Hepatitis C, a life threatening disease, caused by the hepatitis C virus (HCV) currently affects over 170-200 million people worldwide (∼3% of global human population), more than five times the percentage of total HIV infections. HCV infection has been shown to be a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma and is the leading cause of liver transplantation in the U.S. HCV has escaped every therapeutic target to date by means of its error-prone RNA polymerase, which allows it to mutate prolifically. The current standard anti-HCV therapy, which is pegylated interferon α combined with ribavirin, is difficult to tolerate, and more than 50% of HCV patients are refractory to it. No protective vaccine or therapeutic antibody is available, making the need for the development of an efficacious immunoprophylactic and therapeutic agent imperative. HCV is an enveloped virus with a positive sense RNA genome of ∼9.6 kilobases (kb), which carries a large open reading frame (ORF), flanked by 5’- and 3’- untranslated regions (UTRs). Interestingly, within the highly mutational HCV RNA, there are a limited number of 100% conserved and functionally vital motifs, located in the 5’ UTR, coding region and in the 3’ UTR. Within the HCV genome, these motifs have been proposed to be involved in multiple exclusive interactions with each other and furthermore, these interactions have been demonstrated to be essential for HCV replication and/or translation of the viral proteins.
In this study, we used biophysical methods to characterize at the molecular level these proposed HCV RNA-RNA essential interactions involving the highly conserved motifs. Our results indicate that all of these interactions are mediated via kissing complex formation, which have nanomolar dissociation constants. The ability of these conserved motifs to be engaged in multiple interactions suggests the existence of RNA molecular switches, which might regulate the transition between viral replication, translation, packaging and/or other essential processes within HCV life cycle. Given the highly mutational character of HCV genome, analysis of the highly conserved motifs provides valuable information, which aids in identification of new potential therapeutic targets. Subsequently, we designed and analyzed the ability of peptide nucleic acids to function as therapeutic agents by disrupting these essential interactions in HCV and thus, inhibiting the viral replication/translation. Our results show unambiguously that a single PNA molecule designed against a conserved motif located in the 3’UTR effectively disrupts and inhibits all possible interactions involving that motif, thereby opening the possibility of new therapeutic options against HCV replication.
|Commitee:||Armitage, Bruce, Gawalt, Ellen, Seybert, David|
|Department:||Chemistry and Biochemistry|
|School Location:||United States -- Pennsylvania|
|Source:||DAI-B 72/06, Dissertation Abstracts International|
|Subjects:||Genetics, Biochemistry, Virology|
|Keywords:||Genomic dimerization, Hepatitis c virus, Kissing complex, Pna, RNA-RNA interactions, Therapeutic targets|
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