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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Predoctoral Individual National Research Service Award (F31)
Project #
3F31GM064015-03S1
Application #
6864037
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Toliver, Adolphus
Project Start
2002-08-04
Project End
Budget Start
2003-08-04
Budget End
2005-08-03
Support Year
3
Fiscal Year
2004
Total Cost
$26,793
Indirect Cost
Name
Pennsylvania State University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
003403953
City
University Park
State
PA
Country
United States
Zip Code
16802