CELLULAR PROTEINS REQUIRED FOR ADENOASSOCIATED VIRUS-DNA REPLICATION IN THE ABSENCE OF ADENOVIRUS COINFECTION

We previously reported the development of an in vitro adeno-associated virus (AAV) DNA replication system, The system required one of the p5 Rep proteins encoded by AAV (either Rep78 or Rep68) and a crude adeno virus (Ad)-infected HeLa cell cytoplasmic extract to catalyze origin o f replication-dependent AAV DNA replication. However, in addition to f ully permissive DNA replication, which occurs in the presence of Ad, A AV is also capable of partially permissive DNA replication in the abse nce of the helper virus in cells that have been treated with genotoxic agents. Limited DNA replication also occurs in the absence of Ad duri ng the process of establishing a latent infection, In an attempt to is olate uninfected extracts that would support AAV DNA replication, we d iscovered that HeLa cell extracts grown to high density can occasional ly display as much in vitro replication activity as Ad-infected extrac ts, This finding confirmed previous genetic analyses which suggested t hat no Ad-encoded proteins were absolutely essential for AAV DNA repli cation and that the uninfected extracts should be useful for studying the differences between helper-dependent and helper-independent AAV DN A replication, Using specific chemical inhibitors and monoclonal antib odies, as well as the fractionation of uninfected HeLa extracts, we id entified several of the cellular enzymes involved in AAV DNA replicati on, They were the single-stranded DNA. binding protein, replication pr otein A (RFA), the 3' primer binding complex replication factor C (RFC ), and proliferating cell nuclear antigen (PCNA). Consistent with the current model for AAV DNA replication, which requires only leading-str and DNA synthesis, we found no requirement for DNA polymerase alpha-pr imase. AAV DNA replication could be reconstituted with purified Rep78, RPA RFC, and PCNA and a phosphocellulose chromatography fraction (IIA ) that contained DNA polymerase activity. As both RFC and PCNA are kno wn to be accessory proteins for polymerase delta and epsilon, we attem pted to reconstitute AAV DNA replication by substituting either purifi ed polymerase delta or polymerase epsilon for fraction IIA. These atte mpts were unsuccessful and suggested that some novel cellular protein or modification was required for AAV DNA replication that had not been previously identified. Finally, we also further characterized the in vitro DNA replication assay and demonstrated by two-dimensional (2D) g el electrophoresis that all of the intermediates commonly seen in vivo are generated in the in vitro system. The only difference was an accu mulation of single-stranded DNA in vivo that was not seen in vitro. Th e 2D data also suggested that although both Rep78 and Rep68 can genera te dimeric intermediates in vitro, Rep68 is more efficient in processi ng dimers to monomer duplex DNA, Regardless of the Rep that was used i n vitro, we found evidence of an interaction between the elongation co mplex and the terminal repeats, Nicking at the terminal repeats of a r eplicating molecule appeared to be inhibited until after elongation wa s complete.