Hepatitis delta virus mutant: effect on RNA editing.
During the replication cycle of hepatitis delta virus (HDV), RNA editing occurs at position 1012 on the 1679-nucleotide RNA genome. This changes an A to G in the amber termination codon, UAG, of the small form of the delta antigen (delta Ag). The resultant UGG codon, tryptophan, allows the translation of a larger form of the delta Ag with a 19-amino-acid C-terminal extension. Using HDV cDNA-transfected cells, we examined the editing potential of HDV RNA mutated from G to A at 1011 on the antigenome, adjacent to normal editing site at 1012. Four procedures were used to study not only the editing of the A at 1012, but also that of the new A at 1011: (i) nucleotide sequencing, (ii) a PCR-based RNA-editing assay, (iii) immunoblot assays, and (iv) immunofluorescence. Five findings are reported. (i) Even after the mutation at 1011, editing still occurred at 1012. (ii) Site 1011 itself now acted as a novel RNA-editing site. (iii) Sites 1011 and 1012 were edited independently. (iv) At later times, both sites became edited, thereby allowing the synthesis of the large form of the delta Ag (delta Ag-L). (v) Via immunofluorescence, such double editing became apparent as a stochastic event, in that groups of cells arose in which the changes had taken place. Evaluation of these findings and of those from previous studies of the stability of the HDV genomic sequence (H.J. Netter et al., J. Virol. 69:1687-1692, 1995) supports both the recent reevaluation of HDV RNA editing as occurring on antigenomic RNA (Casey and Gerin, personal communication) and the interpretation that editing occurs via the RNA-modifying enzyme known as DRADA.
D000595:Amino Acid Sequence; D000906:Antibodies; D001483:Base Sequence; D002460:Cell Line; D003698:Hepatitis Delta Virus / Q000235:genetics* / Q000502:physiology; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D013728:Terminator Regions, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured; D014779:Virus Replication
D000906:Antibodies; D010455:Peptides; D012367:RNA, Viral