The long term objective of the proposed research is to understand the mechanism of RNA auto-catalytic processing (self-cleavage) associated with the genomic and antigenomic RNA of hepatitis delta virus (HDV). HDV is the agent associated with a serious form of hepatitis and requires concurrent infection by the hepatitis B virus (either chronic or acute). In parts of the world (and populations in the US) where hepatitis B is endemic, HDV can represent a serious health problem. The RNA self-- cleavage is hypothesized to be essential to the replication of the RNA genome of this virus, and therefore, understanding the mechanism of the process may provide insight into a mechanism by which to block viral replication. In addition, the self-cleaving RNA from HDV is the first clear example of an autocatalytic RNA (ribozyme) that has evolved to function in human cells. Therefore, understanding those features that are unique to this RNA will provide an understanding of possible RNA auto-processing in mammals. A trans-acting form of the HDV ribozyme has basic features different from other trans-acting ribozymes and could be developed as a useful tool for cleaving certain RNA sequences.
Specific aims are: (a) to define and test a model for the secondary structure of the self-cleaving sequence by probing the structure in solution with enzymes and chemicals, and to identify regions of tertiary interactions using crosslinking methods; (b) to test the role of specific structures and sequences using site-specific mutagenesis; (c) to characterize the mechanism of the reaction by measuring the rates of individual steps in the cleavage reaction; and (d) to physically characterize the stability of the selfcleaving structure by examining melting profiles of the wild-type and variant sequences in the presence of denaturants and cations which affect activity via the structure. As part of these aims we will initiate experiments directed at preparing sufficient quantities of pure RNA to facilitate the determination of a three dimensional physical model. Many of the proposed experiments will be done with the self-cleaving form of the RNA, however, intermolecular (trans) forms of the reaction have been developed and will facilitate these studies.
