Transfection and vector generation.

<p><b>A) Transfection of DKE1 cells and DKSce cells with linear or circular DNA.</b> For each cell line, the level of transfection, based on GFP detection by flow cytometry, was 4–8% for NotI-AscI or I-SceI digested pCAVGFP or pCAVGFP-Sce. Transfection efficiency (±OHTam) increased 7–10 fold when supercoiled 30 kb plasmids were used. <b>B)</b> DKSce cells were transfected with supercoiled pCAVGFP-Sce, I-SceI digested pCAVGFP-Sce or supercoiled pCAVGFP-Sce+OHTam. The transfected cells were collected 5 days later, and the cleared lysate used to infect 10-cm plates of DKSce cells. At this vector generation step, GFP⁺ cells were quantified by flow cytometry and by scanning for fluorescence by microscopy. No GFP⁺ cells were detected when transfecting supercoiled pCAVGFP-Sce without OHTam (non-digested). A non-representative image showing rare GFP expression in I-SceI digested pCAVGFP-Sce (digested), and a representative image showing the GFP expression in supercoiled pCAVGFP-Sce+OHTam (OHTam). Nuclei are stained with Hoechst (blue). Scale bar = 10 µm. *P value = 0.005. <b>C)</b> A 10-cm plate of DKSce cells was incubated with cleared lysate from the above CAVGFP generation step. No GFP⁺ cells were ever detected in the cells transfected with supercoiled pCAVGFP-Sce (non-digested) and reamplified. Approximately 0.2% of the cells were infected by CAVGFP when using the cleared lysate from cells transfected with I-SceI-digested pCAVGFP-Sce (digested). Greater than 10% of the cells in the 10-cm plate were infected with CAVGFP when using the cleared lysate from cells transfected with supercoiled pCAVGFP-Sce+OHTam Nuclei are stained with Hoechst (blue). Scale bar = 10 µm, *P value = 0.029. <b>D)</b> To determine if we could generate vectors more quickly, we repeated the vector generation step using I-SceI digested pCAVGFP-Sce and supercoiled pCAVGFP-Sce+OHTam. The cells were collected at days 2–5 and the cleared lysate was incubated with a fresh monolayer of DKSce cells. The number of CAVGFP infected cells/million transfected cells was quantified. The assays were performed in duplicate and repeated at least three times. <b>E)</b> To determine if we could inhibit or modify the DSB break response, and in turn increase CAV-2 vector generation, we included drugs (caffeine, KU55933, Z-VAD-FMK, and mirin) that play a role in preventing DSB recognition, repair or downstream events. Z-VAD-FMK and mirin were also combined. No significant difference was seen versus controls. The assays were performed in duplicate and repeated at least twice.</p

Adenovirus Tales: From the Cell Surface to the Nuclear Pore Complex

<p>Adenovirus Tales: From the Cell Surface to the Nuclear Pore Complex</p

Adenovirus structure and trafficking.

<p>A) An illustration of the cross-section of a prototype 90 nm AdV capsid showing the location of the principal capsid proteins (hexon, penton, protein VI, protein IX, protease, and the fibre—the knob is the globular head of the fibre) involved in trafficking. B) An illustration showing the quintessential steps of AdV trafficking in epithelial cells. Via the knob region of the fiber, the capsid engages the cellular receptor. In some cell types, fibres are lost from the metastable* capsid during internalization in clathrin-coated pits. Postinternalization, the capsid continues to dissociate and releases protein VI, which allows the capsid access to the cytosol and interaction with dynein, then dynein-dependent transport along microtubule to the nuclear pore complex. *Metastable is a common term used to describe the biophysical state of fully mature nonenveloped virions. Overall, the particle is stable to the environment; however, it is able to respond to cellular cues to undergo conformational changes during cell entry.</p

Axonal transport of CAV-2 in neurons.

<p>A) A schema showing the assays used to record CAV-2 directionality and speed in murine dorsal root ganglion (DRG) neurons. Neurons are cultivated in microfluidic chambers (top center) in which the microfluidic flow is from left to right. This flow of the medium in the 5-micron-wide and 500-micron-long microgroove prevents diffusion of particles and allows a physical separation between cell bodies (left) and axon termini (right). CAV-2, covalently labeled with a fluorophore (Cy3), were added for 90 min in the axonal compartment before the video was started, and axons in the middle of the microgroove were imaged at one frame/second. The rate of retrograde transport of CAV-2 in these conditions is approximately 1–2 microns/s (insert: ultrastructural electron micrograph of CAV-2 vesicular transport in motor neurons from Salinas et al. [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004821#ppat.1004821.ref031" target="_blank">31</a>]). CAV-2 was mainly present in vesicular structures (white arrow) near microtubule tracks (black arrow). B) Still images of a microgroove of the chambers containing Cy3-labeled CAV-2 (red puncta) 90 min postincubation on the axon termini side. Below is a kymograph, which gives a graphical representation of the spatial position over time, of the corresponding movie (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004821#ppat.1004821.s001" target="_blank">S1 Video</a>). Scale bars in the micrograph = 100 nm.</p

Transduction efficiency of AAV vectors in the mouse cornea.

