The objective of the proposed research is to understand the structure, mechanism, and biological role of self-processing RNA (ribozymes). The ribozymes being studied are self-cleaving sequence associated with the genomic and antigenomic RNA of hepatitis delta virus (HDV). HDV is a human pathogen but is always found in association with hepatitis B virus. The combination leads to serious liver damage with a higher fatality rate that in patients infected only with hepatitis B virus. Thus, in populations where hepatitis B is endemic, HDV can be a serious health threat. It can either be co-transmitted with hepatitis B causing the acute form of the disease or it can infect hepatitis B carriers; the later tends to be more serious. The ribozyme activity is thought to be essential to the replication of the RNA genome of this virus. A greater understanding of the ribozyme structure, cleavage mechanism, and role in replication could lead to a method to block viral replication. In addition because the self-cleaving RNA from HDV is the first clear example of a self-cleaving RNA that functions naturally in human cells; an understanding those features that are unique to this RNA may facilitate the design of ribozymes to be used in therapeutics.
The specific aims of the proposal are: (1) To generate and refine a three dimensional model for the HDV ribozyme and utilize the models to evaluate biochemical and genetic data on the ribozyme structure and mechanism. Molecular modeling programs, some of which are designed Specifically for RNA, will be used to generate models that are consistent with constraints on the structure established experimentally. Data gathered from physical methods will also be used, and towards this end methods for making large quantities of circular RNA will be used to produce RNA for crystallography. (2) To test and refine the structure through biochemical and genetic approaches. Methods include chemical protection, substitution interference, in vitro mutagenesis and in vitro selection. (3) To define the mechanism and requirements of the reaction. A complete kinetic scheme for the trans-reaction will developed and the stereochemistry of the reaction will defined. (4) To characterize and define cis-acting sequences that interfere with cleavage activity and may limit ribozyme cleavage to the nascent transcript. As part of this study templates for making concatameric RNA sequences that mimic replication of the RNA from a circular template will be developed.
