Charged-to-alanine scanning mutagenesis of the N-terminal half of adeno-associated virus type 2 Rep78 protein

Abstract

The adeno-associated virus (AAV) Rep78 and Rep68 proteins are required for site-specific integration of the AAV genome into the AAVS1 locus (19q13.3-qter) as well as for viral DNA replication. Rep78 and Rep68 bind to the GAGC motif on the inverted terminal repeat (ITR) and cut at the trs (terminal resolution site). A similar reaction is believed to occur in AAVS1 harboring an analogous GAGC motif and a trs homolog, followed by integration of the AAV genome. To elucidate the functional domains of Rep proteins at the amino acid level, we performed charged-to-alanine scanning mutagenesis of the N terminus (residues 1 to 240) of Rep78, where DNA binding and nicking domains are thought to exist. Mutants were analyzed for their abilities to bind the GAGC motif, nick at the trs homolog, and integrate an ITR-containing plasmid into AAVS1 by electrophoretic mobility shift assay, trs endonuclease assay, and PCR-based integration assay. We identified the residues responsible for DNA binding: R107A, K136A, and R138A mutations completely abolished the binding activity. The H90A or H92A mutant, carrying a mutation in a putative metal binding site, lost nicking activity while retaining binding activity. Mutations affecting DNA binding or trs nicking also impaired the site-specific integration, except for E66A and E239A. These results provide important information on the structure-function relationship of Rep proteins. We also describe an aberrant nicking of Rep78. We found that Rep78 cuts predominantly at the trs homolog not only between the T residues (GGT/TGG), but also between the G and T residues (GG/TTGG), which may be influenced by the sequence surrounding the GAGC motif.

FIG. 1

Charged-to-alanine scanning mutagenesis of the N-terminal region of the Rep78 protein. All of the charged amino acids, i.e., arginine (R), lysine (K), histidine (H), aspartic acid (D), and glutamic acid (E), in the N-terminal half (residues 1 to 240) were mutated to alanine. Residues replaced are underlined. Because all of the residues for mutation were changed to alanine, for simplicity, mutant Rep proteins were designated by the wild-type amino acids. Most mutant Rep proteins have a single alanine substitution. However, two charged residues were simultaneously changed to alanine where charged residues existed in a cluster (DE16, EK32, EK57, RD61, RR68, EK83, RE113, ER184, KR186, KE204, DK233, and EK239). When these double mutations affected the function of Rep protein, both residues were mutated to alanine independently for further analyses. For example, in the case of DE16, D16A and E17A were also constructed. The K340H mutant, in which the NTP-binding lysine at position 340 was changed to histidine as described before (8), was included in our assay. ∗, all of the mutants and the “wild-type” Rep78 harbor two mutations compared to authentic Rep78: M225G so as not to synthesize small Rep proteins and G17E. The translation start site of the small Rep proteins is indicated by an arrow.

FIG. 2

(A) Wild-type and mutant Rep78 proteins synthesized in vitro. Mutant Rep proteins were produced by using a coupled in vitro transcription-translation system in the presence of [³⁵S]methionine-cysteine. Two microliters of reaction mixture was resolved on an SDS–7.5% polyacrylamide gel. Unprogrammed lysate (no DNA) and lysate programmed with vector (vector) were also loaded. A major single band of about 75 kDa was detected in each lane. The intensities of the bands were not significantly different from one another as revealed by densitometry, indicating that the amount of in vitro-synthesized Rep protein in each reaction was essentially the same. (B) 293 cells were transfected with plasmids expressing mutant Rep proteins under the control of the cytomegalovirus promoter. One day after transfection, cells were harvested, and 5 μg of lysate was electrophoresed on SDS–7.5% polyacrylamide gels. Proteins were transferred onto polyvinylidene difluoride membranes, and Rep proteins were detected with anti-Rep antibody 76.3. Unlike in vitro-synthesized mutant Rep proteins, several mutant Rep proteins (e.g., E34, D149, and E150) were expressed reproducibly at lower levels. ∗, prolonged exposure revealed a band corresponding to Rep protein.

FIG. 3

EMSA of wild-type Rep78 synthesized in vitro. Each lane contained 20,000 cpm of ³²P-labeled RBS oligonucleotide probe. Lane 1 contained unprogrammed lysate. When lysate programmed with pCMVR78 was incubated with the probe, multiple shifted bands (bracket) were observed (lane 2). Shifted bands disappeared gradually as the amount of cold competitor added increased (lanes 3 and 4). The higher-mobility band (arrowhead) is completely depleted when anti-Rep antibody 76.3 is included in the reaction, whereas other slower bands (∗) are not likely to be supershifted (compare lane 5 to lane 2).

FIG. 4

EMSA of mutant Rep78 synthesized in vitro. Three microliters of in vitro-synthesized Rep78 or mutant Rep protein in the absence of radiolabeled amino acids was incubated for 15 min at 30°C with 20,000 cpm of 5′-end-labeled RBS oligonucleotide probe in a 10-μl solution of 10 mM HEPES-KOH (pH 7.9), 50 mM KCl, 0.1 mM EDTA, 0.05% BSA, 10% glycerol, and 1 μg of sheared calf thymus DNA. Reaction products were separated on 4% nondenaturing polyacrylamide gels, dried, and then analyzed on a BAS-1500 imaging analyzer. Unprogrammed lysate (no DNA) and lysate programmed with vector (vector) were also included as negative controls. ∗, complete loss of binding activity; ∗∗, consistently lower binding activity compared to the wild type.

