We report the initial characterization of adeno-associated virus type 5 (AAV5) RNAs generated following viral infection and the construction of a replicating infectious clone of AAV5. While the basic transcription profile of AAV5 was similar to that of AAV2, there were also significant differences. Mapping of the AAV5 transcripts demonstrated an efficient transcription initiation site within the AAV5 inverted terminal repeat (ITR), and mapping of the AAV5 intron revealed that it is considerably smaller than that of AAV2. Furthermore, in contrast to the case for AAV2, neither the Rep protein nor additional adenovirus gene products were required to achieve efficient promoter activity and pre-mRNA splicing following transfection of an AAV5 rep/cap plasmid clone lacking the ITRs into 293 cells. Perhaps most surprisingly, RNAs generated from both the AAV5 P7 and P19 promoters were efficiently polyadenylated at a site lying within the intronic region in the center of the genome. Because P7-and P19-generated transcripts are polyadenylated at this site and not spliced, Rep78 and Rep52 were the only Rep proteins detected during AAV5 infection.The human adeno-associated viruses (AAV) are small, nonenveloped, single-stranded DNA viruses that replicate in mammalian cells best in the presence of larger helper DNA viruses, e.g., adenovirus or herpesvirus (36). Six different serotypes of AAV (AAV type 1 [AAV1] to AAV6) have been characterized. AAV2, the prototypical strain, as well as AAV3 and AAV5 have been isolated directly from human clinical specimens (5, 12, 26). AAV1 and AAV4 have been suggested to be originally of simian origin (5, 26). The more distantly related AAV5 was isolated was from a penile flat condylomatous lesion (2), and epidemiologically, AAV5 transmission appears to follow acquisition of herpesviruses rather than adenovirus (12).At least portions of the genomes of all six serotypes of AAV have been cloned and sequenced (9,10,22,31,37,43). The inverted terminal repeats (ITRs) of AAV1, -2, -3, -4, and -6 are Ͼ95% identical, and the rep genes of these different isolates are approximately 85% identical. In contrast, the rep gene and ITR of AAV5 are only 60% similar to those of other serotypes (9).The Rep proteins of AAV1, -2, -3, -4, and -6 can each support the production of recombinant AAV2 vectors (9, 30), suggesting that they share significant functional homology for replication. In contrast, AAV5 is unable to support replication of AAV2-based vectors (9, 30), most likely because AAV5 Rep processes a novel terminal resolution site (TRS) present only on AAV5 ITR (8).AAV2 is the best characterized of the AAV serotypes. The genome contains three promoters, P5, P19, and P40. The large Rep proteins (Rep78 and Rep68) and the small Rep proteins (Rep52 and Rep40), encoded from a large open reading frame in the left half of the genome, are generated from RNAs which derive from P5 and P19, respectively, and which polyadenylate near the right-hand ITR. Rep78 and -52 are generated from unspliced RNAs, and Rep68 and -40...

Adeno-associated virus type 5 (AAV5) has a linear, single-stranded DNA genome of ca. 5 kb and an overlapping transcription profile featuring multiple promoters and a single intron in the center of the genome. Unlike the situation for the prototype AAV2, AAV5 RNAs transcribed from upstream promoters at map units 7 (P7) and 19 (P19), which encode the viral Rep proteins, are predominantly polyadenylated at a site within the intron [(pA)p]. RNAs generated from the AAV5 capsid gene promoter P41, which is only 78 nucleotides (nt) upstream of the intron donor, and 281 nt upstream of (pA)p, primarily readthrough (pA)p, are polyadenylated at a more distal site at the 3 end of the genome [(pA)d] and ultimately spliced. The intron contains the core sequences sufficient for polyadenylation at (pA)p, which is governed by a G/U-rich downstream element that overlaps with the intron 3 A2 splice acceptor. In addition, polyadenylation of P7-and P19-generated RNAs at (pA)p is influenced by an upstream element that lies 5 to the start of the P41 transcript. Our results also suggest that splicing and polyadenylation of P41-generated RNA can compete for the same pool of precursor pre-mRNA molecules. The cis-acting signals within the A2 3 splice site that govern polyadenylation and splicing of AAV5 RNAs must be optimized to program both (i) the levels of polyadenylation of P7-and P19-generated RNA at (pA)p required to generate the proper levels of the essential Rep proteins and (ii) the splicing of P41-generated RNAs to generate the proper ratio of capsid proteins during AAV5 infection.

