Selective Strand Annealing and Selective Strand Exchange Promoted by the N-terminal Domain of Hepatitis Delta Antigen

Abstract

We have previously shown that the N-terminal domain of hepatitis delta virus (NdAg) has an RNA chaperone activity in vitro (Huang, Z. S., and Wu, H. N. (1998) J. Biol. Chem. 273, 26455-26461). Here we investigate further the basis of the stimulatory effect of NdAg on RNA structural rearrangement: mainly the formation and breakage of base pairs. Duplex dissociation, strand annealing, and exchange of complementary RNA oligonucleotides; the hybridization of yeast U4 and U6 small nuclear RNAs and of hammerhead ribozymes and cognate substrates; and the cis-cleavage reaction of hepatitis delta ribozymes were used to determine directly the role of NdAg in RNA-mediated processes. The results showed that NdAg could accelerate the annealing of complementary sequences in a selective fashion and promote strand exchange for the formation of a more extended duplex. These activities would prohibit NdAg from modifying the structure of a stable RNA, but allow NdAg to facilitate a trans-acting hammerhead ribozyme to find a more extensively matched target in cognate substrate. These and other results suggest that hepatitis delta antigen may have a biological role as an RNA chaperone, modulating the folding of viral RNA for replication and transcription. Hepatitis delta virus (HDV)1 is a satellite virus of hepatitis B virus. The genome of HDV comprises single-stranded circular RNA of ϳ1700 nucleotides, and HDV RNA replicates through a symmetrical rolling circle mechanism (1). Hepatitis delta antigen is the only protein coded by HDV that is critical for viral replication (2) and virion assembly (3), although the molecular mechanisms have not yet been elucidated. HDV RNA, of both genomic and antigenomic senses, contains a ribozyme domain that can adopt a pseudoknot-like structure and undergo ciscleavage in vitro (4 -7). The cis-cleaving activity is essential for generating monomeric size RNA molecules during viral replication (8, 9). Hepatitis delta antigen may enhance, although it is not required for, the processing of multimeric size HDV RNA in vivo (10). Conceivably, hepatitis delta antigen by itself, or together with unidentified cellular factors, acts as an RNA chaperone that modulates the ribozyme activity of HDV RNA.RNA chaperones are defined as proteins that aid in the folding of RNA by preventing misfolding or by resolving misfolded structures (11). The RNA chaperone activities in vitro of several proteins that interact with RNA with broad specificity have been explored through their effect on ribozyme reactions. These proteins, including several Escherichia coli ribosomal proteins (12), the C-terminal domain of heterogeneous nuclear ribonucleoprotein A1 protein (13), and the nucleocapsid protein of human immunodeficiency virus (14, 15), can overcome the general limitations of ribozyme reactions and facilitate ribozyme catalysis. Proteins with RNA chaperone activity are thought to lower the activation energy necessary for breaking and reforming of base pairs, although the molecular mechanism underlying RNA chaperone activity...

HCV NS3 protein helicase domain assists RNA structure conversion

“…To test whether NS3H facilitates dsRNA formation via an action mimicking an RNA chaperon, we replaced NS3H with NdAg, an RNA chaperone derived from the N-terminal domain of hepatitis delta antigen [12], for strand annealing assay. NdAg facilitated the annealing of C2 and C 0 3 RNAs, and the activity was ATP-independent.…”

“…The assay was performed with C2 and C 0 3 RNAs (0.5 nM each), C2 RNA alone (0.5 nM), or C 0 3 RNA alone (0.5 nM), the indicated concentration of NS3H (upper panels) or mNS3H (lower panels), 2.5 mM ATP, and 1.5 mM MnCl 2 at 37°C for 1 h. The reaction mixtures were treated with proteinase K before resolving by gel electrophoresis for detecting dsRNA formation (lanes 1-5). Alternatively, the reaction mixtures were directly resolved by gel electrophoresis for detecting RNA binding (lanes [6][7][8][9][10][11][12][13][14][15][16][17][18][19][20]. Notably, a large fraction of the RNA-protein complex did not migrate into the gel well at 100 nM and 400 nM NS3H or mNS3H.…”

Characterization and application of the selective strand annealing activity of the N terminal domain of hepatitis delta antigen

“…NdAg can accelerate the annealing of complementary sequences selectively and promotes strand exchange selectively for the formation of a more extended duplex among competing sequences [23]. Moreover, NdAg acts on HDV RNA, complicated and simple non-HDV RNAs, and even DNA oligos [23,24]. Therefore, NdAg is a nucleic acid chaperone.…”

“…The RNA chaperone domain of HDAg resides in its N terminal 88 amino acids [22], referred to here as NdAg, which is rich in basic amino acids and interacts with a variety of nucleic acids [24]. NdAg can accelerate the annealing of complementary sequences selectively and promotes strand exchange selectively for the formation of a more extended duplex among competing sequences [23]. Moreover, NdAg acts on HDV RNA, complicated and simple non-HDV RNAs, and even DNA oligos [23,24].…”

“…HDAg has an RNA chaperone activity in vivo [21] and in vitro [22,23]. It can modulate the catalytic activity of HDV genome subfragments by altering RNA structure and stimulate hammerhead ribozyme catalysis by facilitating the annealing of ribozymes to cognate substrates in vitro.…”