A transgenic zebrafish model of hepatocyte function in human Z α1-antitrypsin deficiency.

In human α1-antitrypsin deficiency, homozygous carriers of the Z (E324K) mutation in the gene SERPINA1 have insufficient circulating α1-antitrypsin and are predisposed to emphysema. Misfolding and accumulation of the mutant protein in hepatocytes also causes endoplasmic reticulum stress and underpins long-term liver damage. Here, we describe transgenic zebrafish (Danio rerio) expressing the wildtype or the Z mutant form of human α1-antitrypsin in hepatocytes. As observed in afflicted humans, and in rodent models, about 80% less α1-antitrypsin is evident in the circulation of zebrafish expressing the Z mutant. Although these zebrafish also show signs of liver stress, they do not accumulate α1-antitrypsin in hepatocytes. This new zebrafish model will provide useful insights into understanding and treatment of α1-antitrypsin deficiency.

The antiviral protein viperin inhibits hepatitis C virus replication via interaction with nonstructural protein 5A.

UNLABELLED: The interferon-stimulated gene, viperin, has been shown to have antiviral activity against hepatitis C virus (HCV) in the context of the HCV replicon, although the molecular mechanisms responsible are not well understood. Here, we demonstrate that viperin plays an integral part in the ability of interferon to limit the replication of cell-culture-derived HCV (JFH-1) that accurately reflects the complete viral life cycle. Using confocal microscopy and fluorescence resonance energy transfer (FRET) analysis, we demonstrate that viperin localizes and interacts with HCV nonstructural protein 5A (NS5A) at the lipid-droplet (LD) interface. In addition, viperin also associates with NS5A and the proviral cellular factor, human vesicle-associated membrane protein-associated protein subtype A (VAP-A), at the HCV replication complex. The ability of viperin to limit HCV replication was dependent on residues within the C-terminus, as well as an N-terminal amphipathic helix. Removal of the amphipathic helix-redirected viperin from the cytosolic face of the endoplasmic reticulum and the LD to a homogenous cytoplasmic distribution, coinciding with a loss of antiviral effect. C-terminal viperin mutants still localized to the LD interface and replication complexes, but did not interact with NS5A proteins, as determined by FRET analysis. CONCLUSION: In conclusion, we propose that viperin interacts with NS5A and the host factor, VAP-A, to limit HCV replication at the replication complex. This highlights the complexity of the host control of viral replication by interferon-stimulated gene expression.

Hepatitis B virus and hepatitis C virus interaction in Huh-7 cells.

BACKGROUND/AIMS: Co-infection with hepatitis B virus (HBV) and hepatitis C virus (HCV) increases the risk of development and the severity of chronic liver disease. Although dominant and suppressive effects of each virus over the other have been reported in vivo, in vitro studies of HBV/HCV co-infection have been limited to analysis of the effects of over-expression of HCV proteins on HBV replication. METHODS: We have re-examined HBV/HCV interactions in Huh-7 cells following co-infection with cell culture-propagated HCV (HCVcc; genotype 2a) and a recombinant adenovirus vector capable of delivering a replication-competent HBV genome (AdHBV; genotype A). RESULTS: While intracellular HCV RNA levels were significantly increased when cells were pre-infected with AdHBV, HCV replication and virion secretion were not altered by simultaneous infection with AdHBV or AdHBV superinfection of HCV-infected cells. Likewise intracellular and secreted HBV DNA levels and HBV promoter activities were either unchanged or modestly increased by HCVcc infection. Despite this, HCV E2 and HBsAg proteins colocalized extensively in co-infected cells suggesting shared stages in viral egress. CONCLUSIONS: These studies indicate that there is little direct interaction of HBV and HCV in co-infected hepatocytes and imply that indirect effects of host-viral interactions dictate viral dominance in HBV/HCV co-infected individuals.

A screening method for identifying disruptions in interferon signaling reveals HCV NS3/4a disrupts Stat-1 phosphorylation.

UNLABELLED: Viruses have evolved mechanisms to inhibit the innate immune response to infection. The aim of this study was to develop an efficient screening method to identify viral proteins and their ability to block Jak-Stat signaling using hepatitis C virus (HCV) as an example. The 2FTGH cell assay system was used in combination with transient transfection of HCV proteins in this study. Using 1000U/ml IFN and 30mM 6-TG to treat 2FTGH cells, it was established that transient protein expression in this cell system yielded 39% and 0% cell survival for the positive (HPV E7) and negative controls (GFP expression) respectively. Transient expression of HCV Core-p7 resulted in 22% cell survival, consistent with previous reports, while expression of the HCV serine protease NS3/4a resulted in 54% cell survival. NS3/4a was subsequently shown to inhibit phosphorylation of Stat-1 at the serine residue 727. CONCLUSION: the 2FTGH cell assay system can be adapted for transient screening to examine the ability of viral proteins or other potential inhibitors to block the Jak-Stat signaling pathway. We show that HCV NS3/4a is able to block this pathway at the stage of Stat-1 serine 727 phosphorylation.