IN-VITRO REPLICATION OF ADENOASSOCIATED VIRUS-DNA - ENHANCEMENT BY EXTRACTS FROM ADENOVIRUS-INFECTED HELA-CELLS

Previously we have described an in vitro assay for the replication of adeno-associated virus type 2 (AAV2) DNA. Addition of the AAV2 nonstru ctural protein Rep68 to an extract from uninfected cells supports the replication of linear duplex AAV DNA. In this report, we examine repli cation of linear duplex AAV DNA in extracts from either uninfected or adenovirus (Ad)-infected HeLa cells. The incorporation of radiolabeled nucleotides into full-length linear AAV DNA is SO-fold greater in ext racts from Ad-infected cells than in extracts from uninfected cells. I n addition, the majority of the labeled full-length AAV DNA molecules synthesized in the Ad-infected extract have two newly replicated stran ds, whereas the majority of labeled full-length AAV DNA molecules synt hesized in the uninfected extract have only one newly replicated stran d. The numbers of replication initiations on original templates in the two assays are approximately the same; however, replication in the ca se of the Ad-infected cell extract is much more likely to result in th e synthesis of a full-length AAV DNA molecule, Most of the newly repli cated molecules in the assay using uninfected cell extracts are in the form of stem-loop structures. We hypothesize that Ad infection provid es a helper function related to elongation during replication by a sin gle-strand displacement mechanism. In the assay using the uninfected H eLa cell extract, replication frequently stalls before reaching the en d of the genome, causing the newly synthesized strand to be displaced from the template, with a consequent folding on itself and replication back through the inverted terminal repeat, using itself as a template . In support of this conjecture, replication in the uninfected cell ex tract of shorter substrate molecules is more efficient, as measured by incorporation of radiolabeled nucleotides into full-length substrate DNA, In addition, when shorter substrate molecules are used as the tem plate in the uninfected HeLa cell assay, a greater proportion of the l abeled full-length substrate molecules contain two newly replicated st rands. Shorter substrate molecules have no replicative advantage over full-length substrate molecules in the assay using an extract from Ad- infected cells.