Cellular proteins required for adeno-associated 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 adenovirus (Ad)-infected HeLa cell cytoplasmic extract to catalyze origin of replication-dependent AAV DNA replication. However, in addition to fully permissive DNA replication, which occurs in the presence of Ad, AAV is also capable of partially permissive DNA replication in the absence of the helper virus in cells that have been treated with genotoxic agents. Limited DNA replication also occurs in the absence of Ad during the process of establishing a latent infection. In an attempt to isolate uninfected extracts that would support AAV DNA replication, we discovered that HeLa cell extracts grown to high density can occasionally display as much in vitro replication activity as Ad-infected extracts. This finding confirmed previous genetic analyses which suggested that no Ad-encoded proteins were absolutely essential for AAV DNA replication and that the uninfected extracts should be useful for studying the differences between helper-dependent and helper-independent AAV DNA replication. Using specific chemical inhibitors and monoclonal antibodies, as well as the fractionation of uninfected HeLa extracts, we identified several of the cellular enzymes involved in AAV DNA replication. They were the single-stranded DNA binding protein, replication protein 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-strand DNA synthesis, we found no requirement for DNA polymerase alpha-primase. 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 known to be accessory proteins for polymerase delta and epsilon, we attempted to reconstitute AAV DNA replication by substituting either purified polymerase delta or polymerase epsilon for fraction IIA. These attempts 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) gel electrophoresis that all of the intermediates commonly seen in vivo are generated in the in vitro system. The only difference was an accumulation of single-stranded DNA in vivo that was not seen in vitro. The 2D data also suggested that although both Rep78 and Rep68 can generate dimeric intermediates in vitro, Rep68 is more efficient in processing dimers to monomer duplex DNA. Regardless of the Rep that was used in vitro, we found evidence of an interaction between the elongation complex and the terminal repeats. Nicking at the terminal repeats of a replicating molecule appeared to be inhibited until after elongation was complete.
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Department of Genetics and Molecular Microbiology, State University of New York at Stony Brook, 11794, USA.
McDonald
W F
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Zolotukhin
I
I
Melendy
T
T
Waga
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S
Stillman
B
B
Muzyczka
N
N
D000256:Adenoviridae / Q000502:physiology*; D000818:Animals; D002457:Cell Extracts; D002458:Cell Fractionation; D002460:Cell Line; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis*; D004268:DNA-Binding Proteins / Q000378:metabolism; D000229:Dependovirus / Q000235:genetics* / Q000502:physiology; D019281:Dimerization; D004587:Electrophoresis, Agar Gel; D015180:Electrophoresis, Gel, Two-Dimensional; D006367:HeLa Cells; D006801:Humans; D009532:Nickel; D009692:Nucleic Acid Heteroduplexes; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D011506:Proteins / Q000502:physiology*; D018411:Spodoptera; D013997:Time Factors; D014764:Viral Proteins / Q000378:metabolism; D014779:Virus Replication
D002457:Cell Extracts; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D009692:Nucleic Acid Heteroduplexes; D019384:Nucleic Acid Synthesis Inhibitors; D011506:Proteins; D014764:Viral Proteins; C068734:rep proteins, Adeno-associated virus 2; D009532:Nickel