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Recombinant adenoviruses expressing steatosis-associated hepatitis C virus genotype 3 core protein produce intracellular lipid accumulation in cultured and primary hepatocytes.

ABSTRACT: Steatosis, or "fatty liver", is a common finding in Hepatitis C virus (HCV) infection and reflects dysregulation of lipid homeostasis. HCV Core protein causes lipid accumulation when expressed in cultured cell lines, which data now indicates is a critical step for HCV replication, assembly and release. Previous studies on HCV Core and lipid accumulation have been limited by poor efficiency of conventional transfection techniques or the cell line limitations of the available cell culture system. In this study, we have designed recombinant adenoviruses expressing HCV Core and demonstrate that infection of both cultured and primary cells leads to intracellular lipid accumulation. This system will allow for detailed studies of the mechanisms that drive intracellular lipid accumulation during HCV infection and addresses some of the disadvantages that have hampered previous research.

SUBMITTER: Qiang G

PROVIDER: S-EPMC2634964 | biostudies-literature | 2009 Jan

REPOSITORIES: biostudies-literature

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Recombinant adenoviruses expressing steatosis-associated hepatitis C virus genotype 3 core protein produce intracellular lipid accumulation in cultured and primary hepatocytes.

Qiang Guan G Yang Liu L Witek Rafal P RP Jhaveri Ravi R

Virus research 20081204 1

Steatosis, or "fatty liver", is a common finding in Hepatitis C virus (HCV) infection and reflects dysregulation of lipid homeostasis. HCV Core protein causes lipid accumulation when expressed in cultured cell lines, which data now indicates is a critical step for HCV replication, assembly and release. Previous studies on HCV Core and lipid accumulation have been limited by poor efficiency of conventional transfection techniques or the cell line limitations of the available cell culture system. ...[more]

PMID: 19010361

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Project description:BackgroundIn spite of dozens of clinical trials to establish effective therapeutic and/or preventive vaccine to resolve HCV infection, no real vaccine has been proved to date. Genetic vaccines based on replication-defective adenoviruses have proved to elicit strong and long lasting T-cell responses against a number of viral antigens and are even currently being used for vaccine trials in humans. According to the controversy in the immune modulatory effects of both core and NS3 full length genes, it seemed more practical to employ some parts of these HCV proteins for vaccine design.ObjectivesTo generate recombinant Adenoviral vectors containing new overlapping-truncated region of NS3 gene or both the N- and C-terminal deleted parts of core gene, as well as a fusion fragment derived from both of them.Materials and methodsThe corresponding transfer vectors expressing truncated fragments of core, NS3 or a fusion fragment of both genes were prepared. The integrity and sequence of the transfer vectors were confirmed, and followed by experiments involving homologous recombination between them and the adenovirus backbone plasmid in the bacterial host. Recombinant Ad-pNS3, Ad-pCore and Ad-pNS3pCore viruses were prepared by transfection of these new recombined constructs into 293 packaging cell lines. The virus titer was then calculated by an immunohistochemistry based method. The RT-PCR, Real-Time PCR and western blotting were used to evaluate gene expression by all recombinant constructs. The production of complete virion particles was evaluated by detailed electron microscopy in addition to the appearance of typical cytopathic effects (CPE) and GFP expression patterns in 293 cells. The RT-PCR and GFP detection were employed to monitor the integrity as well as infectivity potency of the viral particles in Hep-G2 cells.ResultsRT-PCR, Real-Time PCR or western blotting confirmed expression of truncated fragment of NS3, core or a fusion fragment of theirs by newly constructed Ad-pNS3, Ad-pCore, Ad- pNS3pCore particles. Electron microscopy, which revealed many adenovirus-like particles and characteristics of CPE in infected cells in addition to GFP detection, confirmed the infectivity, potency and integrity of recombinant adenoviral particles.ConclusionsThese adenoviruses expressing novel fragments of NS3 and core genes may be suitable tools to overcome shortcomings associated with full gene expression in the setting of HCV vaccine therapy.

Project description:Refined methods for maintaining specific functions of isolated hepatocytes under xeno-free and chemical defined conditions is of great importance for the development of hepatocyte research and regenerative therapy. Laminins, a large family of heterotrimeric basement membrane adhesion proteins, are highly cell and tissue type specific components of the extracellular matrix and strongly influence the behavior and function of associated cells and/or tissues. However, detailed biological functions of many laminin isoforms are still to be evaluated. In this study, we determined the distribution of laminin isoforms in human liver tissue and isolated primary human hepatocytes by western blot analysis, and investigated the efficacy of different human recombinant laminin isoforms on hepatic functions during culture. Protein expressions of laminin-chain ?2, ?3, ?4, ?1, ?3, ?1, and ?2 were detected in both isolated human hepatocytes and liver tissue. No ?1 and ?5 expression could be detected in liver tissue or hepatocytes. Hepatocytes were isolated from five different individual livers, and cultured on human recombinant laminin isoforms -111, -211, -221, -332, -411, -421, -511, and -521 (Biolamina AB), matrigel (extracted from Engelbreth-Holm-Swarm sarcoma), or collagen type IV (Collagen). Hepatocytes cultured on laminin showed characteristic hexagonal shape in a flat cell monolayer. Viability, double stranded DNA concentration, and Ki67 expression for hepatocytes cultured for six days on laminin were comparable to those cultured on EHS and Collagen. Hepatocytes cultured on laminin also displayed production of human albumin, alpha-1-antitrypsin, bile acids, and gene expression of liver-enriched factors, such as hepatocyte nuclear factor 4 alpha, glucose-6-phosphate, cytochrome P450 3A4, and multidrug resistance-associated protein 2. We conclude that all forms of human recombinant laminin tested maintain cell viability and liver-specific functions of primary human hepatocytes, and that recombinant laminin is a promising xeno-free and chemical defined strategy for preservation of hepatocyte specific function in vitro.

Dataset Information

Recombinant adenoviruses expressing steatosis-associated hepatitis C virus genotype 3 core protein produce intracellular lipid accumulation in cultured and primary hepatocytes.

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Recombinant adenoviruses expressing steatosis-associated hepatitis C virus genotype 3 core protein produce intracellular lipid accumulation in cultured and primary hepatocytes.

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