Full replication of adeno-associated virus type 5 (AAV5) is sustained by adenovirus type 5 (Ad5) helper functions E1a, E1b, E2a, E4Orf6, and virus-associated (VA) RNA; however, their combined net enhancement of AAV5 replication was comprised of both positive and negative individual effects. Although Ad5 E4Orf6 was required for AAV5 genomic DNA replication, it also functioned together with E1b to degrade de novoexpressed, preassembled AAV5 capsid proteins and Rep52 in a proteosome-dependent manner. VA RNA enhanced accumulation of AAV5 protein, overcoming the degradative effects of E4Orf6, and was thus required to restore adequate amounts of AAV5 proteins necessary to achieve efficient virus production.Efficient replication of adeno-associated viruses (AAVs) requires helper functions that can be supplied by larger DNA viruses such as adenovirus (Ad) or herpes simplex virus (2). There are five adenovirus type 5 (Ad5) functions required to support both AAV2 replication and production of recombinant AAV (E1a, E1b, E2a, E4Orf6, and virus-associated [VA] RNA), and the roles these factors play during both Ad and AAV infection have been extensively characterized (2,3,24,25). Ad5 E2a provides single-stranded DNA (ssDNA) binding activity presumably required during the strand-displacement phase of AAV2 terminal repeat (TR)-mediated genome replication (20). E4Orf6 has been shown to be essential for second strand synthesis, both in vitro and in vivo (7), and likely plays other roles during AAV2 replication as well (22,24,26). VA RNA has been suggested to enhance AAV gene expression at the posttranscriptional level, most likely at the level of protein translation (22,26). The role of VA RNA in the translation of Ad5 proteins during Ad5 infection is well characterized (12, 13). Ad5 E1a and E1b have been reported to perform a number of essential roles during AAV gene expression, including promoter activation (3,11,15,28); however, other than the E1A's well-defined participation in the regulation of expression of AAV2 P5, the role that adenovirus plays in other aspects of AAV2 gene expression is as yet only partially understood (3, 25).Adeno-associated virus type 5 (AAV5), which was first isolated from a penile condylomatous lesion, is the most divergent of the AAV serotypes, sharing only 64% overall nucleotide identity with the prototype AAV2 (1, 8). While the basic transcription profile of AAV5 is similar to that of AAV2, there are also significant differences (17). In contrast to AAV2, RNAs generated from both the AAV5 P7 and P19 promoters are efficiently polyadenylated at a site lying within the intron in the center of the genome, and because these RNAs are not spliced, Rep78 and Rep52 are the only Rep proteins detected during AAV5 infection (17). Furthermore, unlike AAV2, neither the AAV5 Rep protein nor additional adenovirus gene products are required to achieve efficient AAV5 promoter activity and pre-mRNA splicing following transfection of an AAV5 rep/cap plasmid clone lacking the inverted terminal repeats (ITRs) into 293...

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Degradation of de novo-generated adeno-associated virus type 5 (AAV5) Rep52 and capsid proteins is part of the limited target specificity displayed by adenovirus type 5 E4Orf6-E1B-55k as part of a cullin 5-containing E3 ligase complex. Both Rep and capsid proteins can be found in the ligase complex, and their presence is dependent on interaction between E4Orf6 and elongins B and C. Degradation of AAV5 proteins can be inhibited by a dominant-negative ubiquitin that prevents chain elongation or by small interfering RNA directed against cullin 5.