Characterization of the Transcription Profile of Adeno-Associated Virus Type 5 Reveals a Number of Unique Features Compared to Previously Characterized Adeno-Associated Viruses

To better understand the relationship between primate adeno-associated viruses (AAVs) and those of other mammals, we have cloned and sequenced the genome of an AAV found as a contaminant in two isolates of bovine adenovirus that was reported to be serologically distinct from primate AAVs. The bovine AAV (BAAV) genome has 4,693 bp, and its organization is similar to that of other AAV isolates. The left-hand open reading frame (ORF) and both inverted terminal repeats (ITRs) have the highest homology with the rep ORF and ITRs of AAV serotype 5 (AAV-5) (89 and 96%, respectively). However, the right-hand ORF was only 55% identical to the AAV-5 capsid ORF; it had the highest homology with the capsid ORF of AAV-4 (76%). By comparing the BAAV cap sequence with a model of an AAV-4 capsid, we mapped the regions of BAAV VP1 that are divergent from AAV-4. These regions are located on the outside of the capsid and are partially located in exposed loops. BAAV was not neutralized by antisera raised against recombinant AAV-2, AAV-4, or AAV-5, and it demonstrated a unique cell tropism profile in four human cancer cell lines, suggesting that BAAV might have transduction activity distinct from that of other isolates. A murine model of salivary gland gene transfer was used to evaluate the in vivo performance of recombinant BAAV. Recombinant BAAV-mediated gene transfer was 11 times more efficient than that with AAV-2. Overall, these data suggest that vectors based on BAAV could be useful for gene transfer applications.Adeno-associated virus (AAV) was first described as a contaminant of tissue culture-grown simian virus 15 (a simian adenovirus) that is dependent on adenovirus for efficient replication (reviewed in reference 17). This description resulted in its name as well as its classification in the genus Dependovirus. AAV is a member of the Parvoviridae, a virus family characterized by a single-stranded linear DNA genome and a capsid with icosahedral symmetry measuring about 20 nm in diameter. AAV serotype 2 (AAV-2) is the best-characterized isolate and is discussed here as an AAV prototype.The AAV-2 genome consists of a linear single strand of DNA that is 4,780 nucleotides long. Both polarities of DNA are encapsulated by AAV-2 with equal efficiency. The AAV-2 genome contains two open reading frames (ORFs). The rep ORF encodes the nonstructural proteins essential for viral DNA replication, packaging, and AAV targeted integration. The cap ORF encodes three related proteins that assemble to form the virus capsid, while the rep ORF is transcribed from promoters at map units P5 and P19. The rep transcripts contain an intron close to the 3Ј end of the rep ORF that can be alternatively spliced. Thus, the rep ORF is expressed as four partially overlapping proteins, which are referred to according to their molecular weights: Rep78, Rep68, Rep52, and Rep40 (18,20,26). The cap ORF is expressed from a single promoter at P40. By alternative splicing and utilization of an alternative ACG start codon, the cap ORF produces three proteins (VP1 ...

Section: Discussionmentioning

confidence: 91%

Cloning and Characterization of a Bovine Adeno-Associated Virus

Schmidt

Katano

Bossis

et al. 2004

“…A drawback of this uniqueness is that the AAV-5 genotype is hard to pseudotype, which hampered analyses of the contribution of AAV-5 ITRs to vector function in the past and represented a hurdle we had to overcome in the present study (see Results). AAV-5 ITRs are also the only candidates other than AAV-2 for which an intrinsic promoter activity was reported, a property that could be exploited by flanking vector DNAs with AAV-5 ITRs, as was previously suggested (17,52).…”

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confidence: 96%

Liver Transduction with Recombinant Adeno-Associated Virus Is Primarily Restricted by Capsid Serotype Not Vector Genotype

Grimm

Pandey

Nakai

et al. 2006

106

We and others have recently reported highly efficient liver gene transfer with adeno-associated virus 8 (AAV-8) pseudotypes, i.e., AAV-2 genomes packaged into AAV-8 capsids. Here we studied whether liver transduction could be further enhanced by using viral DNA packaging sequences (inverted terminal repeats [ITRs]) derived from AAV genotypes other than 2. To this end, we generated two sets of vector constructs carrying expression cassettes embedding a gfp gene or the human factor IX (hfIX) gene flanked by ITRs from AAV genotypes 1 through 6. Initial in vitro analyses of gfp vector DNA replication, encapsidation, and cell transduction revealed a surprisingly high degree of interchangeability among the six genotypes. For subsequent in vivo studies, we cross-packaged the six hfIX variants into AAV-8 and infused mice via the portal vein with doses of 5 ؋ 10 10 to 1.8 ؋ 10 12 particles. Notably, all vectors expressed comparably high plasma hFIX levels within a dose cohort over the following 6 months, concurrent with the finding of equivalent vector DNA copy numbers per cell. Partial hepatectomies resulted in ϳ80% drops of hFIX levels and vector DNA copy numbers in all groups, indicating genotype-independent persistence of predominantly episomal vector DNA. Southern blot analyses of total liver DNA in fact confirmed the presence of identical and mostly nonintegrated molecular vector forms for all genotypes. We conclude that, unlike serotypes, AAV genotypes are not critical for efficient hepatocyte transduction and can be freely substituted. This corroborates our current model for AAV vector persistence in the liver and provides useful information for the future design and application of recombinant AAV.Gene transfer vectors based on the single-stranded DNA parvovirus AAV (adeno-associated virus) are enormously popular and powerful tools for in vivo delivery of small DNA expression cassettes. The list of human genes carried by such cassettes is growing steadily and spans a wealth of clinically relevant candidates expressing blood coagulation factors (28), the cystic fibrosis transmembrane conductance regulator (18), or dystrophin (32), among many others. Most recently, AAV vectors have begun to attract particular attention for delivery of short hairpin RNAs, and indeed, with a packaging capacity of 5 kb DNA and the promise to mediate safe and persistent gene transfer, they appear as bona fide tools for in vivo RNA interference applications (27).A major reason for AAV's broad and growing appeal is the feasibility to pseudotype the recombinant viral DNA, i.e., to generate particles in which the vector DNA (genotype) and the viral capsid (serotype) differ in their AAV origins (23). The resulting hybrid particles are typically characterized by unique receptor tropisms and are distinctly recognized by the host immune system, as determined by the capsid. Thus, the pseudotyping approach has dramatically extended the array of cells and tissues susceptible to AAV gene transfer and relieved some of the concerns associated with t...

“…However, as shown in Fig. 1B, and as we have shown previously (7,8,(10)(11)(12), substantial levels of a Rep40-like protein product are generated during both AAV5 and goat AAV infections of 293 cells (Fig. 1B, lanes 1 and 2), despite the relatively low levels (Ͻ5%) of splicing from upstream transcripts (Fig.…”

mentioning

confidence: 92%

“…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.…”

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confidence: 99%

See 1 more Smart Citation

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

Farris

Pintel

2010

Self Cite

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...