Isolation and characterization of RNA aptamers specific for the hepatitis C virus nonstructural protein 3 protease

Abstract

Nonstructural protein 3 (NS3) from hepatitis C virus (HCV) is a serine protease that provides an essential function in maturation of the virus by cleaving the nonstructural regions of the viral polyprotein. The goal of this work was to isolate RNA aptamers that bind specifically to the NS3 protease active site in the truncated polypeptide DNS3. RNA aptamers were selected in vitro by systematic evolution of ligands by exponential enrichment (SELEX). The RNA pool for SELEX had a 30-nucleotide randomized core region. After nine selection cycles, a pool of DNS3-specific RNA aptamers were obtained. This RNA pool included 45 clones that divided into three main classes (G9-I, II and III). These classes include the conserved sequence GA(A/U)UGGGAC. These aptamers bind to DNS3 with a binding constant of about 10 nm and inhibit approximately 90% of the protease activity of DNS3 and MBP-NS3 (full-length of NS3 fused with maltose binding protein). In addition, these aptamers inhibited approximately 70% of the MBP-NS3 protease activity in the presence of the NS4A peptide P41. G9-I aptamer appeared to be a noncompetitive inhibitor for DNS3 with a K i < 100 nm in the presence of P41. These results suggest that the pool of selected aptamers have potential as anti-HCV compounds. Mutational analysis of the G9-I aptamer demonstrated that the sequences required for protease inhibition are in stem I, stem III and loop III of the aptamer. These regions include the conserved sequence GA(A/U)UGGGAC.Keywords: HCV; RNA aptamer; SELEX; NS3 serine protease.Hepatitis C virus (HCV) is the major etiological agent of posttransfusion non-A, non-B hepatitis (reviewed in [1]). Chronic HCV infection is a serious disease throughout the world and can also develop into chronic hepatitis, liver cirrhosis or hepatocellular carcinoma [2]. Although the number of HCV carriers has increased to about 300 million worldwide, the principle drugs against HCV are all based on interferon and effective antiviral drugs have not yet been developed.HCV has a single, positive-stranded RNA genome approximately 9.5 kb in length. The genetic organization of HCV is similar to that of flaviviruses and pestiviruses and HCV has been classified as a new genus of the family Flaviviridae. HCV proteins include the core (C), envelope (E), and nonstructural (NS) groups of proteins. The proteins are synthesized as a polyprotein of approximately 3000 amino acids in the order NH 2 ±C±E1±E2±p7±NS2±NS3±NS4A±NS4B±NS5A±NS5B± COOH [3±5]. Mature viral structural and nonstructural proteins are produced from the polyprotein by a series of cotranslational and post-translational cleavages mediated by host signal peptidases [6±8] and two viral proteases, NS2-3 [9,10] and NS3 [11±14]. The NS2-3 protease activity is zinc-dependent and it cleaves at the NS2/NS3 junction. The protease activity of NS3 lies in the N-terminal third of the polypeptide. The NS3 protease is a trypsin-like serine protease which is essential for processing many of the nonstructural proteins of HCV, including NS3, NS4A, NS4...

Aptamers That Preferentially Bind Type IVB Pili and Inhibit Human Monocytic-Cell Invasion by Salmonella enterica Serovar Typhi

“…In order to increase the specificity of selected RNA aptamers, we have utilized a combinatorial library approach by pretreating RNA aptamers with GST-Sepharose 4B before each round of SELEX selection, from the third round to the eighth and final round. Although this combinatorial library approach has been successfully used to isolate aptamers blocking ligand-receptor interactions between eukaryotic cells and viruses (2,5,8) or between eukaryotic cells themselves (17,22,29), the technique has not been applied to the selection of ligands blocking an interaction between bacterial and eukaryotic cells. Our studies indicate that the SELEX approach is a useful tool to study the roles of bacterial proteins in pathogenesis and invasion of host cells, as well as to provide insights into the mechanisms of pathogen-host cell interactions.…”

Toll-like receptor 3 recognizes incomplete stem structures in single-stranded viral RNA

“…Radioisotope labelling of PV-RNAs by in vitro transcription with [a-32 P-ATP] was carried out as previously decribed 49 . Labelled PV-RNAs (2 nM) were mixed with varying concentrations (none or 6.25, 12.5 25, 50, 100 or 200 nM) of human TLR3 (amino acid 27-711; R&D Systems) and adjusted to a total volume of 25 ml using binding buffer (pH 5.0-7.0) containing 100 mM NaCl (20 mM AcONa (pH 5.0-6.0) or 20 mM Tris-HCl (pH 6.5-7.0)).…”

Understanding Helicases as a Means of Virus Control

“…Nishikawa's group first used SELEX to uncover a series of aptamers with specific affinity to the NS3 protease region. The protease-binding aptamers all share the conserved sequence GA(A/U)UGGGAC [191]. These aptamers bind to the NS3 protease with high affinity (K d 10 nM) and are effective at inhibiting the HCV NS3 protease [191,192].…”

“…The protease-binding aptamers all share the conserved sequence GA(A/U)UGGGAC [191]. These aptamers bind to the NS3 protease with high affinity (K d 10 nM) and are effective at inhibiting the HCV NS3 protease [191,192]. The conserved part of the RNA aptamers likely interacts with a cluster of basic amino acid (residues Arg130, Arg161 and Lys165), which is located outside of the serine protease catalytic pocket in the region that links the protease to the helicase [193].…”