Adeno-associated virus type 2 (AAV2) uncoating is a stepwise process and is linked to structural reorganization of the nucleolus

Figures

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  Fig 1. Spatial distribution of AAV2 capsids and DNA over time. NHF cells were infected with wtAAV2 (MOI 20`000). At various time points post infection, the cells were fixed and processed for multicolor IF analysis combined with FISH and CLSM. Nucleoli (Nuc) were visualized using an antibody against fibrillarin (yellow). Intact capsids were stained using an antibody that detects a conformational capsid epitope (green). AAV2 DNA (magenta) was detected with an Alexa Fluor (AF) 647 labeled, amine-modified DNA probe that binds to the AAV2 genome. (A) Spatial and temporal distribution of AAV2 capsids and DNA. The white line represents the edge of the nucleoli (fibrillarin staining), the blue line represents the edge of the nucleus (4‘,6-diamidino-2-phenylindole (DAPI) staining). (B) Orthogonal projections of a z-stack at 24 hpi. The white line represents the edge of the nucleus (DAPI staining). (C) Image-based quantification of the uncoating rate of 50 individual cells per time point in the nucleolus and (D) in the cytoplasm. p-values were calculated using an unpaired Student`s t-test (*—p ≤ 0.05, **—p ≤ 0.01, ***—p ≤ 0.001, ****—p ≤ 0.0001).

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  Fig 2. AAV2 particles on fibronectin coated coverslips. (A) wtAAV2 particles were directly applied to fibronectin coated coverslips and processed for IF analysis combined with FISH and CLSM, exactly as described for Fig 1. Intact capsids were stained using an antibody that detects a conformational capsid epitope (green). AAV2 DNA (red) was detected with an Alexa Fluor (AF) 647 labeled, amine-modified DNA probe that binds to the AAV2 genome. (B) Electron photomicrographs show complete disintegration of the AAV2 capsids at 75°C.

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  Fig 3. Acidification enhances genome accessibility of AAV2. NHF cells were treated with bafilomycine A1 (50 or 200 nM) or DMSO 1 h prior to infection with wtAAV2 (MOI 20`000). At 3 hpi, the cells were fixed and processed for multicolor IF analysis combined with FISH and CLSM. Nucleoli (Nuc) were visualized using an antibody against fibrillarin (yellow). Intact capsids were stained using an antibody that detects a conformational capsid epitope (green). AAV2 DNA (magenta) was detected with an Alexa Fluor (AF) 647 labeled, amine-modified DNA probe that binds to the AAV2 genome. (A) Genome accessibility of AAV2 capsids after inhibition of the acidification of the endosome-lysosome system. The white line represents the edge of the nucleoli (fibrillarin staining), the blue line represents the edge of the nucleus (DAPI staining). (B) Image-based quantification of the genome accessibility (ratio of AAV2 capsid+DNA+/AAV2 capsid+DNA- signal) of 50 individual cells per sample in the cytoplasm and (C) nuclear AAV2 capsid counts. p-values were calculated using an unpaired Student`s t-test (*—p ≤ 0.05, **—p ≤ 0.01, ***—p ≤ 0.001, ****—p ≤ 0.0001).

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  Fig 4. Absence of intact AAV2 capsids in AAV2 genome positive nucleoli points towards complete viral uncoating. NHF cells were infected with wtAAV2 (MOI 20`000). At 24 hpi, the cells were fixed and processed for multicolor IF analysis combined with FISH and CLSM. Nucleoli (Nuc) were visualized using an antibody against fibrillarin (yellow). Intact capsids were stained using an antibody that detects a conformational capsid epitope (green). AAV2 DNA (magenta) was detected with an Alexa Fluor (AF) 647 labeled, amine-modified DNA probe that binds to the AAV2 genome. Nuclei were counterstained with DAPI and illustrated as white lines. (A) Distinct pattern (I—III) of AAV2 capsid signal in cells with AAV2 genome positive nucleoli. (B) Quantification of 50 individual cells with distinct AAV2 capsid signal. (C) Image-based quantification of the integrated intensity of AAV2 capsid signals in dense or dispersed nucleoli of 70 individual cells. p-values were calculated using an unpaired Student`s t-test (*—p ≤ 0.05, **—p ≤ 0.01, ***—p ≤ 0.001, ****—p ≤ 0.0001).

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  Fig 5. Southern analysis of AAV2 DNA in cell fractions. Southern Blot of denatured Hirt DNA extracted at 24 and 48 hpi from isolated nuclei, nucleoplasm, or nucleoli of NHF cells infected with wtAAV2 (MOI 20’000; 1.5x106 cell equivalents per lane). AAV2 DNA was visualized with an AAV2 rep-specific probe disclosing bands representing the single-stranded (ss)AAV2 DNA and the replication form monomer (rfm; product of AAV2 second-strand synthesis). Representative images from two independent experiments are shown.

