Complete in vitro reconstitution of adeno-associated virus DNA replication requires the minichromosome maintenance complex proteins.
Adeno-associated virus (AAV) replicates its DNA exclusively by a leading-strand DNA replication mechanism and requires coinfection with a helper virus, such as adenovirus, to achieve a productive infection. In previous work, we described an in vitro AAV replication assay that required the AAV terminal repeats (the origins for DNA replication), the AAV Rep protein (the origin binding protein), and an adenovirus-infected crude extract. Fractionation of these crude extracts identified replication factor C (RFC), proliferating cell nuclear antigen (PCNA), and polymerase delta as cellular enzymes that were essential for AAV DNA replication in vitro. Here we identify the remaining factor that is necessary as the minichromosome maintenance (MCM) complex, a cellular helicase complex that is believed to be the replicative helicase for eukaryotic chromosomes. Thus, polymerase delta, RFC, PCNA, and the MCM complex, along with the virally encoded Rep protein, constitute the minimal protein complexes required to reconstitute efficient AAV DNA replication in vitro. Interfering RNAs targeted to MCM and polymerase delta inhibited AAV DNA replication in vivo, suggesting that one or more components of the MCM complex and polymerase delta play an essential role in AAV DNA replication in vivo as well as in vitro. Our reconstituted in vitro DNA replication system is consistent with the current genetic information about AAV DNA replication. The use of highly conserved cellular replication enzymes may explain why AAV is capable of productive infection in a wide variety of species with several different families of helper viruses.
Department of Molecular Genetics and Microbiology, University of Florida, 1376 Mowry Rd., Gainesville, FL 32610, USA. nash@ufl.edu
Kevin
K
Nash
Weijun
W
Chen
Nicholas
N
Muzyczka
D002458:Cell Fractionation; D045424:Complex Mixtures / Q000737:chemistry; D004265:DNA Helicases / Q000302:isolation & purification / Q000378:metabolism*; D004261:DNA Replication / Q000502:physiology*; D004279:DNA, Viral / Q000096:biosynthesis*; D000229:Dependovirus / Q000502:physiology*; D020868:Gene Silencing; D066298:In Vitro Techniques
D045424:Complex Mixtures; D004279:DNA, Viral; D004265:DNA Helicases