Hepatitis C virus NS5B RNA replicase specifically binds ribosomes

Hepatitis C virus (HCV) non-structural protein 5B (NS5B) is an RNA replicase. We expressed full-length NS5B (591 amino acid residues) in Escherichia coli as a fusion protein with maltose binding protein (MBP-NS5B). MBP-NS5B was recovered in the soluble fraction after centrifugation at 40,000 x g and affinity-purified with amylose resin. The purified MBP-NS5B had a high-level of poly (A), oligo (U)-dependent UMP incorporation with a Km of 2 microM for UTP. Surprisingly, the enzymatically active MBP-NS5B was sedimented by ultracentrifugation at 160,000 x g. The pellet contained 16S and 23S ribosomal RNAs, suggesting that ribosomes were associated with MBP-NS5B. Ribosomes and MBP-NS5B were subsequently co-purified on amylose resin. Deletion study revealed that either the N-terminal (amino acid residues 1-107) or the C-terminal (amino acid residues 498-591) region of NS5B were sufficient for this association with ribosomes. We further found that NS5B also bound with human ribosomes. Our results implicate a novel mechanism of coupling between replication and translation of the viral genome in the life cycle of HCV.     

The expression of phosphatidic acid phosphatase 2a, which hydrolyzes lipids to generate diacylglycerol, is regulated by p73, a member of the p53 family

p73, a p53-related gene, is essential for a development of animals, while p53 is important for tumor formation. And little is known about the target genes specifically regulated by p73. Identifying the specific targets of p73 is important to understand the physiological roles of p73. To identify the genes specifically regulated by p73, we conducted serial analysis of gene expression to quantitatively evaluate messenger RNA populations. We found that the gene for phosphatidic acid phosphatase 2a (PAP2a), an enzyme that hydrolyzes lipids to generate diacylglycerol, was specifically upregulated by ectopic production of p73beta. The promoter region of this gene contains an element that is functionally responsive to p73beta. And the quantity of PAP2a protein was upregulated by ectopic production of p73beta. These results suggest that the expression of PAP2a is directly regulated by p73.     

In vitro models for analysis of the hepatitis C virus life cycle

Chronic hepatitis C virus (HCV) infection affects approximately 170 million people worldwide. HCV infection is a major global health problem as it can be complicated with liver cirrhosis and hepatocellular carcinoma. So far, there is no vaccine available and the non-specific, interferon (IFN)-based treatments now in use have significant side-effects and are frequently ineffective, as only approximately 50% of treated patients with genotypes 1 and 4 demonstrate HCV clearance. The lack of suitable in vitro and in vivo models for the analysis of HCV infection has hampered elucidation of the HCV life cycle and the development of both protective and therapeutic strategies against HCV infection. The present review focuses on the progress made towards the establishment of such models.