Cloning of an avian adeno-associated virus (AAAV) and generation of recombinant AAAV particles

Abstract

Recent studies have proposed that adeno-associated viruses (AAVs) are not evolutionarily linked to other mammalian autonomous parvoviruses but are more closely linked to the autonomous parvoviruses of birds. To better understand the relationship between primate and avian AAVs (AAAVs), we cloned and sequenced the genome of an AAAV (ATCC VR-865) and generated recombinant AAAV particles. The genome of AAAV is 4,694 nucleotides in length and has organization similar to that of other AAVs. The entire genome of AAAV displays 56 to 65% identity at the nucleotide level with the other known AAVs. The AAAV genome has inverted terminal repeats of 142 nucleotides, with the first 122 forming the characteristic T-shaped palindromic structure. The putative Rep-binding element consists of a tandem (GAGY)(4) repeat, and the putative terminal resolution site (trs), CCGGT/CG, contains a single nucleotide substitution relative to the AAV(2) trs. The Rep open reading frame of AAAV displays 50 to 54% identity at the amino acid level with the other AAVs, with most of the diversity clustered at the carboxyl and amino termini. Comparison of the capsid proteins of AAAV and the primate dependoviruses indicate that divergent regions are localized to surface-exposed loops. Despite these sequence differences, we were able to produce recombinant AAAV particles carrying a lacZ reporter gene by cotransfection in 293T cells and were able to examine transduction efficiency in both chicken primary cells and several cell lines. Our findings indicate that AAAV is the most divergent AAV described to date but maintains all the characteristics unique to the genera of dependovirus.

FIG. 1.

Southern blot analysis of AAAV nuclease-resistant particles in 293T and LMH cells. (A) 293T cells were transfected with pAAAV alone (lane 3), pAAAV plus pAd12 (lane 2), and pAAAV plus infection with wt Ad (lane 1). (B) LMH cells were transfected with pAAAV alone (lane 2) or pAAAV plus infection with FAV1 (lane 1). Viral DNA was isolated as described in Materials and Methods and fractionated on agarose gel before Southern blot analysis with a ³²P-labeled pAAAV DNA.

FIG. 2.

AAAV ITR. The sequence of the ITR is shown in the hairpin conformation. The putative Rep binding site is boxed, while the putative trs is underlined and the cleavage site is indicated by an arrow. RBE, Rep binding element.

FIG. 3.

Sequence of the AAAV genome. The genomes of AAAV, AAV2, AAV4, and AAV5 were aligned by using Clustal W. The sequences of the ITRs are presented in italics. The putative trs is indicated by a vertical arrow, and the putative Rep binding site is underlined. Proposed transcription factor binding sites and the polyadenylation signal are also underlined. Proposed transcription initiation sites of the p5, p19, and p40 promoters and splice donor and acceptor sites are indicated by horizontal arrows. Initiation and termination codons are presented in bold letters. USF, upstream factor.

FIG. 3.

Sequence of the AAAV genome. The genomes of AAAV, AAV2, AAV4, and AAV5 were aligned by using Clustal W. The sequences of the ITRs are presented in italics. The putative trs is indicated by a vertical arrow, and the putative Rep binding site is underlined. Proposed transcription factor binding sites and the polyadenylation signal are also underlined. Proposed transcription initiation sites of the p5, p19, and p40 promoters and splice donor and acceptor sites are indicated by horizontal arrows. Initiation and termination codons are presented in bold letters. USF, upstream factor.

FIG. 3.

Sequence of the AAAV genome. The genomes of AAAV, AAV2, AAV4, and AAV5 were aligned by using Clustal W. The sequences of the ITRs are presented in italics. The putative trs is indicated by a vertical arrow, and the putative Rep binding site is underlined. Proposed transcription factor binding sites and the polyadenylation signal are also underlined. Proposed transcription initiation sites of the p5, p19, and p40 promoters and splice donor and acceptor sites are indicated by horizontal arrows. Initiation and termination codons are presented in bold letters. USF, upstream factor.

FIG. 3.

Sequence of the AAAV genome. The genomes of AAAV, AAV2, AAV4, and AAV5 were aligned by using Clustal W. The sequences of the ITRs are presented in italics. The putative trs is indicated by a vertical arrow, and the putative Rep binding site is underlined. Proposed transcription factor binding sites and the polyadenylation signal are also underlined. Proposed transcription initiation sites of the p5, p19, and p40 promoters and splice donor and acceptor sites are indicated by horizontal arrows. Initiation and termination codons are presented in bold letters. USF, upstream factor.

FIG. 3.

Sequence of the AAAV genome. The genomes of AAAV, AAV2, AAV4, and AAV5 were aligned by using Clustal W. The sequences of the ITRs are presented in italics. The putative trs is indicated by a vertical arrow, and the putative Rep binding site is underlined. Proposed transcription factor binding sites and the polyadenylation signal are also underlined. Proposed transcription initiation sites of the p5, p19, and p40 promoters and splice donor and acceptor sites are indicated by horizontal arrows. Initiation and termination codons are presented in bold letters. USF, upstream factor.

FIG. 3.

Sequence of the AAAV genome. The genomes of AAAV, AAV2, AAV4, and AAV5 were aligned by using Clustal W. The sequences of the ITRs are presented in italics. The putative trs is indicated by a vertical arrow, and the putative Rep binding site is underlined. Proposed transcription factor binding sites and the polyadenylation signal are also underlined. Proposed transcription initiation sites of the p5, p19, and p40 promoters and splice donor and acceptor sites are indicated by horizontal arrows. Initiation and termination codons are presented in bold letters. USF, upstream factor.

FIG. 4.

Comparisons of rep and cap ORFs. The rep and cap ORFs of AAAV, AAV2, AAV4, AAV5, and GPV (GP) were aligned by using Clustal W. Identical amino acids are indicated by a dot. Dashes indicate gaps in the sequence added by the alignment program. (A) Horizontal arrows indicate the initiator codon of the p5 and p19 Rep proteins. The Rep endonuclease site established by Tyr155 and the tetrahedrally coordinated Asp24, Glu83, His90, and His92 are presented in bold letters and are overlined by an asterisk. The region important for Rep multimerization, the ATP binding site, and the basic amino acids of the nuclear localization signal are underlined. The zinc finger motifs in the carboxy terminus are underlined and the coordinating cystine and histidine residues are indicated by dots. (B) The theoretical initiator codons of VP2 and VP3 are indicated in bold letters. Regions that have been proposed to be on the surface of AAV2 are underlined and divergent regions are boxed. The heparin binding region in the capsid of AAV2 is also indicated.

FIG. 5.

Vector constructs for the generation of recombinant AAAV and transduction of chicken fibroblasts. (A) Wild-type AAAV, vector plasmid (pA3Vβ-Gal), and production yields of rAAAV using helper plasmids providing the rep gene under the control of CMV, MMTV, or the native P5 promoter. The helper plasmids pCA3VRC, pMA3VRC, and pA3VRC were individually cotransfected with pA3Vβ-Gal and an adenovirus helper plasmid in 293T cells, and rAAAV was produced as described in Materials and Methods. The number of rAAAV genomes produced in each group was determined by quantitative PCR and is expressed as DNase-resistant particles per cell (DRP/cell). (B) Relative transduction efficiency of primary chicken embryonic fibroblasts (CEF) and immortalized chicken embryonic fibroblasts (DF1) with equal particles of rAAAV expressing LacZ.