Editing of hepatitis delta virus antigenomic RNA by recombinant human adenosine deaminases that act on RNA

The adenosine deaminases that act on RNA (ADARs) are a family of RNA editing enzymes that catalyze the hydrolytic deamination of the C6 position of specific adenosines within the context of RNA. The deamination reaction converts targeted adenosines to inosines. Inosine, like guanosine, prefers to form basepairs with cytidine. Consequently, when ADARs deaminated certain adenosines in the coding regions of cellular pre-messenger RNAs and Viral RNAs, the edited RNAs direct the translation of alternative forms of the encoded protein, often with significant biological consequences. In order to study the action of human ADARs (hADARs) on cognate substrates in vitro, and to compare the enzymatic activity of different hADARs on defined substrates, a source of recombinant hADARs was needed. The first experimental section of this dissertation describes a Saccharomyces cereviasiase-based protein expression system and associated purification protocol that readily yields biochemical quantities of pure, active hADAR1 and hADAR2. The system and protocol represent a significant break-through in the field of ADAR enzymology that has already allowed others to investigate how ADARs catalyze their deamination reaction and target specific adenosines for modification. ADAR-catalyzed editing of hepatitis delta virus (HDV) antigenomic RNA allows two forms of HDV antigen to be made from a single open reading frame. Edition converts an amber stop codon (UAG) to a tryptophan (W) codon (UIG) at the so-called amber/W site. Edited RNAs direct the translation of antigen that is 19 amino acids longer than that encoded by unedited RNAs. The shorter from of the antigen is required for viral replication whereas the longer form inhibits replication and is required for packaging. Hence, the life cycle of HDV is dependent upon editing by hASARs. The second experimental section of this dissertation describes the results of side-by-side in vitro amber/W site editing reactions catalyzed by recombinant hADAR1 and hADAR2. These studies revealed significant differences in the abilities of the two hADARs to edit substrates bearing mutations proximal to the amber/W site, and differences in the selectivity of editing by the two enzymes. These data, along with others published results, implicate hADAR1 as being responsible for amber/W site editing in vivo.