The role of a metastable RNA secondary structure in hepatitis delta virus genotype III RNA editing

Abstract

RNA editing plays a critical role in the life cycle of hepatitis delta virus (HDV). The host editing enzyme ADAR1 recognizes specific RNA secondary structure features around the amber/W site in the HDV antigenome and deaminates the amber/W adenosine. A previous report suggested that a branched secondary structure is necessary for editing in HDV genotype III. This branched structure, which is distinct from the characteristic unbranched rod structure required for HDV replication, was only partially characterized, and knowledge concerning its formation and stability was limited. Here, we examine the secondary structures, conformational dynamics, and amber/W site editing of HDV genotype III RNA using a miniaturized HDV genotype III RNA in vitro. Computational analysis of this RNA using the MPGAfold algorithm indicated that the RNA has a tendency to form both metastable and stable unbranched secondary structures. Moreover, native polyacrylamide gel electrophoresis demonstrated that this RNA forms both branched and unbranched rod structures when transcribed in vitro. As predicted, the branched structure is a metastable structure that converts readily to the unbranched rod structure. Only branched RNA was edited at the amber/W site by ADAR1 in vitro. The structural heterogeneity of HDV genotype III RNA is significant because not only are both conformations of the RNA functionally important for viral replication, but the ratio of the two forms could modulate editing by determining the amount of substrate RNA available for modification.

Identification of Cis-Acting Elements in the 3′-Untranslated Region of the Dengue Virus Type 2 RNA That Modulate Translation and Replication

“…These metastable intermediates as well as stable and best fit structures predicted by MPGAfold have been shown to have biological relevance in a number of previous studies (61)(62)(63)(64)(65)72). Another advantage of MPGAfold is that besides computing the free energy of the RNA, it also provides us with information on the relative frequencies of occurrence of local secondary structural motifs.…”

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

“…These isolates differ at 17-nucleotide (nt) positions in the z300-nt region required for editing. Previously, we used a 320-nt miniaturized version of the Ecuadorian isolate RNA, referred to here as mE, that can form both the branched structure required for editing at the amber/W site and the unbranched rod structure characteristic of HDV RNA (Linnstaedt et al 2006). Stem-flip (SF) mutations were introduced in the stems of stem-loops SL1 and SL2 (Fig.…”

“…Here, we compare RNA folding dynamics (in silico and in vitro) and editing (in vitro and in vivo) in two HDV-3 isolates, one from Peru (Casey et al 1993;Casey 2002), the other from Ecuador (Manock et al 2000;Linnstaedt et al 2006). We find that HDV-3 controls RNA editing levels via (1) the fraction of the RNA that folds, following transcription, into the metastable branched structure required for editing and (2) the efficiency with which ADAR1 edits this branched substrate RNA.…”

“…However, we previously observed that HDAg-S is not an effective inhibitor of amber/W site editing for HDV-3, which differs from HDV-1 by z40% in nucleic acid sequence (Cheng et al 2003), thus indicating that an alternative mechanism must exist for the regulation of editing for this genotype. Unlike HDV-1, amber/W site editing in HDV-3 requires a branched secondary structure comprised of a central base-paired region containing the amber/W site flanked by two z25-base-pair stem-loops, designated SL1 and SL2 (Casey 2002;Linnstaedt et al 2006). This structure is energetically less stable than the unbranched rod structure that is characteristic of HDV RNA and is required for RNA replication (Linnstaedt et al 2006).…”

“…Unlike HDV-1, amber/W site editing in HDV-3 requires a branched secondary structure comprised of a central base-paired region containing the amber/W site flanked by two z25-base-pair stem-loops, designated SL1 and SL2 (Casey 2002;Linnstaedt et al 2006). This structure is energetically less stable than the unbranched rod structure that is characteristic of HDV RNA and is required for RNA replication (Linnstaedt et al 2006). Because only the metastable branched structure can be edited, the distribution of the antigenomic RNA between the two structures following transcription could be an important determinant of the amount of editing that occurs during viral RNA replication.…”