The fraction of RNA that folds into the correct branched secondary structure determines hepatitis delta virus type 3 RNA editing levels

Abstract

RNA editing by the host RNA adenosine deaminase ADAR1 at the amber/W site of hepatitis delta virus RNA plays a central role in the viral replication cycle by affecting the balance between viral RNA synthesis and packaging. Previously, we found that HDV genotype III (HDV-3) RNA can form two secondary structures following transcription: an unbranched rod structure, which is characteristic of HDV, and a metastable branched structure that serves as the substrate for editing. The unstable nature of the branched editing substrate structure raised the possibility that structural dynamics of the RNA following transcription could determine the rate at which editing occurs. Here, editing and its control are examined in two HDV-3 isolates, from Peru and Ecuador. Analysis of editing in vitro by ADAR1 indicated that the branched structure formed by RNA derived from the Peruvian isolate is edited more efficiently than that from the Ecuadorian isolate. In contrast, in the context of replication, Peruvian RNA is edited less efficiently than RNA containing Ecuadorian sequences. Computational analyses of RNA folding using the massively parallel genetic algorithm (MPGAfold) indicated that the Peruvian RNA is less likely to form the branched structure required for editing than the Ecuadorian isolate. This difference was confirmed by in vitro transcription of these RNAs. Overall, our data indicate that HDV-3 controls RNA editing levels via (1) the fraction of the RNA that folds, during transcription, into the metastable branched structure required for editing and (2) the efficiency with which ADAR1 edits this branched substrate RNA.

RNA Editing and its Control in Hepatitis Delta Virus Replication

“…Computational analysis of the secondary structures formed by HDV-3E and HDV-3P [36] suggested that this apparent paradox might be explained by differences in the RNA structural dynamics of these two RNAs - HDV-3E RNA could form the branched structure required for editing more readily than HDV-3P RNA. Consistent with the predictions, following transcription in vitro HDV-3E and HDV-3P RNAs formed both the branched structure required for editing and the unbranched rod (which is not edited), but HDV-3E RNA formed the branched editing structure 3–4 fold more efficiently than did HDV-3P [13]. Based on these results, our model for down-modulation of editing of the HDV type 3 editing site is that substrate availability is limited by the fraction of the RNA that folds into the branched structure following transcription (Figure 7).…”

“…No additional factors aside from HDV RNA and ADAR1 are required [9,13]. ADAR1 contains a catalytic deaminase domain along with three dsRNA binding motifs (DRBMs) [14,15].…”

Enhancement of Replication of RNA Viruses by ADAR1 via RNA Editing and Inhibition of RNA-Activated Protein Kinase

“…85, 2011 MINIREVIEW 8461 change produced by this editing event, the editing site is called the amber/W site. HDV-editing activity is accomplished by ADAR1 p110 in the nuclei of HuH-7 hepatoma cells, and the secondary structure of HDV RNA determines the level of editing (10,44,63,88). Experiments using knockdown or overexpression of ADAR1 were conducted to determine to what extent the editing activity is essential for viral RNA replication.…”

When you can’t trust the DNA: RNA editing changes transcript sequences

“…Strikingly, some of these editing sites are conserved across very wide phylogenetic distances, e.g., ranging from insect to squid in the case of a potassium channel [203]. Among the notable other targets of the ADAR-based A-to-I editing machinery are the transcripts of the primate-specific Alu repeats [204] and genes of the immune system [205], as well as viral RNAs, e.g., of HDV or HIV [206,207]. Even micro RNAs (miRNAs) may be edited, hence suggesting a ''crosstalk between editors and silencers'' for gene regulation via the RNA interference machinery [208].…”