Hepatitis delta virus (HDV) is a human pathogen found co-infecting hepatitis B virus (HBV) patients and linked to worsened symptoms of infection. HDV has been shown to employ an RNA enzyme, or ribozyme, in its replication cycle. The ribozyme cleaves the multimeric genome from rolling circle replication into unit length RNAs by self- cleavage of a specific phosphodiester bond. The proposed mechanism for this cleavage involves general acid-base catalysis with Cytosine 75 (C75) acting as the general acid and a hydrated metal hydroxide acting as the general base. The crystal structure of the HDV ribozyme supports the general-acid role of C75. However, the inferred metal is absent, presumably because the structure is of the cleaved foam and lacks a putative scissile phosphate-hydrated magnesium ion ligand.
The aim of the proposed work is to test the mechanism by identifying the hydrated metal-hydroxide-binding site and measuring the pKa of C75's imino nitrogen. The main technique is nuclear magnetic resonance (NMR) spectroscopy. Cobalt hexammine will be used as a competitive inhibitor of the active-site magnesium. Its location will be determined through chemical shift perturbations induced on nearby imino protons. 13C and 15N isotopic labeling of the short piece of a two-piece construct will be used to simplify assignment of C75. This system will also be used to investigate the pKa of C75 by 13C NMR pH titration. As interferon treatment of chronic HBV HDV carriers has not been particularly successful, the understanding of the HDV ribozyme coming from the proposed work will provide useful information for anti-hepatitis drug design.
