983. Inhibition of Hepatitis C Virus Replication by In Vitro Selected Specific RNA Aptamers

Abstract

Top of pageAbstract Hepatitis C virus (HCV) is the main etiological agent of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Although the HCV infection causes worldwide health problem, efficient and specific antiviral therapy has not yet been developed. Thus, development of more specific and effective therapeutics against HCV is warranted. The RNA dependent RNA polymerase (NS5B) of HCV plays a central role in the viral replication. Therefore, NS5B is a good target for anti-HCV agents. Characteristics of RNAs which can adopt complex structures to bind target proteins with high affinities and encode amplificable genetic information make RNA a potentially very useful diagnostic and/or therapeutic lead compounds. Such short RNA ligands, termed RNA aptamers, have been identified from a random RNA library to bind several proteins with high affinity and specificity using in vitro selection techniques, called systemic evolution of ligands by exponential enrichment (SELEX). In the present study, we isolated RNA aptamers to NS5B from a combinatorial RNA library using SELEX techniques and analyzed the biochemical and biological functions of the RNA aptamers. The RNA aptamers were found to specifically and avidly bind NS5B with an apparent Kd of 1.3|[sim]|4 nM. Through enzymatic probing and footprinting analysis, critical regions of the RNAs for NS5B binding have been identified. Moreover, in vitro RNA synthesis by NS5B was significantly inhibited by the RNA aptamers. Interestingly, the RNA aptamers can be utilized as substrates for de-novo RNA synthesis by NS5B. Specific interaction between the RNAs and NS5B was confirmed by Maldi-Tof analysis of the aptamer-bound proteins in liver cells with HCV replicon. Importantly, the replication of HCV replicon in liver cells was efficiently inhibited by cytoplasmic expression of the RNA aptamers, but not by nuclear expression of the RNAs. In addition, we showed that these aptamers could function as decoys in HCV replicon cells, explaining the mechanism underlying inhibition phenomena of HCV replicon replication observed in cells by the specific RNA aptamers against HCV NS5B. These results suggest that the RNA aptamers could be useful not only as therapeutic and diagnostic agents of HCV infection but also as a powerful tool for the study of HCV replication mechanism.