Mechanism Of Antiviral Activity Of Aptamers Directed To Hepatitis C Virus Nonstructure Protein5A

Hepatitis C virus (HCV) infects170million people worldwide and up to80%ofthose infected will become chronic infection. HCV infection can cause chronichepatitis, liver cirrhosis and even hepatocellular carcinoma. There is no vaccineavailable and the current standard treatment for the patients which costs long treatmentperiod can cause resistance to IFN in patients with HCV infection. To overcome IFNresistance in patients with HCV infection, the development of safe, well-tolerated andeffective therapeutic agents against HCV infection that inhibit HCV virus productionhas been undertaken.NS5A is a membrane-associated phosphoprotein that plays an important role inmodulating HCV RNA replication and particle formation including assembly andrelease. So it is great potential to be a target for drug discovery. Aptamer is a new typeof recognition molecules which has a strong affinity, high selectivity, good stability,easy modification and other advantages. Aptamer is widely used in biologicalmonitoring and drug development.In this thesis, we screened the functional ssDNA aptamers which can specificallybound to NS5A protein using SELEX technique and investigated the influences of theirantiviral activity. The full-length NS5A gene of HCV was successfully amplified byPolymerase chain reaction (PCR) using pJFH1as a template and the targeted gene wascloned into the prokaryotic expression vector pET28b. The recombinant plasmidpET28b-NS5A was transferred to BL21competent cells to express the recombinantfusion protein in E.coli.. We purified the histidine-tagged protein with Ni-NTA agarose.After8rounds of seletion, we selected five ssDNA aptamers with high-affinity toNS5A protein (namely NS5A-1, NS5A-2, NS5A-3, NS5A-4and NS5A-5). ThesessDNA aptamers can bind specifically to NS5A protein other than control protein LacZ.Molecular biological and immunologic methods were employed to detect HCV virusRNA replication and virion production in HCV-infected hepatocytes with aptamertreatment. We cloned different NS5A domains to eukaryotic vector to obtain NS5Afractions. Competition assay in cells was employed to determine which domain NS5Aaptamers bind to. Then we explored the antiviral mechanism of aptamer. Our resultsshowed that the DNA aptamer against NS5A protein can inhibit viral replication andrelease, among these aptamers, NS5A-4which combined to NS5A Domain I can obviously inhibit viral RNA replication, and disturb localization between NS5A andlipid drops. NS5A-5has inhibitory effect on virus particles assemble or release, andcan disrupted the interaction of NS5A with core, and the combination of HCV RNAwith viral protein.All the data indicate that aptamers against NS5A protein exert antiviral effectsthrough disrupting the localization of NS5A with lipid droplets and preventing theinteraction of NS5A with core protein. Aptamers for NS5A may be used to understandthe mechanisms of virus replication and assembly and served as potential therapeuticagents for hepatitis C.