Adeno‐Associated Virus RNA Transcription in vivo

Abstract

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 x lo3, 3.3 x lo3 and 2.3 x lo3 bases respectively. The nuclear precursors of these RNAs appear to be the coterminal unspliced poly(A)-rich RNAs containing 4.2 x lo3, 3.6 x lo3 and 2.6 x lo3 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 x lo3 bases. In addition, the Hirt pellet fraction of the nuclear RNA contained two discrete AAV poly(A)-free RNAs having sizes of 2.5 x lo3 and 2.8 x lo3 bases.Thc 4.2 x lo3 and 3.6 x 103-base unspliced RNAs are more abundant than the coterminal 3.9 x lo3 and 3.3 x 103-base spliced RNAs whereas the 2.3 x 103-base spliced RNA is much more abundant than the 2.6 x lo3-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.We previously showed that transcription of the defective parvovirus, adeno-associated virus (AAV), genome in human K B cells using a human adenovirus as the helper yielded several cytoplasmic AAV RNA species [l]. All of these RNAs were complementary to overlapping regions of the AAV DNA minus strand. Using DNA probes labeled in vivo we detected three unspliced RNAs (4.2 x lo3, 3.6 x lo3 and 2.6 x lo3 bases) having common 3' termini at map position 96.0 and 5' termini at approximate map positions of 5, 19 and 39 respectively. There were also three co-terminal spliced RNAs (3.9 x lo3, 3.3 x lo3 and 2.3 x lo3 bases respectively) with a single spliced-out region of about 350 nucleotides between map position 40 and 49. Other workers also identified and mapped the unspliced 4.2 x lo3, 3.6 x lo3 and 2.6 x lo3-base RNAs and the spliced 2.3 x 103-base RNA but did not detect the 3.9 x lo3 and 3.3 x 103-base spliced RNAs [2,3]. To resolve these discrepancies we have now performed a more extensive analysis of AAV poly(A)-rich and poly(A)-free RNAs present in both the cytoplasmic and nuclear fractions using both one-dimensional and two-dimensional gel electrophoresis.We show directly the presence of the spliced 3.9 x lo3 and 3.3 x 103-base RNAs in the cytoplasm. In addition, our data shows that splicing of these two RNAs or their accum...

High-titer recombinant adeno-associated virus production utilizing a recombinant herpes simplex virus type I vector expressing AAV-2 Rep and Cap

“…The p5, p19 and p40 promoters drive expression of the AAV-2 proteins Rep 78 and 68, Rep 52 and 40 and the capsid structural proteins VP1, VP2 and VP3, respectively. [33][34][35][36][37] Homologous recombination into the tk gene was confirmed by Southern blot analysis of restriction digests of d27.1-rc infected cell DNA (data not shown). In addition, d27.1-rc plaque formation on V27 cells, a complementing cell line, was not affected by 5-bromo-deoxycytidine.…”

Negative and positive regulation in trans of gene expression from adeno-associated virus vectors in mammalian cells by a viral rep gene product.

“…1. The AAV2 genome contains three promoters, P5, plg, and P40, which each yield two transcripts differing in the presence or absence of the intron (11,21,39,40,42). All of the transcripts terminate at the same 3' polyadenylation site (39,57).…”

Producing Recombinant Adeno-Associated Virus in Foster Cells: Overcoming Production Limitations Using a Baculovirus–Insect Cell Expression Strategy

“…The stoichiometry of VP1, VP2, and VP3 within the assembled virion likely reflects the expression levels of the three capsid proteins. The AAV cap gene promoter, p40, produces a single primary 2.6-kb transcript (Marcus et al, 1981). Using alternate splice acceptors results in mRNAs lacking the AUG initiation codon for VP1.…”