The Adeno-Associated Virus Type 5 Small Rep Proteins Expressed via Internal Translation Initiation Are Functional

Although precluded from using splicing to produce multiple small Rep proteins, adeno-associated virus type 5 (AAV5) generates a Rep40-like protein by alternative translation initiation at an internal AUG. A defined region upstream of the internal AUGwas both required and sufficient to program internal initiation within AAV5 andmay act similarly in heterologous contexts. The internally initiated AAV5 Rep40-like protein was functional and had helicase activity similar to that of AAV2 Rep40. Surpris-ingly, both the AAV5 Rep40-like protein and Rep52 were able to be translated from the AAV5 upstream P7-generated RNAs; however, the relative level of small to large Rep proteins was reduced compared to that of the wild type. A P19mutant AAV5 in-fectious clone generated near-wild-type levels of the double-strandedmonomer replicative form (mRF) replicative intermediate but reduced levels of virus, consistent with the previously defined role of Rep40-like proteins in genome encapsidation. Levels of mutant virus were dramatically reduced upon amplification. The transcription maps of adeno-associated virus type 5(AAV5) and many of the animal AAVs are quite different from those of AAV2 and other human AAVs. While all AAV2 RNAs extend to a polyadenylation site near the right-hand end of the genome and are exported to the cytoplasm as both spliced and unspliced species, AAV5 RNAs generated by the viral P7 and P19 promoters are predominately polyadenylated at a site within th