FIG. 5

trs endonuclease activity of in vitro-synthesized Rep78 protein. Three different substrates were used. γ-³²P-end-labeled AAV hairpin DNA was prepared as described previously with minor modification (see Materials and Methods). The SmaI- and StyI-XhoI fragments (113 and 198 bp, respectively) derived from AAVS1 harbored a minimum sequence element required for site-specific integration (Fig. 6A and B). Three microliters of in vitro-translated Rep78 protein was mixed with 15,000 cpm of probe in a 10-μl solution containing 25 mM HEPES-KOH (pH 7.5), 10 mM MgCl₂, 1 mM dithiothreitol, 2% glycerol, and 0.1 μg of BSA, in the presence or absence of 0.4 mM ATP, and then incubated for 1 h at 37°C. Instead of Rep78, lysate programmed with blank vector (vector) or with luciferase DNA (Luc) was also used. After addition of 3× loading buffer (0.5% SDS, 50 mM EDTA, 40% [vol/vol] glycerol, 0.1% [wt/vol] xylene cyanol, 0.1% [wt/vol] bromophenol blue), samples were boiled for 5 min and then applied to 8% nondenaturing polyacrylamide gels. Arrows indicate the nicking products. Note the products that were present even when ATP was not included (arrowheads). However, when the AAVS1 fragment (SmaIXhoI or StyI-XhoI) was incubated with Rep78 in the absence of ATP, the amount of released fragment was small.

FIG. 6

Lengths of nicking fragments produced by in vitro-synthesized Rep78. (A) The StyI-XhoI (PvuII) fragment was used for the nicking reaction. The major nicking site of Rep protein reported elsewhere is shown (arrowhead). The sequencing primer (nt 311 to 337) to produce DNA ladders is indicated by an arrow. (B) The other substrate for the trs nicking reaction. (C) The nicking products derived from the StyI-XhoI fragment (lane N) were resolved on a 6% denaturing sequencing gel along with sequencing ladders. The sequencing ladders were prepared by using the Takara Taq cycle sequencing kit with some modifications: the reaction mixture contained dGTP instead of 7-deaza dGTP, 10% dimethyl sulfoxide, and ³²P-5′-end-labeled 27-nt oligonucleotide (see panel A). Arrowheads indicate the cutting sites. (D) The nicking products derived from the StyI-XhoI fragment (lane N) were also separated on a 6% gel with sequence ladders produced by a chemical reaction. The deduced cut sites are indicated by arrowheads. There is about a 1-base difference between ladders produced by primer extension and by chemical reaction. (E) The nicking products derived from the SmaI-XhoI fragment were resolved on a 6% denaturing sequence gel with chemically prepared sequence ladders. Arrowheads indicates the deduced nicking sites.

FIG. 7

trs endonuclease activities of mutant Rep proteins synthesized in vitro. The ³²P-5′-end-labeled StyI-XhoI fragment derived from AAVS1 was used as a template (S). Reaction mixtures were the same as described in the legend to Fig. 5. Substrates and nicking products (P) were separated on 8% nondenaturing gels. When nicking products were observed, we concluded that mutant Rep proteins retained trs endonuclease activity even if the amounts of the products were small. Nicking products below a background level are indicated by closed circles.

FIG. 8

Ability of mutant Rep proteins to introduce ITR plasmid into AAVS1. Two micrograms of pCMVR78, mutant Rep expression plasmids, or blank vector was transfected into 2 × 10⁵ 293 cells/well in six-well plates along with 2 μg of pW1, harboring a lacZ expression cassette flanked by ITRs, by a standard calcium phosphate precipitation method. Twenty-four hours later, total cellular DNA was isolated and suspended finally in 200 μl of TE. PCR to detect site-specific integration was carried out as reported previously with minor modifications: 1 μl of isolated genomic DNA was subjected to a thermal cycling reaction in a 20-μl reaction mixture containing 1× thermophilic DNA polymerase buffer [10 mM KCl, 20 mM Tris-HCl (pH 8.8), 10 mM (NH₄)₂SO₄, 4 mM MgSO₄, 0.1% Triton X-100 (NEB)], 1 μM 5′-CGGCCTCAGTGAGCGAGCGAGC and 5′-CGGGGAGGATCCGCTCAGAGGACA, and 2 U of Deep Vent Exo(−) DNA polymerase (NEB). The cycling conditions were 99°C for 1 min followed by 35 cycles of 99°C for 10 s and 72°C for 4 min. Ten microliters of the PCR mixture was transferred to a hybridization membrane (Hybond-N⁺; Amersham) by using a dot blot apparatus and hybridized with a ³²P-labeled AAVS1 probe. The membranes were then analyzed on a BAS-1500 imaging analyzer. The assay was repeated at least four times. p, positive control for hybridization.

FIG. 9

Summary of mutant Rep78 proteins and their functions. The abilities of mutant Rep78 proteins to bind to the GAGC motif, nick at the trs, and mediate integration into AAVS1 were compared to those of wild-type Rep78. Open circles indicate that the activities are comparable to those of the wild type. Shaded circles indicate reduced activities. Closed circles indicate that activities are below background levels. EMSA was performed in triplicate, and the assay to detect site-specific integration was repeated at least four times. When nicking products were observed in the trs endonuclease assay, we concluded that the mutant Rep proteins retained nicking activity even if the amounts of the products were small. If data obtained by using single-substitution mutants are available, results for double mutants are omitted.