Adeno-associated virus DNA replication: Nonunit-length molecules

Abstract

Nonunit-length adeno-associated virus (AAV) DNA molecules which incorporate radiolabel during a short pulse were isolated from infected cells and characterized with regard to structure and to their potential role as replicative intermediates. Two classes of molecules were observed. (1) Dimer length duplex molecules containing the entire AAV genome were shown to be precursors to mature unit length AAV DNA. About one-third of the single strands from these molecules were dimers, the rest being of unit length. Some of these dimeric duplex molecules contain a hairpinned terminal structure formed by folding over of the terminal repetition from the right end of the AAV genome. (2) Smaller than unit-length molecules contain only the terminal 10–25% of the AAV genome and are not processed to unit-length genomes. They have one natural AAV terminus and a hairpinned internal terminus. Both classes of AAV molecules can be generated from a replication model utilizing the 3′-OH end of a hairpinned palindromic AAV terminal sequence as a primer for daughter-strand synthesis. The short, defective molecules result from a template strand switch to either the complementary strand of the parental duplex or to the newly synthesized complementary strand.