B AAV Genome Structure

The AAV2 DNA genome (22,63) is 4681 nucleotides long, and includes 1 copy of the 145-nucleotide long inverted terminal repeat (ITR) at each end and a unique sequence region of 4391 nucleotides long that contains 2 main open reading frames for the rep and cap genes (Fig. 2). The unique region contains 3 transcription promoters, p5, p19, and p40, which are used to express the rep and cap genes. The ITR sequences are required in cis to provide functional origins of replication (ori), as well as signals for encapsidation, integration into the cell genome, and rescue from either host cell chromosomes or recombinant plasmids. The genomes of other AAV serotypes have been sequenced and appear to have a structure similar to AAV2 (24-26,30).

The rep gene is transcribed from 2 promoters, p5 and p19, to generate 2 families of transcripts and 2 families of rep proteins (Fig. 1). In addition, splicing of these mRNAs yields 2 different carboxyl terminal regions in the rep proteins. The capsid gene is expressed from transcripts from the p40 promoter that accumulate as two 2.3-kb mRNAs that are alternately spliced. The majority 2.3-kb transcript codes for the VP3 protein initiated from a consensus AUG initiation codon. However, at about a 10-fold lower frequency, translation of this transcript also occurs slightly upstream at a nonconsensus ACG initiation codon to yield VP2. The minority 2.3-kb mRNA is spliced to an alternate 3' donor site 30 nucleotides upstream, and this retains an AUG codon that is used to initiate translation of VP1. Thus, VP1 and VP2 have the same poly-peptide sequence as VP3 but have additional amino-terminal sequences. This elegant arrangement results in generation of VP1, VP2, and VP3 in ratios of about 1:1:8, which is the same as the ratio of these proteins in the viral particle.

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