Fig 1.
The AAV2 Rep protein domains and the Rep expression plasmids analyzed in this study.
(A) Schematic representation of the main protein domains within the rep ORF (grey bars). The promoters p5 and p19 responsible for the expression of the different Rep proteins are indicated. The common splicing site at the C-terminal end of the ORF is depicted with an up-facing arrow head. (B) Schematic representation of the AAV2 Rep proteins analyzed in this study; (i) the wild-type (wt) Rep, (ii) the helicase-deficient mutant Rep-K340H and (iii) the helicase mutant Rep-D371Y. The ATPase/helicase domain (grey box) and the corresponding mutations (vertical black lines) are indicated. A detailed representation of the helicase domain (aa 225–490) is shown below. The mutation K340H is located within the Walker motif A whereas the mutation D371Y is located between the Walker motifs A and B. The corresponding locations within the Rep aa-sequence are indicated. (C) Rep protein levels. Plasmids encoding either the wt Rep, the mutant Rep-K340H, the mutant Rep-D371Y or no Rep (pcDNA) were transfected into Vero cells. At 24 hrs after transfection, the cells were harvested and processed for Western analysis using a Rep-specific antibody. The different Rep protein variants are indicated on the right. Detection of actin served as a loading control.
Fig 2.
Effects of the different Rep constructs on HSV-1 and AAV2 DNA replication.
(A) Replication of an HSV-1 replicon is visualized with plasmid pHSV-TetO containing an HSV-1 origin of DNA replication (oriS) and a TetO-cassette consisting of 35x TetR binding sites. In presence of HSV-1 replication factors (HSV-1), the accumulation of pHSV-TetO replication products is visualized by binding of an eYFP-TetR fusion protein (yellow fluorescent RCs). (B) Cells expressing the different Rep constructs (indicated on top) were analyzed for the ability to inhibit HSV-1 replication. The presence of Rep proteins was confirmed by staining with a Rep-specific antibody (red; bottom panels). A cell transfected with pcDNAmRFP expressing the monomeric red fluorescent protein (mRFP) was used as a positive control. DAPI was used to stain the nuclei. Scale bar; 5μm. (C) Effects of the different Rep constructs on HSV-1 amplicon vector production. Vero 2–2 cells were transfected with the HSV-1 amplicon DNA (pHSVGFP), packaging-defective HSV-1 helper DNA (fHSVΔpacΔ27Δkn), HSV-1 ICP27 encoding plasmid (pEBHICP27) and the different Rep encoding plasmids. At 72 hrs post transfection the pHSVGFP amplicon vector particles were harvested and titrated on Vero cells. The data are shown as means ± standard errors (SE) from three independent experiments. Asterisks indicate statistically significant differences based on a paired two-tail Student t-test (** = p<0.01). (D) Replication of an AAV2 replicon is visualized with plasmid pAV2-LacO, which is harboring a LacO-cassette consisting of 40x LacI binding sites flanked by AAV2 ITRs. In presence of the different Rep constructs and HSV-1 helper factors (HSV-1), the accumulation of pAV2-LacO replication products is visualized by binding of an eYFP-LacI fusion protein (yellow fluorescent RCs, top panels). (E) Cells expressing the different Rep constructs (indicated on top) were analyzed for the ability to replicate pAV2-LacO and accumulate corresponding RCs. A Rep-specific antibody was used to confirm the synthesis of Rep proteins in the transfected cells (red; bottom panels). A cell expressing mRFP was used as a negative control. DAPI was used to stain the nuclei. Scale bar; 5μm. (F) Vero 2–2 cells were transfected with pAV2GFP and the different Rep constructs as indicated. At 24 hrs after transfection, the cells were infected with HSV-1 (MOI 2) and subjected to Hirt DNA extraction 48hrs later. Extrachromosomal DNA was digested with DpnI and analyzed by Southern blotting with a DIG-labeled probe specific for GFP. The ITR ssDNA, the monomeric (ITRm) and the dimeric (ITRd) AAV2 replication intermediates are indicated on the left.
