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. 1998 Apr;72(4):2777-87.
doi: 10.1128/JVI.72.4.2777-2787.1998.

Cellular proteins required for adeno-associated virus DNA replication in the absence of adenovirus coinfection

T H Ni  1 , W F McDonald, I Zolotukhin, T Melendy, S Waga, B Stillman, N Muzyczka
Affiliations

Cellular proteins required for adeno-associated virus DNA replication in the absence of adenovirus coinfection

T H Ni et al. J Virol. 1998 Apr.

Abstract

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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Figures

FIG. 1
FIG. 1
Mechanism of AAV DNA replication. The diagram at the top illustrates a working model for the formation of replicative AAV DNA intermediates when NE DNA is used as a template for replication in vitro. Designations mE, mT, dE, dT, and ss stand for monomer extended, monomer turnaround, dimer extended, dimer turnaround, and single-stranded DNA, respectively. The series of reactions on the left depict steps involved in the generation of monomer duplex and single-stranded DNA species, and those on the right depict the steps involved in the formation of dimer duplex DNA. The boxed region illustrates the steps in terminal resolution on one or both ends of NE DNA. In contrast to the in vitro reaction with NE substrate, in a normal virus infection a single-stranded input DNA molecule (ss) is converted to a monomer turnaround (mT) form, which is then converted via terminal resolution to a monomer extended form (mE). See text for further details.
FIG. 2
FIG. 2
Abilities of Ad(S), HeLa(S), and HeLa(H) extracts to support AAV DNA replication in the standard in vitro AAV DNA replication assay containing partially purified Rep78 (ssDNA fraction; 0.25 μg). Two different high-density extracts are shown. The amount of incorporation into DNA product was measured by DE-81 filter binding assay.
FIG. 3
FIG. 3
Effects of neutralizing monoclonal antibodies on AAV DNA synthesis in vitro. Standard DNA replication reactions mixtures containing uninfected HeLa cell crude extract (255 μg) and crude baculovirus-expressed Rep78 (8 μg) were incubated with monoclonal antibodies against T antigen (TAg; lanes 2 to 4; 0.4, 0.8, and 2.4 μg), RFC (lanes 5 to 8; 0.5, 1.0, 2.0, and 3.0 μg), RPA (lanes 10 to 13; 0.75, 1.5, 3.0, and 4.5 μg), PCNA (lanes 14 to 17; 0.4, 0.8, 1.6, and 2.4 μg), or DNA pol α (lanes 18 to 21; 0.4, 0.8, 1.6, and 2.4 μg) for 2 h at 37°C. DNA products were digested with DpnI and analyzed on a 0.8% agarose gel. md and dd indicate monomer duplex and dimer duplex DNA species that are resistant to DpnI digestion, respectively. DNA products that are sensitive to DpnI digestion are marked with a line on the left. Lanes 1 and 9 represent reaction mixtures incubated without antibody.
FIG. 4
FIG. 4
Time course of in vitro AAV DNA replication comparing purified and crude Rep78. The standard DNA replication assay contained 200 μg of Ad-infected HeLa cell extract and either crude baculovirus extract containing Rep78 (8 μg) or partially purified Rep78 (ssDNA fraction; 0.4 μg). Shown is the amount of dAMP incorporated per 30-μl standard reaction as determined from counting of the DpnI-resistant monomer and dimer RF species at each time point.
FIG. 5
FIG. 5
Scheme for fractionation of crude uninfected HeLa cell extracts by phosphocellulose chromatography. The presence or absence of previously characterized replication factors in each fraction is indicated (57).
FIG. 6
FIG. 6
(A) Reconstitution of AAV DNA replication in vitro using fractionated HeLa cell extracts. Standard DNA replication reaction mixtures (15 μl) contained the following concentrations of the indicated P-cell fractions and purified DNA replication factors: 50 μg of human RPA per ml, 8 μg of PCNA per ml, 72 μg of RFC (fraction IV per ml, 5 mg of fraction II per ml, 0.62 mg of fraction IIA per ml, 0.19 mg of fraction IIB per ml, 0.31 mg of fraction IIC per ml, and 0.11 mg of fraction IID per ml. Where indicated, the reaction mixtures contained, in 15 μl, 0.003 U of pol α, 0.02 U of pol δ, and 0.014 U of pol ɛ. Reaction products were processed and fractionated on 0.8% agarose gels as described in Materials and Methods. md and dd indicate monomer duplex and dimer duplex DNA species that are resistant to DpnI digestion, respectively. DNA products sensitive to DpnI digestion are denoted with a line at the left. Molecular weight markers are λ DNA molecules digested with BstEII. (B) Same as Fig. 6A except that fraction I was used at a final concentration of 5.3 mg/ml, topo I was used at 4 μg/ml, and topo II was used at 1.8 μg/ml. AQ, mono-Q fraction derived from P-cell fraction IIA.
FIG. 7
FIG. 7
2D agarose gel analysis of in vitro-synthesized AAV DNA. Crude uninfected HeLa cell extracts were incubated with NE DNA and Rep78 (B; ssDNA-cellulose fraction) or Rep68 (C; ssDNA-cellulose fraction) in 30-μl reactions under standard DNA replication conditions. DpnI-digested DNA products (1/10 of each reaction) were fractionated on a 0.8% agarose gel under neutral conditions in the first dimension (1D) and alkaline conditions in the second dimension (2D). Panel A is a diagram of the RF species generated by Rep78, shown in panel B, and indicates relevant replicative DNA species (see the legend to Fig. 1 legend and text for a description). The relative molecular weights of each DNA species shown were derived from the migration pattern of BstEII-digested lambda DNA (not shown).
FIG. 8
FIG. 8
2D agarose gel analysis of DNA isolated from cells coinfected with Ad5 and AAV. 293 cells were infected with Ad5 and wild-type AAV at multiplicities of infection of 5 and 10, respectively. At 48 h postinfection, cultures were harvested and low-molecular-weight DNA was isolated by Hirt extraction. AAV DNA replicative forms were fractionated on a 0.8% agarose under neutral conditions in the first dimension and alkaline conditions in the second dimension. Panels B and C are 30-min and 2-day exposures of the same blot, respectively. Panel A is a schematic representation of the data. mE and dE represent linear monomer and dimer duplex DNA products with extended or open ends, respectively. mT and dT represent linear monomer and dimer duplex DNA products with a single covalently closed end or turn, respectively. cc denotes linear DNA products that have both ends covalently closed, and ss indicates single-stranded genomic DNA. The relative molecular weights of each DNA species shown were derived from the migration pattern of HindIII-digested lambda DNA (not shown). n indicates monomer size duplex DNA; ss indicates monomer size ssDNA.
FIG. 9
FIG. 9
Diagram of potential Rep cuts at trs sites that might occur during strand displacement synthesis on monomer extended (mE) and monomer turnaround (mT) replicative forms. See text for discussion.
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