The Hepatitis delta virus (HDV) is unique: HDV is the only single-stranded circular RNA virus known in animals, and is a serious human pathogen, lethal in 30% of infections. Both the genomic and the antigenomic RNAs of human Hepatitis delta virus are capable of self-processing in vitro, cleaving at a specific site in the absence of protein. To characterize the basis of the self-cleavage reaction, minimal self-cleaving genomic and antigenomic molecules will be derived in vitro. These molecules will be modified for use in a trans-cleavage reaction in vitro, first against HDV sequences, and then against HIV sequences, such as a site located in the TAR RNA. Finally, mutagenesis of individual nucleotides will be carried out to define the essential components of the self-cleavage reaction and to broaden the range of HIV target RNAs. Since the ribozymes differ from all those previously described, elucidation of their structures and catalytic sites will broaden our knowledge of such RNA enzymes and will provide a new range of RNA enzymes for therapeutic applications. The purposes of the proposed research are: first, to define minimal self-cleaving genomic and antigenomic Delta ribozymes; second, to delineate enzyme-substrate relationships for the Delta ribozymes and to expand these to targets in HIV1, notably the TAR RNA; and third, to detail the essential components of the self-cleavage reaction of these unique RNA enzymes.
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