Mimicry of the hepatitis delta virus replication cycle mediated by synthetic circular oligodeoxynucleotides

Abstract

Hepatitis delta virus (HDV) is a circular single-stranded RNA pathogen whose monomeric form results from self-processing. Although studies have examined minimal HDV ribozyme activities, the mechanism for forming the circular virus remains unclear, and the trans catalytic properties of self-processed forms of HDV ribozymes have not been studied. In addition, HDV ribozymes have not previously been engineered to cleave a non-HDV sequence. Long repeating RNAs have been produced from in vitro rolling-circle transcription of synthetic circular oligodeoxynucleotides encoding catalytically active subsets of the entire antigenomic RNA virus. Like full-length HDV, these multimeric RNAs undergo self-processing to monomer length; importantly, cyclization is found to occur efficiently, but only in the presence of the circular template. Linear and circular monomer ribozymes and engineered variants are shown to be active in cleaving HDV and HIV RNA targets in trans, despite having self-binding domains. Mimicry of the rolling-circle replication pathway for HDV replication has led to a new proposal for cyclization of HDV RNA. Under these conditions, cyclization is mediated by the complementary circular template. In addition, it has been shown that self-processed HDV ribozymes can be catalytically active in trans despite the presence of antisense sequences built into their structure.