Cloning and Characterization of Adeno-Associated Virus Type 5

The adeno-associated viruses (AAV) are members of the parvovirus family and are helper virus dependent for replication and gene expression. Of the six serotypes of AAV which have been identified in primates, only three (AAV type 2 [AAV2], AAV3, and AAV5) have been isolated from humans. While AAV4 can infect human and rat cells in culture, serological data suggest that its natural host is the African green monkey (2). Several of these serotypes have been recently cloned, expanding our knowledge of basic AAV biology. The most extensively studied of these serotypes is AAV type 2 (AAV2). The genome of AAV2 consists of a single strand of DNA 4,680 nucleotides in length (29, 44). The terminal sequence is organized as interrupted palindromes that can form a characteristic hairpin structure which is energetically stable. Two large open reading frames (ORFs) have been identified within the genome. The left ORF encodes the nonstructural replication initiator polypeptides (Rep). The Rep proteins regulate AAV DNA replication, transcription, and the accumulation of single-stranded progeny genomes (4, 5, 8, 20, 32, 39, 46, 47). The importance of the Rep proteins is emphasized by their high degree of conservation. The Rep ORFs of AAV2, AAV3, AAV4, and AAV6 are approximately 90% identical, with most of the changes being conserved amino acid substitutions. The Rep ORF can encode four different polypeptides from two promoters, with a splice variant in each. The p5-promoted Rep proteins (Rep78 and Rep68) can bind to DNA in a sequence-specific manner and nick duplex inverted terminal repeats (ITRs) in a site- and strand-specific manner. This cleavage reaction occurs at the terminal resolution site (trs) within the ITR and permits the replication of the ends of the linear genome. The DNA-nicking activity requires the binding of the Rep proteins to tandem repeats of a GAGY motif present in the ITRs, structures at the ends of the viral genome (6, 8, 34, 36, 42). DNA binding and terminal resolution are central to the preferential integration of the AAV genome into the AAVS1 locus on the q arm of human chromosome 19 (26, 38, 48, 50). The ITR serves as the origin of viral DNA replication. Within the ITR, two elements are described which are central for this function: a GAGC repeat motif and the trs. While a number of sequence differences within the ITR have been reported in the different serotypes, the ability to fold into a hairpin conformation and the existence of a Rep binding site and trs are conserved. Two additional Rep proteins are produced from an internal promoter in the Rep ORF at map unit 19 and are referred to by their apparent molecular masses: Rep52 and Rep40. While these two proteins lack the DNA binding ability of Rep68 and Rep78 they have recently been shown to possess ATP-dependent DNA helicase activity (40). All four of the Rep proteins possess nucleoside triphosphate (NTP) binding activities, DNA and RNA helicase activities (22, 23, 25, 51), and nuclear localization signals (25, 54). In addition to the role of Rep proteins in the life cycle of AAV, Rep expression results in a characteristic phenotype. Overexpression of Rep68 and Rep78 can either negatively or positively affect transcription of heterologous promoters, inhibit cellular transformation by papillomavirus or by adenovirus E1a plus an activated ras oncogene (17–19, 24, 27, 28, 40), and inhibit progression through the cell cycle (16, 53). While some of these activities may be mediated by the direct interaction of Rep with DNA, other effects may result from interactions with cellular regulatory proteins. Rep52 and Rep78 have recently been shown to bind to the cyclic AMP-dependent protein kinase (PKA) and to a novel PKA-like kinase (PrKX). This interaction results in an inhibition of the protein kinase activity and an alteration in gene expression in vivo (11). The AAV2 virion is a nonenveloped, icosahedral particle approximately 25 nm in diameter. It consists of three related proteins referred to as VP1, VP2, and VP3. These proteins are found in a ratio of approximately 1:1:10, respectively (3), and are each derived from the right-hand ORF. These proteins differ from each other as a result of alternative splicing and from the use of an atypical start codon. The cap ORF is less conserved among serotypes than the rep ORF. Protein sequence alignment of the different serotypes indicates blocks of divergent and conserved sequences. Comparison with the X-ray crystallographic structure determined for canine parvovirus indicates that some of these sequences may exist on the exterior of the virion, thus accounting for the altered tissue tropism among the different serotypes (10, 33, 35) and the hemagglutination activity of AAV4. Recent work has demonstrated that heparan sulfate proteoglycans (HSP) can play a role in virus binding and uptake, presumably through an interaction with the capsid proteins (45). AAV has a number of characteristics which make it an attractive vector for gene therapy (21). Transduction with AAV vectors results in stable, long-term transgene expression in vivo in skeletal muscle, photoreceptors, liver, and central nervous system neuronal cells (14, 31, 43, 52). The ITRs have been shown to be the only cis element required for rescue, replication, packaging, and integration (37). Since no viral genes are included in the recombinant particle, a host immune response directed against viral genes expressed in vivo is prevented. In contrast to other viral vectors, AAV is naturally replication defective and is classified as nonpathogenic. A comparison of human AAV2, AAV3, and AAV5 by DNA Southern blot hybridization indicates a high degree of homology between AAV2 and AAV3 compared to that between both types 2 and 3 and AAV5 (1). In addition, serological data have shown that seroconversion occurs in early childhood with AAV2 and AAV3 but not until 15 to 20 years of age with AAV5 (15), further suggesting that AAV5 is a more distantly related member of the AAV family. While the seroconversion pattern for AAV2 and AAV3 closely follows that of adenovirus, the later seroconversion for AAV5 temporally occurs closer to and follows that for herpes simplex virus (HSV). This suggests that HSV, instead of adenovirus, may serve as the natural helper virus for AAV5. While adenovirus type 12 has been shown to function as a helper virus, the circumstantial association of AAV5 and HSV suggests an altered tissue tropism for AAV5 compared to that of types 2 and 3. To better understand the nature of AAV5 and to determine its usefulness as a vector for gene transfer, the viral genome was cloned and sequenced and recombinant virus was produced using the AAV5 rep and cap genes.