HCV NS5A Protein Research Articles

High amino acid variability within the NS5A of hepatitis C virus (HCV) is associated with hepatocellular carcinoma in patients with HCV-1b–related cirrhosis

Interferon therapy may decrease the risk of hepatocellular carcinoma in patients with hepatitis C virus (HCV)-related liver cirrhosis. Interaction of the cellular protein kinase PKR with the PKR-binding domain (PKR-bd) of HCV-NS5A protein may affect cellular growth control and viral resistance to interferon therapy. Mutations within the PKR-bd, which comprises the interferon sensitivity determining region (ISDR), have been associated with interferon sensitivity. To determine whether or not there is an association between HCV heterogeneity and the presence of hepatocellular carcinoma, HCV-1b genomic regions were amplified and directly sequenced from serum samples obtained from 82 patients with liver cirrhosis, 53 with, and 29 without hepatocellular carcinoma. None of them had received antiviral therapy. When compared with the deduced consensus sequence, the median number of amino acid changes in the PKR-bd was higher among samples from patients with (4.22) than from those without hepatocellular carcinoma (1.62; P < .001), and isolates with 3 or more amino acid changes were significantly more common among the former (60%) than among the later (6%, P < .001). No such differences were observed in other viral regions, including Core, E2-HVR-1, E2-PePHD, NS3, and the 5′ and 3′ PKR-bd flanking regions. In addition, amino acid variation in viral regions other than HVR-1 did not accumulate over time in the analyzed sequential serum samples obtained from patients with or without hepatocellular carcinoma. Therefore, a mutated HCV-PKR-bd phenotype is very common in cirrhotic patients with hepatocellular carcinoma. (HEPATOLOGY 2001;34:158-167.)

Priming of hepatitis C virus-specific cytotoxic T lymphocytes in mice following portal vein injection of a liver-specific plasmid DNA.

The immunology of hepatitis C virus (HCV) infection should be studied in the context of HCV antigen expression in the liver, because HCV primarily infects this organ. Indeed, the nature, function, and fate of T cells primed after antigen expression in the liver might differ from those primed when antigens are expressed systemically or in other organs, because the nature of the antigen-presenting cells (APCs) involved may be different. In addition, the normal liver contains a resident population of lymphocytes that differ from those present at other sites. Thus, we investigated whether HCV-specific CD8(+) cytotoxic T cells (CTLs) could be elicited following portal vein (PV) injection of plasmid DNA in mice whose hepatic veins were transiently occluded. We show that PV injection of mice with "naked" DNA expressing the HCV-NS5a protein, under the control of a liver-specific enhancer/promoter, resulted in NS5a expression in the liver and the priming of HCV-specific CTLs. These results suggested that such a model might be relevant to the study of HCV-specific immune responses primed during natural infection.

Specific interaction between the hepatitis C virus NS5B RNA polymerase and the 3' end of the viral RNA.

Hepatitis C virus (HCV) NS5B protein is the viral RNA-dependent RNA polymerase capable of directing RNA synthesis. In this study, an electrophoretic mobility shift assay demonstrated the interaction between a partially purified recombinant NS5B protein and a 3' viral genomic RNA with or without the conserved 98-nucleotide tail. The NS5B-RNA complexes were specifically competed away by the unlabeled homologous RNA but not by the viral 5' noncoding region and very poorly by the 3' conserved 98-nucleotide tail. A 3' coding region with conserved stem-loop structures rather than the 3' noncoding region of the HCV genome is critical for the specific binding of NS5B. Nevertheless, no direct interaction between the 3' coding region and the HCV NS5A protein was detected. Furthermore, two independent RNA-binding domains (RBDs) of NS5B were identified, RBD1, from amino acid residues 83 to 194, and RBD2, from residues 196 to 298. Interestingly, the conserved motifs of RNA-dependent RNA polymerase for putative RNA binding (220-DxxxxD-225) and template/primer position (282-S/TGxxxTxxxNS/T-292) are present in the RBD2. Nevertheless, the RNA-binding activity of RBD2 was abolished when it was linked to the carboxy-terminal half of the NS5B. These results provide some clues to understanding the initiation of HCV replication.

HCV NS5A protein: subcellular localization by fluorescence and electron microscopy

HCV NS5A protein: subcellular localization by fluorescence and electron microscopy