatitis, alcohol, and metabolic disorders, in the context of increased cellular turnover induced by chronic liver injury, regeneration and cirrhosis. Activation of cellular oncogenes, inactivation of tumour suppressor genes, genomic instability, including DNA mismatch repair defects and impaired chromosomal segregation, overexpression of growth and angiogenic factors, and telomerase activation may contribute to the development of HCC. Overall, HCCs are genetically very heterogeneous tumours. New technologies, including gene expression profiling and proteomic analyses, should allow us to further elucidate the molecular events underlying HCC development and identify novel diagnostic markers as well as therapeutic targets.
Carcinoma, Hepatocellular↗
Stable human lymphoblastoid cell lines constitutively expressing hepatitis C virus proteins.
The cellular immune response plays a central role in virus clearance and pathogenesis of liver disease in hepatitis C. The study of hepatitis C virus (HCV)-specific immune responses is limited by currently available cell-culture systems. Here, the establishment and characterization of stable human HLA-A2-positive B-lymphoblastoid x T hybrid cell lines constitutively expressing either the NS3-4A complex or the entire HCV polyprotein are reported. These cell lines, termed T1/NS3-4A and T1/HCVcon, respectively, were maintained in continuous culture for more than 1 year with stable characteristics. HCV structural and non-structural proteins were processed accurately, indicating that the cellular and viral proteolytic machineries are functional in these cell lines. Viral proteins were found in the cytoplasm in dot-like structures when expressed in the context of the HCV polyprotein or in a perinuclear fringe when the NS3-4A complex was expressed alone. T1/NS3-4A and T1/HCVcon cells were lysed efficiently by HCV-specific cytotoxic T lymphocytes from patients with hepatitis C and from human HLA-A2.1 transgenic mice immunized with a liposomal HCV vaccine, indicating that viral proteins are processed endogenously and presented efficiently via the major histocompatibility complex class I pathway. In conclusion, these cell lines represent a unique tool to study the cellular immune response, as well as to evaluate novel vaccine and immunotherapeutic strategies against HCV.
Animals↗
T cells with a CD4+CD25+ regulatory phenotype suppress in vitro proliferation of virus-specific CD8+ T cells during chronic hepatitis C virus infection.
Chronic hepatitis C virus (HCV) infection is associated with impaired proliferative, cytokine, and cytotoxic effector functions of HCV-specific CD8(+) T cells that probably contribute significantly to viral persistence. Here, we investigated the potential role of T cells with a CD4(+)CD25(+) regulatory phenotype in suppressing virus-specific CD8(+) T-cell proliferation during chronic HCV infection. In vitro depletion studies and coculture experiments revealed that peptide specific proliferation as well as gamma interferon production of HCV-specific CD8(+) T cells were inhibited by CD4(+)CD25(+) T cells. This inhibition was dose dependent, required direct cell-cell contact, and was independent of interleukin-10 and transforming growth factor beta. Interestingly, the T-cell-mediated suppression in chronically HCV-infected patients was not restricted to HCV-specific CD8(+) T cells but also to influenza virus-specific CD8(+) T cells. Importantly, CD4(+)CD25(+) T cells from persons recovered from HCV infection and from healthy blood donors exhibited significantly less suppressor activity. Thus, the inhibition of virus-specific CD8(+) T-cell proliferation was enhanced in chronically HCV-infected patients. This was associated with a higher frequency of circulating CD4(+)CD25(+) cells observed in this patient group. Taken together, our results suggest that chronic HCV infection leads to the expansion of CD4(+)CD25(+) T cells that are able to suppress CD8(+) T-cell responses to different viral antigens. Our results further suggest that CD4(+)CD25(+) T cells may contribute to viral persistence in chronically HCV-infected patients and may be a target for immunotherapy of chronic hepatitis C.
Adult↗
Characterization of nonstructural protein membrane anchor deletion mutants expressed in the context of the hepatitis C virus polyprotein.
Protein-protein interactions involved in formation of the membrane-associated hepatitis C virus (HCV) replication complex are poorly understood. Here, we investigated nonstructural proteins with deletions in their membrane anchor domains when expressed in the context of the entire HCV polyprotein. Interactions among cytosolic domains of HCV nonstructural proteins were found not to be sufficiently strong to rescue such mutants to the membrane. Thus, the membrane anchor domains of nonstructural proteins are essential for incorporation of these proteins into the HCV replication complex while interactions among the cytosolic domains appear to be relatively weak. This feature may provide the nonstructural proteins with a certain flexibility to perform their multiple functions during HCV replication.
Cell Line, Tumor↗
Increased expression of the NK cell receptor KLRG1 by virus-specific CD8 T cells during persistent antigen stimulation.
The killer cell lectin-like receptor G1 (KLRG1) is a natural killer cell receptor expressed by T cells that exhibit impaired proliferative capacity. Here, we determined the KLRG1 expression by virus-specific T cells. We found that repetitive and persistent antigen stimulation leads to an increase in KLRG1 expression of virus-specific CD8+ T cells in mice and that virus-specific CD8+ T cells are mostly KLRG1+ in chronic human viral infections (human immunodeficiency virus, cytomegalovirus, and Epstein-Barr virus) but not in resolved infection (influenza virus). Thus, by using KLRG1 as a T-cell marker, our results suggest that the differentiation status and function of virus-specific CD8+ T cells are directly influenced by persistent antigen stimulation.