<p>EGFP expression (indicated by arrows) in the mouse cornea detected by <i>in vivo</i> epifluorescence microscopy 1-wk post-injection of AAV2/1 (<b>a</b>), AAV2/2 (<b>e</b>), AAV2/5 (<b>i</b>) and AAV2/8 (<b>m</b>) vectors. (<b>b</b>, <b>f</b>, <b>j</b>, <b>n</b>) Higher magnification of <b>a</b>, <b>e</b>, <b>i</b> and <b>m</b>, respectively. (<b>c</b>, <b>g</b>, <b>k</b>, <b>o</b>) EGFP expression in the same corneas detected 4-wk post-injection. (<b>d</b>, <b>h</b>, <b>l</b>, <b>p</b>) Higher magnification of <b>c</b>, <b>g</b>, <b>k</b> and <b>o</b>, respectively. Magnifications <b>a</b>, <b>e</b>, <b>g</b>, <b>i</b>, <b>k</b>, <b>m</b>, <b>o</b>: 20×; <b>b</b>: 43×; <b>c</b>: 25×, <b>d</b>: 53×; <b>f</b>, <b>j</b>: 35×; <b>h</b>: 44×, <b>l</b>: 33×; <b>n</b>: 40×; <b>p</b>: 45×. For reference, the diameter of an adult mouse eye is ∼3.5 mm. The large green spot in centre of photos is the pupil of the mouse eye. The asterisk in panels <b>i</b> to <b>l</b> indicates an opaque lesion on the mouse eye that was present from the beginning of the experiments.</p

Transduction efficiency of the AAV vectors in the human corneal explants.

<p>One-wk post-injection, using <i>in vivo</i> microscopy, EGFP expression can be seen in throughout the cornea following intra-stromal injection of the vectors AAV2/1 (<b>a</b>) and AAV2/8 (<b>i</b>). (<b>b</b>, <b>j</b>) Higher magnification of the boxed regions in <b>a</b> and <b>i</b>, respectively. (<b>e</b>) Three-weeks post transduction, EGFP expression can be detected following AAV2/2 injection. (<b>f</b>) Higher magnification of the boxed area in <b>e</b>. (<b>c</b>, <b>g</b>, <b>k</b>) EGFP expression on histological sections of each cornea 8-wk post-injection of AAV2/1, −/2, −/8, respectively. (<b>d</b>, <b>h</b>, <b>l</b>) Imaris-treated images of <b>c</b>, <b>g</b>, <b>k</b>, respectively, showing EGFP-expressing cells.</p

Cell specificity of AAV2/8 transduction.

<p>(<b>a</b>–<b>d</b>) Anti-CD34 staining (in red) of a non-injected mouse cornea shows abundant CD34+ cells. (<b>e</b>–<b>h</b>) Anti-CD34 labelling (in red) of a mouse cornea 24 h post-injection with 3×10⁹ vg of AAV2/8. Intra-stromal injection results in a decrease in the proportion of CD34+ cells (in red) due to an augmentation in the number of cell nuclei (in blue). The EGFP signal co-localises (arrows and arrowheads) with the CD34-labelled cells. Inset in <b>h</b> shows a higher magnification of the cells indicated by arrowheads minus the Hoechst filter. (<b>i</b>–<b>l</b>) Anti-F4/80 staining (in red) of a non-injected mouse cornea showing a low number of F4/80+ cells. (<b>m</b>–<b>p</b>) Anti-F4/80 labelling (in red) of a mouse cornea 24 h post-injection with 3×10⁹ vg of AAV2/8. The EGFP signal does not co-localise (arrows) with the F4/80-labelled cells.</p

Intra-stromal injection of AAV2/8 in the human cornea results in transduction of keratocytes.

<p>(<b>a–d</b>) Anti-CD34 labelling (in red) of a human cornea co-localises with EGFP-expressing cells (in green) and identifies these cells as quiescent keratocytes. (<b>e–h</b>) Anti-α–SMA labelling (in red) of a human cornea co-localises with EGFP-expressing cells (in green) and identifies these cells as activated keratocytes.</p

EGFP-expressing cell population following PBS injection in the transduced mouse cornea.

<p>(<b>a</b>–<b>d</b>) Anti-CD34 staining (in red) of a mouse cornea 24 h post-PBS injection (same eye shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035318#pone-0035318-g004" target="_blank">Fig. 4C</a>). The CD34 signal co-localises with the EGFP signal (arrows and arrowheads). (<b>e</b>–<b>f</b>) Anti-F4/80 staining (in red) of a mouse cornea 24 h post-PBS injection. The F4/80 signal does not co-localise with the EGFP signal (arrows).</p

Schematic representation of the events following PBS injection and hyper-activation of EGFP expression.

<p>(<b>a</b>) Following initial AAV2/8 injection, a large number of cells harbour viral particles or vector genomes but a small number of cells express EGFP (in green). (<b>b</b>) Twenty-four h post-PBS injection, the disruption of the epithelial basement membrane results in the release of cytokines that induce the repair process. This process involves cell death in the stroma immediately underlying the injection site, which reduces the vector genome levels (∼2-fold decrease), and cell migration from the limbal region. The ensuing events also result in either <i>de novo</i> uncoating of encapsidated virions or reactivation of transcriptionally-silenced genomes leading to activated EGFP expression (consistent with the 65-fold increase in EGFP mRNA levels). (<b>c</b>) One-week post-PBS injection, the cornea has returned to its basal state and is no longer expressing EGFP (low mRNA levels). The cell death process has removed a number of transduced keratocytes (4-fold decrease in vector genome levels). However, cells harbouring the virions are still present (persisting DNA levels) as a subsequent PBS injection still can activate EGFP expression but less extensively.</p