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  Fig 6. Co-detection of AAV2 DNA with AAV2 capsids and AAV2 capsid proteins. NHF cells were infected with wtAAV2 (MOI 20`000). At 24 hpi, the cells were fixed and processed for multicolor IF analysis combined with FISH and CLSM. (A) Intact capsids (green) or capsid proteins (yellow) were detected using either an antibody against intact AAV2 capsids (conformational capsid epitope) or an antibody (linear epitope) against VP1, VP2 and VP3. AAV2 DNA (red) was detected with an Alexa Fluor (AF) 647 labeled, amine-modified DNA probe that binds to the AAV2 genome. Nuclei were counterstained with DAPI (blue). The wtAAV2 infected cells shown were assigned as pattern I and III according to the intensity of the capsid staining and following a similar classification as defined in Fig 4. (B) AAV2 capsid proteins (green) were detected using an antibody (linear epitope) against VP1 and VP2. AAV2 DNA (magenta) was detected by linking the amine-modified DNA to AF647. Nucleoli (Nuc) were visualized using an antibody against fibrillarin (yellow). Nuclei were counterstained with DAPI and illustrated as white lines.

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  Fig 7. Nucleolar reorganization during cell cycle progression. Image-based analysis of the ratios of dense to dispersed nucleoli of 100 individual mock- or AAV2 infected cells in different cell cycle phases (G1, S and G2). p-values were calculated using an unpaired Student`s t-test (*—p ≤ 0.05, **—p ≤ 0.01, ***—p ≤ 0.001, ****—p ≤ 0.0001).

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  Fig 8. G1 cell cycle arrest obstructs complete uncoating in nucleoli. NHF cells were arrested in G1 cell cycle phase by a double thymidine block before and during infection with wtAAV2 (MOI 20`000). As control, the cells were released 8 h prior to infection by washing out the thymidine. At 24 hpi, the cells were fixed and processed for multicolor IF analysis combined with FISH, CLSM and image-based analysis of (A) the cell cycle profile after continuous thymidine block or release, respectively. (B) Quantification of the total uncoating rate in nucleoli. (C) Image-based quantification of the uncoating rate in nucleoli in G1 cell cycle phase. (D) Image-based quantification of the nucleolar area after continuous thymidine block or release, respectively. p-values were calculated using an unpaired Student`s t-test (*—p ≤ 0.05, **—p ≤ 0.01, ***—p ≤ 0.001, ****—p ≤ 0.0001).

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  Fig 9. Actinomycin D treatment overcomes thymidine-mediated obstruction of AAV2 uncoating. NHF cells were arrested in G1 cell cycle phase by a double thymidine block before and during infection with wtAAV2 (MOI 20`000). At 24 hpi, the cells were treated with actinomycin D for 1 h, fixed and processed for multicolor IF analysis combined with FISH and CLSM. (A) Nucleoli (Nuc) were visualized using an antibody against fibrillarin (yellow). Intact capsids were stained using an antibody that detects a conformational capsid epitope (green). AAV2 DNA (red) was detected with an Alexa Fluor (AF) 647 labeled, amine-modified DNA probe that binds to the AAV2 genome. (B) Image-based quantification of 50 individual cells per condition for nucleolar capsid, total nuclear capsid or total nuclear AAV2 genome signals. p-values were calculated using an unpaired Student`s t-test (*—p ≤ 0.05, **—p ≤ 0.01, ***—p ≤ 0.001, ****—p ≤ 0.0001).

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  Fig 10. Capsid disassembly coincides with cell cycle progression. NHF cells were infected with wtAAV2 (MOI 20`000). At 24 hpi, the cells were fixed and processed for multicolor IF analysis combined with FISH and CLSM. (A) Intact capsids (green) or capsid proteins (yellow) were detected using either an antibody against intact AAV2 capsids (conformational capsid epitope) or an antibody (linear epitope) against VP1, VP2 and VP3. AAV2 DNA (magenta) was detected with an Alexa Fluor (AF) 647 labeled, amine-modified DNA probe that binds to the AAV2 genome. Nuclei were counterstained with DAPI and illustrated as white lines. The cells shown were assigned as pattern I and III according to the intensity of the capsid staining and following a similar classification as defined in Fig 4. (B) Quantification of at least 70 nuclei positive for intact AAV2 capsids or capsid proteins during cell cycle progression.