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...

The specific interaction between R17 coat protein and its target of translational repression at the initiation site of the R17 replicase gene was studied by synthesizing variants of the RNA binding site and measuring their affinity to the coat protein by using a nitrocellulose filter binding assay. Substitution of two of the seven single-stranded residues by other nucleotides greatly reduced the Ka, indicating that they are essential for the RNA-protein interaction. In contrast, three other single-stranded residues can be substituted without altering the Ka. When several of the base-paired residues in the binding site are altered in such a way that pairing is maintained, little change in Ka is observed. However, when the base pairs are disrupted, coat protein does not bind. These data suggest that while the hairpin loop structure is essential for protein binding, the base-paired residues do not contact the protein directly. On the basis of these and previous data, a model for the structural requirements of the R17 coat protein binding site is proposed. The model was successfully tested by demonstrating that oligomers with sequences quite different from the replicase initiator were able to bind coat protein.

Age and fatty liver in both sexes were found to be risk factors for GSD in the study population. The finding of a correlation between fatty liver and GSD is an important addition to the literature concerning the risk factors of GSD. Diabetes mellitus, history of GSD in the first-degree relatives, and use of oral contraceptives were also risk factors for GSD in women.

PriB is a primosomal protein required for replication restart in Escherichia coli. PriB stimulates PriA helicase activity via interaction with single-stranded DNA (ssDNA), but the molecular details of this interaction remain unclear. Here, we report the crystal structure of PriB complexed with a 15 bases oligonucleotide (dT15) at 2.7 Å resolution. PriB shares structural similarity with the E.coli ssDNA-binding protein (EcoSSB). However, the structure of the PriB–dT15 complex reveals that PriB binds ssDNA differently. Results from filter-binding assays show that PriB–ssDNA interaction is salt-sensitive and cooperative. Mutational analysis suggests that the loop L45 plays an important role in ssDNA binding. Based on the crystal structure and biochemical analyses, we propose a cooperative mechanism for the binding of PriB to ssDNA and a model for the assembly of the PriA–PriB–ssDNA complex. This report presents the first structure of a replication restart primosomal protein complexed with DNA, and a novel model that explains the interactions between a dimeric oligonucleotide-binding-fold protein and ssDNA.

a b s t r a c tThe helicase domain of dengue virus NS3 protein (DENV NS3H) contains RNA-stimulated nucleoside triphosphatase (NTPase), ATPase/helicase, and RNA 5 0 -triphosphatase (RTPase) activities that are essential for viral RNA replication and capping. Here, we show that DENV NS3H unwinds 3 0 -tailed duplex with an RNA but not a DNA loading strand, and the helicase activity is poorly processive. The substrate of the divalent cation-dependent RTPase activity is not restricted to viral RNA 5 0 -terminus, a protruding 5 0 -terminus made the RNA 5 0 -triphosphate readily accessible to DENV NS3H. DENV NS3H preferentially binds RNA to DNA, and the functional interaction with RNA is sensitive to ionic strength.

The translational operator of the R17 replicase gene contains a bulged A residue that is essential for the specific binding to R17 coat protein. A large number of operator variants have been synthesized to more precisely examine the role of the bulged A residue on this specific protein-RNA interaction. By use of RNA ligase and transcription of synthetic DNA templates by T7 RNA polymerase, 14 different nucleotides were introduced to the bulged A position of three different coat protein binding fragments. The affinity between coat protein and each fragment was determined by a nitrocellulose filter binding assay. The data indicate that while functional groups on N1, C2, C6, N7, and 2'OH of the bulged A can be substituted without greatly changing protein binding, bulky substituents cannot be tolerated at these positions. Data from additional fragments that have base-pair changes adjacent to the bulged A suggest that the propensity of the bulged A to intercalate into the helix can affect protein binding.

We conducted extensive mutagenesis analysis on a hepatitis delta virus (HDV) genomic ribozyme to study the sequence specificity of certain region and to derive the secondary structure associated with the catalytic core. The results confirmed that the autocatalytic domain of HDV genomic RNA contained four basepairing regions as predicted in the 'pseudo-knot' model [Perrotta & Been (1990) Nature 350,[434][435][436].The size and sequence of one of the basepairing regions, i. e. stem-and-loop, could be flexible. Helix 3 and the first basepair of helix 1 required specific sequence to retain self-cleavage activity. The structural requirement of helix 2 was less stringent than the other basepairing regions. Moreover, the size of helix 1 affected self-cleavage whereas the length of hinge could be variable even though the first three residues of hinge had stringent sequence requirement.

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