Wild-type is the optimal sequence of the HDV ribozyme under cotranscriptional conditions

RNA viruses are responsible for a variety of human diseases, and the pathogenicity of RNA viruses is often attributed to a high rate of mutation. Self-cleavage activity of the wild-type hepatitis delta virus (HDV) ribozyme as measured in standard divalent ion renaturation assays is biphasic and mostly slow and can be improved by multiple rational changes to ribozyme sequence or by addition of chemical denaturants. This is unusual in the sense that wild type is the most catalytically active sequence for the majority of protein enzymes, and RNA viruses are highly mutable. To see whether the ribozyme takes advantage of fast-reacting sequence changes in vivo, we performed alignment of 76 genomic and 269 antigenomic HDV isolates. Paradoxically, the sequence for the ribozyme was found to be essentially invariant in nature. We therefore tested whether three ribozyme sequence changes that improve self-cleavage under standard divalent ion renaturation assays also improve self-cleavage during transcription. Remarkably, wild type was as fast, or faster, than these mutants under cotranscriptional conditions. Slowing the rate of transcription or adding the hepatitis delta antigen protein only further stimulated cotranscriptional self-cleavage activity. Thus, the relative activity of HDV ribozyme mutants depends critically on whether the reaction is assayed under in vivo-like conditions. A model is presented for how wild-type ribozyme sequence and flanking sequence work in concert to promote efficient self-cleavage during transcription. Wild type being the optimal ribozyme sequence under in vivo-like conditions parallels the behavior of most protein enzymes.