Definition of a novel promoter for the major adenovirus-associated virus mRNA

Abstract

A 660 nucleotide adenovirus-associated virus type 2 (AAV2) DNA fragment which encodes the 5′ terminal leader and the entire intervening sequence of the major viral mRNA has been cloned into pBR322, and its primary sequence has been determined. The 5′ terminal viral mRNA sequence was deduced by sequencing the reverse-transcriptase cDNA extension product of a 5′ end-labeled DNA primer complementary to the RNA 5′ terminal region. From combined DNA and RNA sequence analyses (which confirm our previous mapping data) we conclude that the major AAV2 transcript contains a 5′ terminal leader sequence about 55 nucleotides in length encoded from a continuous region of DNA (near position 39 on the viral genome) 320 bases from the RNA body. The DNA sequences of the splice junctions are similar to those found for other class II genes. No other nucleotide sequence, indicative of promotion at another (upstream) site, is present at the 5′ terminus. The DNA region encoding and flanking the leader sequence displays structural features expected for a class II gene promoter, including the canonical ATATAA sequence 23–25 bases upstream from the presumed initiation site. When the cloned viral DNA fragment is transcribed in vitro by RNA polymerase II in a cell-free system, a transcript is produced with a 5′ end that is similar or identical to that found on the in vivo mRNA. Taken together these data strongly suggest that the major polysomal RNA may be generated from a transcription unit with a promoter at position 39, even though this transcription unit is part of a larger transcription unit with an upstream promoter near position 6. This indication of overlapping transcription units with independent promoters provides a major new insight into parvovirus gene expression.