T_mstudies of a tertiary structure from the human hepatitis delta agent which functionsin vitroas a ribozyme control element

Abstract

Viroids and other circular subviral RNA pathogens, such as the hepatitis delta agent, use a rolling circle replication cycle requiring an intact circular RNA. However, many infectious RNAs have the potential to form self-cleavage structures, whose formation must be controlled in order to preserve the circular replication template. The native structure of delta RNA contains a highly conserved element of local tertiary structure which is composed of sequences partially overlapping those needed to form the self-cleavage motif. A bimolecular complex containing the tertiary structure can be made. We show that when it is part of this bimolecular complex the potential cleavage site is protected and is not cleaved by the delta ribozyme, demonstrating that the element of local tertiary structure can function as a ribozyme control element in vitro. Physical studies of the complex containing this element were carried out. The complex binds magnesium ions and is not readily dissociated by EDTA under the conditions tested; >50%/o of the complexes remain following incubation in 1 mM EDTA at 600C for 81 min. The thermal stability of the complex is reduced in the presence of sodium ions. A DNA complex and a perfect RNA duplex studied in parallel showed a similar effect, but of lesser magnitude. The RNA complex melts at temperatures -10°C lower in buffers containing 0.5 mM MgCI2 and 100 mM NaCI than in buffers containing 0.5 mM MgCI2 with no NaCI (78.1 compared with 87.70C). The element of local tertiary structure in delta genomic RNA appears to be a molecular clamp whose stability is highly sensitive to ion concentration in the physiological range.

Recurring features of local tertiary structural elements in RNA molecules exemplified by hepatitis D virus RNA

“…Our laboratory has worked out techniques for producing specific UV crosslinks in RNA molecules at sites of preexisting local tertiary structure (Branch et al 1985a(Branch et al , 1989c, isolating the RNAs containing them by altered behavior on polyacrylamide gels, and their specific regions by partial RNase T1 digestion (Branch et al 1985b(Branch et al , 1989c, and mapping their exact positions in the RNA sequence (Branch et al 1989a(Branch et al , 1989b(Branch et al , 1989c(Branch et al , 1990aLyons et al 2001). For antigenomic HDV, a transcript containing the conserved domain, spanning residues 482-963 (shown in Fig.…”

“…Portions of the antigenomic RNA containing the crosslinked site and flanking sequences, along with the UVcrosslinkable site in the HDV genomic RNA (Branch et al 1990a), are shown in Figure 2. After adjusting their orientations to facilitate comparison, they reveal a number of interesting similarities.…”

“…The highly structured RNA genome of the hepatitis D virus (HDV, or the delta agent) is an ideal test molecule for studying this idea. We already know that the genomic strand of HDV contains a tertiary structural element that undergoes UV-crosslinking between two bases at a specific site (Branch et al 1989a(Branch et al , 1990a(Branch et al , 1990b. Figure 1 is a schematic diagram of the HDV RNA genome.…”

“…1) of the ribozyme cleavage site at base 685 in the collapsed rod structure (Branch et al 1990a), and that it helps to form the binding site for PKR (Circle et al 1997), a cellular protein kinase that contains two copies of the double-stranded RNA-binding motif (dsRBM; Green and Mathews 1992). The HDV antigen protein is phosphorylated in vivo during viral infection, and recent studies indicate a role for PKR in this process (Yeh et al 1996;Bichko et al 1997;Chen et al 2002).…”

3-D models of the antigenomic ribozyme of the hepatitis delta agent with eight new contacts suggested by sequence analysis of 188 cDNA clones

“…Within these 25 bases, only a single mutation (at C-13) has been reported in sequenced isolates of the delta agent (41). During the replication cycle, the ribozyme sequences are constrained by factors in addition to those directly related to their catalytic capability; such as their ability to function as templates for replication, which may impose sequence restrictions (42); ability to form the rodlike structure (9); and their ability to generate the UV-sensitive tertiary structure (43,44) proposed as a control element for the genomic ribozyme (45,46). In our current in vitro study, in which none of these factors had an effect, the ranking of nucleotides from the least conserved to the most conserved was as follows: (the number of clones with a mutation at a particular position is given in parenthesis): G (47), nucleotides contributing to tertiary structure are known to be highly conserved.…”