NUCLEOTIDE-SEQUENCE STABILITY OF THE GENOME OF HEPATITIS-DELTA VIRUS

Cultured cells were cotransfected with a fully sequenced 1,679-base cD NA clone of human hepatitis delta virus (HDV) RNA genome and a cDNA fo r the genome of woodchuck hepatitis virus (WHV). The HDV particles rel eased were able to infect a woodchuck that was chronically infected wi th WW. The HDV so produced was passaged a total of six times in woodch ucks in order to determine the stability of the HDV nucleotide sequenc e. During a final chronic infection with such virus, liver RNA was ext racted, and the HDV nucleotide sequence for the 352-base region, posit ions 905 to 1256, was obtained. By means of PCR, we obtained double-st randed cDNA both for direct sequencing and also for molecular cloning followed by sequencing. By direct sequencing, we found that a consensu s sequence existed and was identical to the original sequence. From th e sequences of 31 clones, we found 32% (10 of 31) to be identical to t he original single nucleotide sequence. For the remainder, there were neither insertions nor deletions but there was a small number of singl e-nucleotide changes. These changes were predominantly transitions rat her than transversions. Furthermore, the transitions were largely of j ust two types, uridine to cytidine and adenosine to guanosine. Of the 40 changes detected on HDV, 35% (14 of 40) occurred within an eight-nu cleotide region that included position 1012, previously shown to be a site of RNA editing. These findings may have significant implications regarding both the stability of the HDV RNA genome and the mechanism o f RNA editing.