Mapping of the cohesive overlap of duck hepatitis B virus DNA and of the site of initiation of reverse transcription

Abstract

The hepatitis B-like viruses have a approximately 3.2 kilobase, partially double-stranded DNA genome that is held in a circular conformation by a cohesive overlap between the 5' ends of the two strands. In addition, a protein is covalently bound to the 5' end of the minus strand of virion DNA. The sequence of the cohesive overlap region and its location relative to open reading frames and to the initiation site for minus-strand DNA synthesis, which occurs by reverse transcription of viral RNA, were investigated in duck hepatitis B virus. The 5' ends of virion DNA were mapped by restriction endonuclease analysis of labeled virion DNA, S1 nuclease digestion, and primer extension, using avian myeloblastosis virus DNA polymerase. The cohesive overlap region was shown to be 69 +/- 4 base pairs in length. It contained a 10-base pair inverted repeat in approximately the middle and a 12-base pair direct repeat near each end. The apparent initiation site of reverse transcription was determined by partial sequence analysis of dideoxynucleotide-truncated minus-strand DNA intermediates and comparison of their lengths with the length of a known DNA sequence. It mapped within two to four nucleotides of the 5' end of the minus strand of virion DNA. The results are consistent with the interpretation that the 5' end of the minus strand of virion DNA is the origin of reverse transcription and that the protein covalently bound to virion DNA is the primer of reverse transcription.