Kotaro Fukuda

A hepatitis C virus (HCV) internal ribosome entry site (IRES) domain III–IV-targeted aptamer inhibits translation by binding to an apical loop of domain IIId

The hepatitis C virus (HCV) has a positive single-stranded RNA genome, and translation starts within the internal ribosome entry site (IRES) in a cap-independent manner. The IRES is well conserved among HCV subtypes and has a unique structure consisting of four domains. We used an in vitro selection procedure to isolate RNA aptamers capable of binding to the IRES domains III–IV. The aptamers that were obtained shared the consensus sequence ACCCA, which is complementary to the apical loop of domain IIId that is known to be a critical region of IRES-dependent translation. This convergence suggests that domain IIId is preferentially selected in an RNA–RNA interaction. Mutation analysis showed that the aptamer binding was sequence and structure dependent. One of the aptamers inhibited translation both in vitro and in vivo. Our results indicate that domain IIId is a suitable target site for HCV blockage and that rationally designed RNA aptamers have great potential as anti-HCV drugs.

Increased inhibitory ability of conjugated RNA aptamers against the HCV IRES

Hepatitis C virus (HCV) translation begins within the internal ribosome entry site (IRES). We have previously isolated two RNA aptamers, 2-02 and 3-07, which specifically bind to domain II and domain III-IV of the HCV IRES, respectively, and inhibit IRES-dependent translation. To improve the function of these aptamers, we constructed two conjugated molecules of 2-02 and 3-07. These bound to the target RNA more efficiently than the two parental aptamers. Furthermore, they inhibited IRES-dependent translation about 10 times as efficiently as the 3-07 aptamer. This result indicates that combining aptamers for different target recognition sites potentiates the inhibition activity by enhancing the domain-binding efficiency.

Modulation of Double-stranded RNA Recognition by the N-terminal Histidine-rich Region of the Human Toll-like Receptor 3

Toll-like receptors (TLRs) are an essential component of the innate immune response to microbial pathogens. TLR3 is localized in intracellular compartments, such as endosomes, and initiates signals in response to virus-derived double-stranded RNA (dsRNA). The TLR3 ectodomain (ECD), which is implicated in dsRNA recognition, is a horseshoe-shaped solenoid composed of 23 leucine-rich repeats (LRRs). Recent mutagenesis studies on the TLR3 ECD revealed that TLR3 activation depends on a single binding site on the nonglycosylated surface in the C-terminal region, comprising H539 and several asparagines within LRR17 to -20. TLR3 localization within endosomes is required for ligand recognition, suggesting that acidic pH is the driving force for TLR3 ligand binding. To elucidate the pH-dependent binding mechanism of TLR3 at the structural level, we focused on three highly conserved histidine residues clustered at the N-terminal region of the TLR3 ECD: His39 in the N-cap region, His60 in LRR1, and His108 in LRR3. Mutagenesis of these residues showed that His39, His60, and His108 were essential for ligand-dependent TLR3 activation in a cell-based assay. Furthermore, dsRNA binding to recombinant TLR3 ECD depended strongly on pH and dsRNA length and was reduced by mutation of His39, His60, and His108, demonstrating that TLR3 signaling is initiated from the endosome through a pH-dependent binding mechanism, and that a second dsRNA binding site exists in the N-terminal region of the TLR3 ECD characteristic solenoid. We propose a novel model for the formation of TLR3 ECD dimers complexed with dsRNA, which incorporates this second binding site.

Inhibition of hepatitis C virus serine protease in living cells by RNA aptamers detected using fluorescent protein substrates.

Hepatitis C virus is one of the causative agents of non-A non-B hepatitis. Since one of viral proteins, NS3, has serine protease activity indispensable for virus maturation. NS3 serine protease is considered to be a suitable target for anti-HCV reagents. We report an assay of HCV NS3 protease in living cells. We designed peptide substrates bearing one of the sequences of HCV NS3 protease cleavage sites sandwiched with fluorescent proteins CFP and YFP. Substrates were expressed and cleaved efficiently in HeLa cells by cotransfection with HCV NS3 protease. The relationship between the progress of cleavage reaction and the change in fluorescence of the substrate emitted from living cells was confirmed. As a group of candidates for inhibitor of HCV NS3 protease, we chose RNA aptamers, nucleic acid ligands selected from a completely random RNA pool by in vitro selection. We found that 3 classes of aptamers, G9-I, II and III, bound NS3 protease specifically and inhibited cleavage in vitro. We studied the effect of RNA aptamers introduced into HeLa cells. The addition of G9-II RNA in the medium at a concentration of 2.5 micro g/ml reduced cleavage by one-third that of control.

AN RNA APTAMER CONTAINING TWO BINDING SITES AGAINST THE HCV MINUS-IRES DOMAIN I

The higher order structure of HCV (−)IRES containing five stem-loop structures (domain I) is essential for HCV replication because the viral RNA-dependent RNA polymerase, NS5B, recognizes it as the initiation site for plus-strand synthesis. To inhibit a de novo synthesis of plus-strand RNA molecules, in vitro selection against (−)IRES domain I was performed. One of the obtained aptamers, AP30, contained two consensus sequences within a random sequence region. Two consensus sequences form two apical loops and mutational analysis showed that both sequences were essential for binding to the target and for inhibiting NS5B-mediated RNA synthesis in vitro.