MAB8694 Sigma-AldrichAnti-Hepatitis C Virus Antibody, NS5A

This study identified specific and avid RNA aptamers consisting of 2'-hydroxyl- or 2'-fluoropyrimidines against hepatitis C virus (HCV) NS5B replicase, an enzyme that is essential for HCV replication. These aptamers acted as potent decoys to competitively impede replicase-catalyzed RNA synthesis activity. Cytoplasmic expression of the 2'-hydroxyl aptamer efficiently inhibited HCV replicon replication in human liver cells through specific interaction with, and sequestration of, the target protein without either off-target effects or escape mutant generation. A selected 2'-fluoro aptamer could be truncated to a chemically manufacturable length of 29 nucleotides (nt), with increase in the affinity to HCV NS5B. Noticeably, transfection of the truncated aptamer efficiently suppressed HCV replication in cells without escape mutant appearance. The aptamer was further modified through conjugation of a cholesterol or galactose-polyethylene glycol ligand for in vivo availability and liver-specific delivery. The conjugated aptamer efficiently entered cells and inhibited genotype 1b subgenomic and genotype 2a full-length HCV JFH-1 RNA replication without toxicity and innate immunity induction. Importantly, a therapeutically feasible amount of the conjugated aptamer was delivered in vivo to liver tissue in mice. Therefore, cytoplasmic expression of 2'-hydroxyl aptamer or direct administration of chemically synthesized and ligand-conjugated 2'-fluoro aptamer against HCV NS5B could be a potent anti-HCV approach.

Functional sequestration of microRNA 122 (miR-122) by treatment with an oligonucleotide complementary to the miRNA results in long-lasting suppression of hepatitis C virus (HCV) viremia in primates. However, the safety of the constitutive miR-122 silencing approach to HCV inhibition is unclear, since miR-122 can modulate the expression of many host genes. In this study, a regulation system capable of specifically inhibiting miR-122 activity only upon HCV infection was developed. To this end, an allosteric self-cleavable ribozyme capable of releasing antisense sequence to miR-122 only in the presence of HCV nonstructural protein 5B was developed using in vitro selection method. The activity of the reporter construct with miR-122 target sequences at its 3' untranslated region and the expression of endogenous miR-122 target proteins were specifically stimulated through sequestration of miR-122 only in HCV replicon Huh-7 cells, but not in naïve Huh-7 cells, when transfected with expression vector encoding the specific allosteric ribozyme. These findings indicate that miR-122 function can be specifically inhibited by the allosteric ribozyme only in HCV-replicating cells. Importantly, HCV replicon replication was efficiently inhibited by the allosteric ribozyme. This ribozyme could be useful for the specific, safe, and efficacious anti-HCV modulation.

Hepatitis C virus (HCV) infection causes not only intrahepatic diseases but also extrahepatic manifestations, including type 2 diabetes. We previously reported that HCV replication suppresses cellular glucose uptake by downregulation of cell surface expression of glucose transporter 2 (GLUT2) (D. Kasai et al., J. Hepatol. 50:883-894, 2009). GLUT2 mRNA levels were decreased in both HCV RNA replicon cells and HCV J6/JFH1-infected cells. To elucidate molecular mechanisms of HCV-induced suppression of GLUT2 gene expression, we analyzed transcriptional regulation of the GLUT2 promoter using a series of GLUT2 promoter-luciferase reporter plasmids. HCV-induced suppression of GLUT2 promoter activity was abrogated when the hepatocyte nuclear factor 1α (HNF-1α)-binding motif was deleted from the GLUT2 promoter. HNF-1α mRNA levels were significantly reduced in HCV J6/JFH1-infected cells. Furthermore, HCV infection remarkably decreased HNF-1α protein levels. We assessed the effects of proteasome inhibitor or lysosomal protease inhibitors on the HCV-induced reduction of HNF-1α protein levels. Treatment of HCV-infected cells with a lysosomal protease inhibitor, but not with a proteasome inhibitor, restored HNF-1α protein levels, suggesting that HCV infection promotes lysosomal degradation of HNF-1α protein. Overexpression of NS5A protein enhanced lysosomal degradation of HNF-1α protein and suppressed GLUT2 promoter activity. Immunoprecipitation analyses revealed that the region from amino acids 1 to 126 of the NS5A domain I physically interacts with HNF-1α protein. Taken together, our results suggest that HCV infection suppresses GLUT2 gene expression via downregulation of HNF-1α expression at transcriptional and posttranslational levels. HCV-induced downregulation of HNF-1α expression may play a crucial role in glucose metabolic disorders caused by HCV.

The hepatitis C non-structural protein 5A (NS5A) is a Zn(2+)-binding phosphoprotein essential for viral replication. Expression of NS5A perturbs intracellular Ca(2+) levels by an undefined mechanism, activating transcription factors implicated in the chronic pathogenesis of hepatitis infections. Here, we demonstrate that regulated expression of NS5A enhanced the passive leak of Ca(2+) from a subset of the endoplasmic reticulum (ER) Ca(2+) stores. This action was not replicated by expression of the amphipathic NH(2)-membrane anchoring domain of NS5A alone, despite targeting to intracellular membranes. Depletion of the NS5A-targeted ER Ca(2+) store was prevented under conditions of ample ATP supply suggesting compensatory Ca(2+) ATPase activity, but observed under conditions of ATP insufficiency and in intact cells expressing NS5A.