Human hepatitis delta virus RNA subfragments contain an autocleavage activity.

Abstract

Hepatitis 8 virus (HDV) contains a singlestranded circular RNA genome of 1.7 kilobases. In this report we demonstrate that subfragments of HDV RNA can undergo autocatalytic cleavage. This cleavage requires at least 500 pM of Mg2+ or Ca2+, is not affected by varying the pH from 5.0 to 9.1, and occurs with RNA fragments as small as 133 nucleotides. The larger RNA fragments containing additional HDV sequences have a lower efficiency of cleavage. Deletion analysis at both ends of RNA subfragments suggested that the catalytic ability of HDV RNA resides in a stretch of no more than 117 nucleotides around the cleavage site. The cleavage occurs at the phosphodiester bond between nucleotides 688 and 689 on the HDV genomic map, generating a 5' fragment with a terminal uridyl 2',3'-cyclic monophosphate residue and a 3' fragment with a guanosyl residue with a 5'-hydroxyl group. The smallest autocleaving RNA does not contain the "hammerhead" sequence required for the autocleavage of other known selfcleaving RNA. The cleavage of HDV RNA occurs at a much faster rate, even at a very low Mg2+ concentration, than that of other "ribozymes." Thus, HDV RNA represents a distinct class of ribozyme.Human hepatitis 8 virus (HDV) is a defective virus often associated with fulminant hepatitis in hepatitis B virus carriers. The virus particles have an envelope in which hepatitis B virus surface antigen is located. Internal to the envelope are HDV-specific 6 antigen and a single-stranded circular RNA genome of 1.7 kilobases (kb) (1-3). This animal virus is distinctive because it has such a mixed structure that resembles somewhat the virus coat protein-encapsidated plant virus satellite and virusoid RNAs. The HDV RNA possesses several additional properties similar to those of circular RNA of viroids; for instance, the presence of a high degree of intramolecular self-complementarity and conservation of the consensus sequences known to be important for viroid RNA replication (4,5). However, HDV RNA is much larger than viroid RNAs and encodes at least one structural protein (6 antigen), whereas viroid RNA does not encode any protein. Despite these differences HDV RNA and viroid RNA appear to replicate through a similar mechanism-i.e., a rolling circle mechanism (6)-since a larger-than-genomic size RNA intermediate has been detected in HDV-infected cells (7,8). Thus a mechanism must exist to cleave these RNA intermediates into monomeric RNA and to circularize them.One viroid and several virusoid and satellite RNAs have been shown to possess an autocleaving activity (9-11). The cleavage sites of these RNAs correspond to sites of in vivo RNA processing, suggesting that these autocleavage reactions have physiological roles. A linear dimer cDNA of HDV genome can be processed into a monomeric RNA when transfected into a monkey kidney (COS) cell line (unpublished observation), suggesting either that HDV RNA has an unusual secondary structure that allows for specific attack by a cellular RNase or that HDV RNA has a specific autocleavage activit...

HDV Family of Self-Cleaving Ribozymes

“…Several such catalytic motifs exist, including the hammerhead, hairpin, hepatitis delta virus (HDV), the Neurospora Varkud satellite motifs, and the cofactor-dependent glmS ribozyme. [10][11][12][13][14] The HDV family of ribozymes is the topic of this chapter.…”

How RNA acts as a nuclease: some mechanistic comparisons in the nucleolytic ribozymes

“…In the next group I am placing the hepatitis D virus (HDV) ribozyme (61)(62)(63) and the TS ribozyme. Structural and mechanistic evidence indicates that the HDV ribozyme uses metal ion-bound water as the general base, and a cytosine nucleobase as a general acid (15,(64)(65)(66)(67).…”

X-ray crystallographic observation of “in-line” and “adjacent” conformations in a bulged self-cleaving RNA/DNA hybrid

“…Following the discovery of the catalytic properties of RNA some 20 years ago (Kruger et al+, 1982;GuerrierTakada et al+, 1983), many more self-splicing introns belonging to two distinct groups along with three types of small self-cleaving ribozymes have been identified+ These are the hammerhead (Hutchins et al+, 1986;Prody et al+, 1986;Uhlenbeck, 1987;Verma et al+, 1997), the hairpin (Buzayan et al+, 1986;Hampel & Tritz, 1989;Hampel, 1998;Walter & Burke, 1998), and the delta ribozymes (Sharmeen et al+, 1988;Wu et al+, 1989)+ Two types of reactions are mediated by these ribozymes, namely RNA splicing and site-specific RNA cleavage (Cech, 1987)+ In the case of the three above classes of self-cleaving ribozymes, the autolytic processing reaction proceeds with inversion of configuration of the phosphate group, as initially established with hammerhead-type ribozymes (van Tol et al+, 1990;Koizumi & Ohtsuka, 1991;Slim & Gait, 1991)+ A general feature of ribozymes is that they either require or are at least stimulated by divalent metal cations (Dahm & Uhlenbeck, 1991;Dahm et al+, 1993)+ Many of the natural ribozymes show rather broad metalion specificity+ Thus, hammerhead-type ribozymes are active with several earth alkali cations (Mg 2ϩ , Ca 2ϩ , Sr 2ϩ ) or transition metal cations (Mn 2ϩ , Co 2ϩ , Zn 2ϩ ) (Dahm & Uhlenbeck, 1991)+ Detailed analysis of the metal-ion dependence of cleavage reactions mediated by ribozymes provides evidence that the precise role of the metal ions varies considerably between different classes of ribozymes+ For example, RNA catalysis in the case of the hairpin ribozyme does not involve direct coordination of magnesium to the phosphate ester, nor activation of a bound water molecule, as demonstrated by efficient pH-independent cleavage in the presence of cobalt hexammine (Hampel & Cowan, 1997;Nesbitt et al+, 1997;Young et al+, 1997;Fedor, 2000)+ In addition, it was shown that aminoglycoside antibiotics or spermine alone can promote efficient hairpin cleavage with rates similar to the magnesium-dependent reaction (Earnshaw & Gait, 1998)+ Similarly, the Hepatitis delta virus ribozyme does not require high concentrations of metal ions for activity, perhaps due to a particular cytosine that could act as the general base in the cleavage reaction (Ferré-D'Amaré et al+, 1998;…”