Adeno-Associated Virus Type 5 Utilizes Alternative Translation Initiation To Encode a Small Rep40-Like Protein

Abstract

Alternative splicing of adeno-associated virus type 2 (AAV2) P19-generated pre-mRNAs generates the small Rep proteins Rep52 and Rep40, which differ in their carboxyl termini. Both proteins are required for optimal packaging of AAV2 genomes. AAV5 Rep-encoding P19-generated transcripts are primarily polyadenylated within the central intron and not efficiently spliced; however, surprisingly, AAV5 was found to generate high levels of a Rep40-like protein. The AAV5 Rep40-like protein was generated by internal initiation and has the same C terminus as Rep52. Although precluded from using alternative splicing to generate multiple Rep isoforms, AAV5 ensures the production of a Rep40-like protein by utilizing a novel internal translation initiation event.The adeno-associated virus type 2 (AAV2) P19 promoter generates pre-mRNAs that are alternatively spliced to yield the small nonstructural proteins Rep52 and Rep40 from unspliced and spliced mRNAs, respectively. These Rep proteins (as well as the overlapping large Rep proteins) share a common Walker-type domain common to the superfamily 3 (SF3)-type helicases (3, 4). Both small Rep proteins are required for efficient packaging of the AAV2 genome; Rep52 and Rep40 both possess 3Ј-to-5Ј helicase activity and are required for unwinding of the double-stranded replicative forms of AAV for insertion into preformed capsids (5). Rep52 and Rep40 possess some functional redundancy in their helicase activities, ATPase activities, and DNA binding activities; however, their precise mechanisms of action are different. Structural analyses have indicated that when bound to DNA, AAV2 Rep52 remains monomeric (14), while Rep40 assumes a hexameric ringlike structure (1, 4), which has been suggested to be important for its function. Although Rep52 and Rep40 both are capable of unwinding DNA substrates with single-stranded DNA ends in a 3Ј-to-5Ј fashion, Rep40, but not Rep52, efficiently unwinds double-stranded DNA (1). Therefore, it has been suggested that during AAV replication, the Rep40 hexamer may be necessary for the initial unwinding of the first few bases of the double-stranded DNA intermediate, and further unwinding may then be accomplished by Rep52 alone or by Rep52 in conjunction with Rep40 and other Rep proteins (1).In contrast to AAV2, pre-mRNA transcripts derived from the AAV5 and goat AAV P19 promoter are preferentially polyadenylated in the viral central intron and thus spliced at low efficiency (10-12). Thus, these viruses would not be expected to generate significant quantities of Rep40. However, immunoblot analyses have consistently demonstrated the presence of significant levels of a Rep40-like (but not a Rep 68-like) protein during AAV5 and goat AAV coinfection with adenovirus, as well as during transient transfection (7,8,11). This suggested that AAV5 may generate a Rep40-like protein using a mechanism distinct from the alternative splicing utilized by AAV2. Here we show that, unlike AAV2 Rep40, the AAV5 Rep40-like protein has the same C terminus as Rep52 and that these tw...

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

“…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.…”

“…2C). We have previously shown that when the 150-nt region between the two AAV5 small-Rep AUGs (here termed AUG1 and AUG2, respectively) was replaced with the analogous region from AAV2, the downstream AAV5 AUG (AUG2) was not used at detectable levels (7). As can be seen in Fig.…”

“…For these experiments, we utilized a system in which the small rep genes from AAV5 and AAV2 were expressed from a CMV expression vector and were tagged at their carboxyl termini with HA, as previously described (7). Genes within these constructs were expressed similarly to their expression from within full-length viral constructs (7) or from the isolated P19 transcription unit (data not shown). The Ϫ6 to ϩ3 extended AUG1 and AUG2 motifs of AAV2 are somewhat different from those of AAV5 (Fig.…”

“…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 little evidence to suggest that it exists other than as a monomer in solution (22). Interestingly, the AAV5 Rep40-like protein lacks the helical bundle at the amino terminus present in AAV2 Rep40 (7,9). Like AAV5, AAV2 contains both a similarly positioned internal polyadenylation site and an in-frame AUG downstream of its Rep52 initiating codon (23); however, neither the internal polyadenylation signal nor the internal AUG is used in the AAV2 context.…”

Highly Divergent Integration Profile of Adeno-Associated Virus Serotype 5 Revealed by High-Throughput Sequencing

“…For example, AAV-5 transcription and RNA processing is divergent, involving alternative polyadenylation and distance-dependent RNA processing (8)(9)(10). Several unique transcripts are created from the AAV-5 genome, such as functional truncated versions of the viral replication proteins (11)(12)(13). In addition, in contrast to other serotypes, transcription of AAV-5 capsid proteins does not require Rep expression (14).…”