Animals↗
Scavenger receptor class B type I and hepatitis C virus infection of primary tupaia hepatocytes.
Hepatitis C virus (HCV) is a major cause of chronic hepatitis worldwide. The study of early steps during HCV infection has been hampered by the lack of suitable in vitro or in vivo models. Primary Tupaia hepatocytes (PTH) have been shown to be susceptible to HCV infection in vitro and in vivo. Human scavenger receptor class B type I (SR-BI) represents an HCV receptor candidate mediating the cellular binding of E2 glycoprotein to HepG2 hepatoma cells. However, the function of SR-BI for viral infection of hepatocytes is unknown. In this study, we used PTH to assess the functional role of SR-BI as a putative HCV receptor. Sequence analysis of cloned tupaia SR-BI revealed a high homology between tupaia and human SR-BI. Transfection of CHO cells with human or tupaia SR-BI but not mouse SR-BI cDNA resulted in cellular E2 binding, suggesting that E2-binding domains between human and tupaia SR-BI are highly conserved. Preincubation of PTH with anti-SR-BI antibodies resulted in marked inhibition of E2 or HCV-like particle binding. However, anti-SR-BI antibodies were not able to block HCV infection of PTH. In conclusion, our results demonstrate that SR-BI represents an important cell surface molecule for the binding of the HCV envelope to hepatocytes and suggest that other or additional cell surface molecules are required for the initiation of HCV infection. Furthermore, the structural and functional similarities between human and tupaia SR-BI indicate that PTH represent a useful model system to characterize the molecular interaction of the HCV envelope and SR-BI on primary hepatocytes.
12E7 Antigen↗
A mouse model of oral-esophageal carcinogenesis.
Squamous cancers of the oral cavity and esophagus are common worldwide. A number of environmental factors as well as genetic alterations have been identified. However, the specific combination of genetic events and their interplay with environmental carcinogens are largely un-known. Furthermore, no good animal model existed to study the molecular changes important in the induction and progression of the disease. Here we summarize the efforts made to establish a mouse model of oral-esophageal carcinogenesis. Cyclin D1 overexpressing(L2D1+) mice were generated using an EBV promoter to specifically target the oral cavity and the esophageal squamous epithelium. Besides analyzing different environmental factors, such as nitrosamines and zinc deficiency, cyclin D1 transgenic mice were crossbred with p53-deficient mice. While L2D1+ mice exhibited a phenotype of dysplasia, different combinations of mice result-ed in invasive oral-esophageal cancer. This mouse model provides a well-defined and reproducible model of oral-esophageal cancer that should be useful for testing chemopreventive, diagnostic, and therapeutic strategies.
Animals↗
Comment on "Inhibition of hepatitis B virus replication by APOBEC3G".
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APOBEC-3G Deaminase↗
Structure and function of the membrane anchor domain of hepatitis C virus nonstructural protein 5A.
Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a membrane-associated, essential component of the viral replication complex. Here, we report the three-dimensional structure of the membrane anchor domain of NS5A as determined by NMR spectroscopy. An alpha-helix extending from amino acid residue 5 to 25 was observed in the presence of different membrane mimetic media. This helix exhibited a hydrophobic, Trprich side embedded in detergent micelles, while the polar, charged side was exposed to the solvent. Thus, the NS5A membrane anchor domain forms an in-plane amphipathic alpha-helix embedded in the cytosolic leaflet of the membrane bilayer. Interestingly, mutations affecting the positioning of fully conserved residues located at the cytosolic surface of the helix impaired HCV RNA replication without interfering with the membrane association of NS5A. In conclusion, the NS5A membrane anchor domain constitutes a unique platform that is likely involved in specific interactions essential for the assembly of the HCV replication complex and that may represent a novel target for antiviral intervention.
Amino Acid Sequence↗
DDB treatment of patients with chronic hepatitis.
We report 13 patients (10 with chronic hepatitis C, 1 with chronic hepatitis B, 2 with nonalcoholic steatohepatitis) with persistently elevated alanine aminotransferase (ALT) levels who were treated with dimethyl-4,4'-dimethoxy-5,6,5',6-dimethylenedioxybiphenyl-2,2' dicarboxylate (DDB). ALT rapidly normalized in 12/13 patients and remained normal during treatment. Unlike ALT levels, aspartate aminotransferase, gamma-glutamyl transferase and glutamate dehydrogenase levels were not affected. Furthermore, there was no beneficial effect on the histological grade and stage of liver disease. In vitro experiments with hepatocytes resulted in a significant decrease of hepatocellular ALT levels in the DDB treated cells, suggesting, that DDB affects the synthesis and/or degradation of ALT in liver cells. In conclusion, the normalization of ALT during DDB treatment does not indicate therapeutic efficacy. In view of the wide use of DDB in patients with chronic liver diseases who participate in clinical studies DDB use should be excluded.
Dioxoles↗
Tracking cccDNA in chronic HBV infection.
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Animals↗
Out of control: hepatitis B reactivation by transarterial treatment of hepatocellular carcinoma.
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Antineoplastic Agents↗