Purification of Host Cell Enzymes Involved in Adeno-Associated Virus DNA Replication

Abstract

Adeno-associated virus (AAV) replicates its DNA by a modified rolling-circle mechanism that exclusively uses leading strand displacement synthesis. To identify the enzymes directly involved in AAV DNA replication, we fractionated adenovirus-infected crude extracts and tested them in an in vitro replication system that required the presence of the AAV-encoded Rep protein and the AAV origins of DNA replication, thus faithfully reproducing in vivo viral DNA replication. Fractions that contained replication factor C (RFC) and proliferating cell nuclear antigen (PCNA) were found to be essential for reconstituting AAV DNA replication. These could be replaced by purified PCNA and RFC to retain full activity. We also found that fractions containing polymerase ␦, but not polymerase or ␣, were capable of replicating AAV DNA in vitro. This was confirmed when highly purified polymerase ␦ complex purified from baculovirus expression clones was used. Curiously, as the components of the DNA replication system were purified, neither the cellular single-stranded DNA binding protein (RPA) nor the adenovirus-encoded DNA binding protein was found to be essential for DNA replication; both only modestly stimulated DNA synthesis on an AAV template. Also, in addition to polymerase ␦, RFC, and PCNA, an as yet unidentified factor(s) is required for AAV DNA replication, which appeared to be enriched in adenovirus-infected cells. Finally, the absence of any apparent cellular DNA helicase requirement led us to develop an artificial AAV replication system in which polymerase ␦, RFC, and PCNA were replaced with T4 DNA polymerase and gp32 protein. This system was capable of supporting AAV DNA replication, demonstrating that under some conditions the Rep helicase activity can function to unwind duplex DNA during strand displacement synthesis.

Identification of Rep-Associated Factors in Herpes Simplex Virus Type 1-Induced Adeno-Associated Virus Type 2 Replication Compartments

“…Importantly, the final list of proteins also included several HSV-1 proteins, some of which were previously described as implicated in HSV-1-induced AAV replication. Interestingly, a significant number of proteins listed in Table 1 were previously found in a similar proteomic analysis performed during AAV and adenovirus coinfection, and some of them were shown to be involved directly in AAV replication (40)(41)(42).…”

“…Adenovirus-induced AAV replication is performed mainly by cellular factors, and previous studies have shown that PCNA, RFC, Pol ␦, RPA, and MCM constitute the minimal set of cellular proteins able to drive AAV DNA replication in vitro (40,41,44). Most of these factors were also found in a recent proteomic analysis of Rep-associated proteins in adenovirus-and AAV-coinfected cells (42) (Fig.…”

“…More recently, the adenovirus proteins E1b55k and E4orf6 were shown to stimulate AAV genome replication by degrading the cellular Mre11/ Rad50/Nbs1 (MRN) complex that restricts AAV genome replication during adenovirus coinfection (32). The concept that AAV genome replication can rely mostly, if not uniquely, on direct help from cellular factors was further strengthened by the demonstration that purified proteins such as replication protein A (RPA), replication factor C (RFC), proliferating cell nuclear antigen (PCNA), minichromosome maintenance (MCM) proteins, and DNA polymerase ␦ (Pol ␦) were sufficient to replicate the AAV genome in vitro in the presence of Rep (40)(41)43). The involvement of these cellular proteins during AAV genome replication was also confirmed by the proteomic analysis of factors associated with Rep proteins during adenovirus-induced AAV replication (42).…”

Promyelocytic Leukemia Protein Is a Cell-Intrinsic Factor Inhibiting Parvovirus DNA Replication

“…PML affects the replication of many viruses through sequestration of viral components (17,18) or prevention of transcription (15,16); however, we are not aware of any other studies demonstrating a PML effect specifically on genome replication. AAV genome replication relies on the cellular replication machinery, including DNA polymerase ␦, replication factor C, proliferating cell nuclear antigen, and replication protein A (RPA) (36,37), although second-strand synthesis has not been examined directly. In the future, it will be interesting to investigate whether any of these factors are involved in PML's effect on rAAV second-strand synthesis.…”

The Role of DNA Repair Pathways in Adeno-Associated Virus Infection and Viral Genome Replication / Recombination / Integration

“…Its dephosphorylation by T-cell protein tyrosine phosphatase (TC-PTP) dissociates FKBP52 from AAV-ITR, allowing the formation of ds genomes (Qing et al, 2003). In vitro AAV replication studies have identified the DNA polymerase that catalyzes second-strand synthesis as DNA polymerase δ (Nash et al, 2007), which is a polymerase that fills a single-stranded DNA gap created during the nuclear excision repair (Torres-Ramos et al, 1997). Physical association of DNA polymerase δ and AAV genome has also been demonstrated (Jurvansuu et al, 2005).…”