IN-VITRO REPLICATION OF ADENOASSOCIATED VIRUS-DNA

The study of eukaryotic viral DNA replication in vitro has led to the identification of cellular enzymes involved in DNA replication. Adeno- associated virus (AAV) is distinct from previously reported systems in that it is believed to replicate entirely by leading-strand DNA synth esis and requires coinfection with adenovirus to establish completely permissive replication. In previous work, we demonstrated that two of the AAV nonstructural proteins, Rep78 and -68, are site-specific endon ucleases and DNA helicases that are capable of resolving covalently cl osed AAV termini, a key step in AAV DNA replication. We have now clone d the AAV nonstructural proteins Rep78, Rep68, and Rep52 in the baculo virus expression system. Using the baculovirus-expressed proteins, we have developed an efficient in vitro AAV DNA replication system which mimics the in vivo behavior of AAV in every respect. With no-end AAV D NA as the starting substrate, the reaction required an adenovirus-infe cted cell extract and the presence of either Rep78 or Rep68. Rep52, as expected, did not support DNA replication. A mutant in the AAV termin al resolution site (Irs) was defective for DNA replication in the in v itro assay. Little, if any, product was formed in the absence of the a denovirus-infected HeLa cell extract. In general, uninfected HeLa extr acts were less efficient in supporting AAV DNA replication than adenov irus-infected extracts. Thus, the requirement for adenovirus infection in vivo was partially duplicated in vitro. The reduced ability of uni nfected HeLa extracts to support complete DNA replication was not due to a defect in terminal resolution but rather to a defect in the reini tiation reaction or in elongation. Rep78 produced a characteristic mon omer-dimer pattern of replicative intermediates, but surprisingly, Rep 68 produced little, if any, dimer replicative form. The reaction had a significant lag (30 min) before incorporation of P-32-deoxynucleoside triphosphate could be detected in DpnI-resistant monomer replicative form and was linear for at least 4 h after the lag. The rate of incorp oration in the reaction was comparable? to that in tile simian virus 4 0 in vitro system. Replication of the complete AAV DNA molecule was de monstrated by the following criteria. (i) Most of the monomer and dime r product DNAs were completely resistant to digestion with DpnI. (ii) Virtually all of the starting substrate was converted to heavy-light o r heavy-heavy product DNA in the presence of bromo-dUTP when examined on CsCl density gradients. (iii) Both flip and flop orientations of th e terminal repeat were present in the product DNA, suggesting that eac h of the terminal repents underwent at least two rounds of terminal re solution. The availability of an efficient in vitro AAV DNA replicatio n assay should help to identify cellular proteins required exclusively for leading-strand DNA synthesis.