Adeno-Associated Virus RNA Transcription in vivo.

We have studied RNA transcripts of the defective parvovirus, adeno-associated virus (AAV) present in poly-(A)rich and poly-(A)free fractions of nuclear and cytoplasmic RNA prepared from cells infected together with a helper adenovirus. Cytoplasmic poly-(A)rich RNA contains three overlapping spliced AAV RNAs having sizes of 3.9 × 10³, 3.3 × 10³ and 2.3 × 10³ bases respectively. The nuclear precursors of these RNAs appear to be the coterminal unspliced poly(A)-rich RNAs containing 4.2 × 10³, 3.6 × 10³ and 2.6 × 10³ bases respectively. These unspliced RNAs were also found in the cytoplasm. The nuclear poly(A)-free RNA contained a heterogenous population of AAV RNAs that were generally smaller than 2.3 × 10³ bases. In addition, the Hirt pellet fraction of the nuclear RNA contained two discrete AAV poly(A)-free RNAs having sizes of 2.5 × 10³ and 2.8 × 10³ bases. The 4.2 × 10³ and 3.6 × 10³-base unspliced RNAs are more abundant than the coterminal 3.9 × 10³ and 3.3 × 10³-base spliced RNAs whereas the 2.3 × 10³-base spliced RNA is much more abundant than the 2.6 × 10³-base unspliced RNA. Thus, the cytoplasmic abundance of the AAV spliced RNAs appears to be controlled in part by the post-transcriptional events of splicing or message stability. We also analysed the effects of AAV defective-interfering genomes upon AAV transcription. These studies showed that when synthesis of standard AAV genomes was inhibited more than 10-fold by defective-interfering genomes there was no significant effect on the types or amounts of AAV RNA transcripts which accumulated. These observations indicate that interference by defective-interfering genomes occurs mostly at the level of DNA replication rather than transcription. [ABSTRACT FROM AUTHOR]