Figure 1.
Characterization of the HeLaAAVtCR cell clone.
(A) Schematic representation of the wt AAV-2 genome and of the AAVtCR derivative. In the AAVtCR construct the rep gene is fused at its N-terminus with a sequence coding for the calmodulin and streptavidin binding peptides (C and S) and the fluorescent mCherry protein. p5, p19, and p40 are the AAV promoters and the dotted line indicates the alternative splicing in the rep gene. The black box indicates the BGH polyA. (B) Synthesis of Rep proteins in HSV-1-infected HeLaAAVtCR and HA-16 cells. The cells were infected or not with wt HSV-1 at a multiplicity of infection (MOI) of 5 plaque forming units (pfu)/cell and analyzed for the synthesis of Rep proteins by Western blot. tCR indicates the tagCherryRep78 and 68 proteins. (C) Analysis of AVVtCR genome rescue and replication. Replication was assessed by Southern blot using a rep probe. mRF and dRF indicate the monomeric and dimeric replicative forms, respectively.
Figure 2.
Analysis of Rep protein synthesis upon transfection with plasmids encoding HSV-1 immediate-early proteins.
HeLaAAVtCR cells were transfected with the indicated plasmids and harvested 48 h later. (A) Cell lysates were analyzed by Western blot with an anti-Rep antibody (303.9) and, after stripping of the membrane, with an anti-tubulin antibody. (B) Comparison of Rep synthesis following either co-transfection with ICP0, ICP4, and ICP22 expressing plasmids or with the pTF3 construct. After analysis with an anti-Rep antibody, the membranes were stripped and re-probed with anti-ICP0, anti-ICP4, and anti-tubulin antibodies.
Figure 3.
Induction of Rep protein synthesis and AAVtCR genome replication by combinations of HSV-1 immediate-early and early genes.
(A) Schematic view of the HSV-1 constructs pTF3, pRF, and pTF3Pol. Each plasmid encodes three to four HSV-1 orfs expressed under the control of heterologous promoters and polyA signals. The arrow heads indicate the following heterologous promoters: CMV (black), TK (light grey), PGK (dark grey), short eIF1-α (striped box). The rectangles correspond to the following polyA signals: SV40 (black), TK (light grey), β-Globin (dark grey), and synthetic polyA (striped box). The grey areas between each gene correspond to the Gateway® recombination signals used to obtain these constructs. CcdB is a selection marker used to screen bacterial colonies. The white box indicates the ampicillin resistance gene. (B) HeLaAAVtCR cells were either transfected with different combinations of HSV-1 derived plasmids, or infected with wt HSV-1 or HSVΔUL30 (MOI of 5 pfu/cell). (C) Analysis of AAVtCR genome replication. Genomic DNA was extracted and analyzed by Southern blot with a rep probe. Upper image: undigested DNA; lower image: Pst I digested DNA. Digestion with Pst I was used to resolve all the AAVtCR replication products into a unique 1.4 kb band. (D) Analysis of AAVtCR genome replication by qPCR using primers located in the AAV rep gene. The quantity of rep sequences in each condition was determined as described in the Materials and Methods and presented as the fold enrichment relative to a cellular genomic sequence on chromosome X. The data shown are mean values with error bars representing the standard error of the mean for three independent experiments.
Figure 4.
Effect of a mutated HSV-1 DNA polymerase on Rep protein synthesis and AAV DNA replication.
(A) Intracellular localization of the wt and mutated HSV-1 polymerase in HeLa cells. HeLa cells were co-transfected with pTF3 and pRF and either the pPol or pPolG885A plasmid. 48 h post-transfection the cells were fixed, stained with an anti-UL30 antibody and observed under a confocal microscope. Cell nuclei were stained with Topro3. Bars: 2 µm (B) Analysis of Rep protein synthesis. HeLaAAVtCR cells were transfected with the indicated plasmids and, where indicated, infected 24 h later with the appropriate HSV-1 strains at an MOI of 2 pfu/cells and harvested 24 h later. Cell lysates were analyzed by Western blot with an anti-Rep antibody (303.9) and, after stripping of the membrane, with an anti-tubulin antibody. (C) Genomic DNA was analyzed by qPCR using primers located in the AAV rep gene to measure AAVtCR replication. The amount of rep sequence in each condition was determined as described in the Materials and Methods and presented as the fold enrichment relative to a cellular genomic sequence on chromosome X. The data shown are mean values with error bars representing the standard error of the mean for three independent experiments.
Figure 5.
Visualization and analysis of AAVtCR and rAAV replications centers.
(A) Co-visualization of Rep and ICP8. HeLaAAVtCR cells, transfected with the indicated plasmids, were fixed and stained with an anti-ICP8 antibody. Cell nuclei were stained with Topro3. The Rep signal was provided by the expression of Rep proteins fused to mCherry (CRep). Bars: 2 µm. (B) Co-visualization by immuno-FISH analysis of AAVtCR DNA and Rep proteins. HeLaAAVtCR cells were either infected with HSVΔUL30 at an MOI of 5 pfu/cell or co-transfected with the pTF3pol and pRF plasmids and fixed 48 or 24 h later, respectively. The AAVtCR DNA was visualized using a combination of four non overlapping DIG-labeled rep probes followed by incubation with an anti-DIG antibody; Rep proteins were detected using an anti-Rep antibody (76.3). Cell nuclei were stained with Topro3. Bars: 2 µm. (C) Co-visualization of rAAV DNA with either Rep, ICP4, UL29, or UL30 proteins. Vero cells were co-transfected with the indicated combinations of HSV-1 plasmid, pAAVlacO, pSV2-EYFP/lacI, and p5CR (upper line) or p5Rep (middle and bottom line). Two days later cells were fixed and either directly observed under a confocal microscope (top line) or stained for detection of ICP8, ICP0, ICP4, and UL30 using antibodies. Cell nuclei were stained with DAPI. CRep: Cherry-Rep. Bars: 5 µm.
Figure 6.
Proposed model for HSV-I induced AAV replication from a latently infected cell line.
See the Discussion section for details.