Adeno-associated virus RNAs appear in a temporal order and their splicing is stimulated during coinfection with adenovirus

We have used a quantitative RNase protection assay to characterize the rela tive accumulation and abundance of individual adeno-associated virus type 2 (AAV) RNAs throughout the course of AAV-adenovirus coinfections and preinf ections. We have demonstrated that there is a previously unrecognized tempo ral order to the appearance of AAV RNAs, First, unspliced P5-generated tran scripts, which encode Rep78, were detectable prior to the significant accum ulation of other AAV RNAs, Ultimately, as previously demonstrated, P19-gene rated products accumulated to levels greater than those generated from P5, and P40-generated transcripts predominated in the total RNA pool. Second, t he percentage of each class of AAV RNA that was spliced increased during in fection, and the degree of this increase was different for the P5/P19 produ cts than for those generated by P40. At late times postcoinfection, approxi mately 90% of P40 products, but only approximately 50% of RNAs generated by P5 and P19, were seen to be spliced; thus, the AAV intron was removed to d ifferent final levels from these different RNA species. We have shown that each of the AAV RNAs is quite stable; the majority of each RNA species pers isted 6 h after treatment with actinomycin D, Quantification of the accumul ation of individual AAV RNAs, over intervals during which degradation was n egligible, allowed us to infer that at late times during infection the rela tive strength of P5, P19, and P40 was approximately 1:3:18, respectively, c onsistent with the steady-state accumulated levels of the RNAs generated by each promoter. All AAV RNAs exited to the cytoplasm with similar efficienc ies in the presence or absence of adenovirus; however, adenovirus coinfecti on appeared to stimulate total splicing of AAV RNAs and the relative use of the downstream intron acceptor. Our results confirm and extend previous ob servations concerning the appearance and processing of AAV-generated RNAs.