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 AUG was both required and sufficient to program internal initiation within AAV5 and may act similarly in heterologous contexts. The internally initiated AAV5 Rep40-like protein was functional and had helicase activity similar to that of AAV2 Rep40. Surprisingly, 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 P19 mutant AAV5 infectious clone generated near-wild-type levels of the double-stranded monomer 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 the central intron, and thus, splicing is precluded (1, 2) ( Fig. 1). AAV2 encodes two versions of its large (Rep78 and Rep68) and small (Rep52 and Rep40) Rep proteins from unspliced and spliced P5-and P19-generated RNAs, respectively (3-6). Because AAV5 P7 and P19 RNAs are not spliced, they were predicted to encode only Rep78 and Rep52 (Fig. 1); however, we have shown that while an AAV5 Rep68 protein was absent, as expected, AAV5 encodes an abundant Rep40-like protein from an in-frame internal initiation AUG 150 nucleotides (nt) downstream of the Rep52 initiator (7). That AAV5 uses another genetic mechanism to generate a Rep40-like protein is consistent with it playing an essential role during infection.Work from a number of labs has demonstrated that the small replication proteins of AAV2 are essential for packaging the genome into the viral capsids (8-10). This is likely to be dependent upon their potent 3= to 5= helicase activity (10, 11). All AAV Rep proteins have a central helicase domain characterized by a 100-amino-acid stretch of residues containing Walker motifs A, B, B=, and C (12, 13). AAV Rep proteins are classified as SF3 helicases (11,14), which also include a number of other viral proteins involved in DNA replication and packaging (14-17). The AAV2 Rep78 and Rep68 proteins have been shown to exist as hexamers in solution in the presence of double-stranded DNA, similar to other SF3 helicases (18-21). AAV2 Rep40 is a bimodular protein with a small helical bundle at the amino terminus and a large ␣/ domain at the C terminus (9, 11); there is l...