Fig 3.
Impact of the different Rep constructs on the induction of a Rep-specific DNA damage response and apoptosis.
(A-C) Assessment of Rep induced DNA-damage responses. Vero cells were transfected with plasmids encoding wt Rep, Rep-K340H, Rep-D371Y or eGFP as a negative control. Two days later, the cells were fixed and stained with antibodies specific for Rep (green; insets) and (red) either pRPA32-S4/8 (A), pATM-S1981 (B) or γH2A.X-S139 (C). The cell nuclei were visualized by staining with DAPI (blue; insets). Rep+- or GFP+-cells were scored for staining of the DDR markers using a confocal laser scanning microscope (graphs in A-C). Bars represent mean values (% positive cells) and SEs from 3 individual experiments. Asterisks indicate statistically significant differences between the positive control (wt Rep) and the corresponding mutant Rep constructs in a paired two-tail Student t-test (*, P<0.05; **, P<0.01). Scale bars, 10μm. (D) Screening of the Rep constructs for their ability to induce apoptosis in transfected cells. Vero cells were co-transfected with an eGFP expressing plasmid and a plasmid encoding either wt Rep, Rep-K340H, Rep-D371Y or no Rep (pcDNA). Three days later, the cells were stained with Cy5-conjugated annexin V and analyzed by flow cytometry with filters specific for eGFP (transfected cells) and Cy5 (apoptotic cells). The data are shown as means ± SE from three independent experiments (*, P<0.05).
Fig 4.
The mutant rep-D371Y gene is not compatible with the production of recombinant AAV2 virus stocks.
(A) Wild-type (wt AAV2, a) and recombinant (rAAV2-D371Y, b) virus stocks were visualized and analyzed with transmission electron microscopy. Red arrows: empty particles (electron dense), green arrow: fully packaged particle (electron light). Scale bars; 50nm. (B) Southern analysis to assess replication of wt and recombinant AAV2 virus stocks. Vero 2–2 cells were co-infected with either wt (wt AAV2) or recombinant (rAAV2-D371Y) (MOI 20) and HSV-1 (MOI 1) followed Hirt DNA extraction 48hrs later. Southern blotting was performed as described in Fig 2F with a DIG-labeled probe specific for Rep. The ITR ssDNA, the monomeric (ITRm) and the dimeric (ITRd) AAV2 replication intermediates are indicated on the right. (C, D) The capability of the mutant Rep68-D371Y proteins to bind ssDNA is reduced, whereas specific binding of dsDNA is not affected. Gel filtration chromatography profiles of either wt Rep68 or mutant Rep68-D371Y proteins were utilized to assess the binding capacity to (C) unspecific ssDNA or (D) specific dsDNA (RBS) templates. V0 is the void volume where aggregates are eluted with a molecular mass larger than the exclusion limit of the column (indicating complex Rep multimers). The other species at P1 represent the Rep-dsDNA or Rep-ssDNA complexes respectively. The P2 species represent unbound Rep proteins.
Fig 5.
Virus titers of HSV/AAV hybrid vectors harboring the mutant rep68/78-D371Y gene are significantly higher than titers of hybrid vectors harboring the wt rep68/78 gene.
(A) Packaging and titration of the HSV/AAV hybrid vectors pHyRaNGFPa and pHyRD371YaNGFPa was performed as described for the HSV-1 vector pHSVGFP (Fig 2C). The data are shown as means ± SE from three independent experiments (*, P<0.05). (B) Western analysis to confirm Rep expression in HSV/AAV hybrid vector producing Vero-2-2 cells. Production of vectors was performed as described for Fig 2C and Fig 5A. Instead of harvesting vector particles, the cells were processed for and subjected to Western analysis. In addition to the Rep-specific antibody to detect Rep expression, the blots were stripped and re-stained with an antibody specific for GFP as a loading and expression control.
Table 1.
Summary of AAV2 Rep activities.