MDV cell-to-cell spread is enhanced upon inhibition of the Rac signaling pathway.

<p>Monolayers of CESCs were infected or not with the BAC20-EGFP for 6 hrs, and then treated for 4 days with the indicated concentrations of inhibitors or activator. (A) The effect of a Rac inhibitor, NSC23766, on MDV cell-to-cell spread. Upper panel, F-actin staining of non-infected CESCs treated with 60 µM of NSC23766 (bar, 20 µm). Middle panel, a representative BAC20-EGFP infection plaque obtained following treatment with 60 µM of NSC23766 (bar, 200 µm). Lower panel, the graph shows a quantitative analysis of plaques size in one of three representative experiments. Percentages on graphs indicate the increase (+) in plaques size compared to the untreated cells (***, p<0.001). The error bars represent the SEM of 50 plaques size. (B) The effect of S1P, a Rac1 activator, on F-actin (upper panel), on plaques shape (middle panel) and plaques size (lower panel), as in A (***, p<0.001). (C) Effect of IPA-3, an inhibitor of group I-PAKs, on F-actin (upper panel), plaques shape (middle panel) and plaques size (lower panel), as in A (***, p<0.001). (D) One of three representative experiments showing the relative cell viability after a 4-day treatment with the highest concentration of NSC23766, S1P and IPA-3 are presented. Values were measured and normalized as presented above. Error bars represent the SEM. (E) Control of IPA-3 on CESCs. F-actin was stained on sparsely seeded CESCs, untreated (left), or treated for 2 hrs with 10 µM of IPA-3 (right). For each condition, a two fold magnification of the box is shown next to the lower magnification. Solid arrowheads point to filopodia while open arrowheads point to lamellipodia. All bars, 20 µm.</p

Opposing effects of Rho/Rac pathways in MDV cell-to-cell spread is conserved for two MDV strains.

<p>Monolayers of CESCs were infected or not with one of the two following viruses: (A) the attenuated parental strain BAC20 and (B) the virulent wild-type BAC RB-1B. Six hrs post-infection, one of the three agents, TAT-C3 transferase, Y-27632 or NSC23766, was added to the cells until the end of the spread assay. The spread assay lasted 4 days for BAC RB-1B and 3 days for BAC20, due to its fast growth. The graphs show a quantitative analysis of infection plaques size for each virus in one of two representative experiments (***, p<0.001; **, 0.001</p

Modulators of actin polymerization decrease MDV cell-to-cell spread.

<p>CESCs monolayers were infected or not with the BAC20-EGFP for 6 hrs, and then treated for 4 days at the indicated drug concentration. (A) Upper panel, F-actin staining (AlexaFluor594-Phalloidin) in non-infected cells. Lower panel, representative example of a BAC20-EGFP plaque which area was measured in µm². Bar, 200 µm. (B) Effect of CytD on actin polymerization and MDV cell-to-cell spread. Upper panel, F-actin staining of non-infected CESCs treated with 100 nM of CytD. Lower Panel, the graph shows a quantitative analysis of fifty plaques size in one of three representative experiments. Error bars represent the SEM. Percentages on graphs indicate the decrease (−) in plaques size compared to the untreated cells (***, p<0.001). (C) Effect of Jaspl. F-actin staining and plaques size are shown, as in (B). (D) The relative cell viability after a 4-day treatment with each drug at the highest concentration used. Values were normalized as compared to untreated cells. Error bars represent the SEM. Percentages on the graph indicate the decrease (−) in viability. The dashed line represents the acceptable CESCs viability limit (0.75) that was set in this study. For F-actin detection, bars, 20 µm.</p

LPA and S1P effects on MDV cell-to-cell spread are mediated through Rho-ROCK and Rac-PAK respectively.

<p>Monolayers of CESCs were infected or not with the BAC20-EGFP for 6 hrs, and then treated for 4 days at the indicated molecule concentrations. (A) Specificity of the LPA effect on MDV spread through the Rho-ROCK pathway. In the left panel, a representative picture of both F-actin staining of non-infected CESCs (bar, 20 µm) and BAC20-EGFP infection plaque (bar, 200 µm) obtained after each double treatment. In the right panel, the graph shows a quantitative analysis of plaques size in one representative experiment over two (ns, non significant; ***, p<0.001). The number at the top of each histogram indicates the ratio between plaques size after treatment compared to the untreated condition. The error bars represent the SEM of 50 plaques size. (B) Specificity of the S1P effect on MDV spread through the Rac-PAK pathway. The effect of the indicated double treatments on F-actin and on the plaques size are presented like in A.</p

Inhibition of the Rho-ROCK signaling pathway decreases MDV cell-to-cell spread.

<p>(A) Effect of TAT-C3 transferase, an inhibitor of Rho proteins. Upper panel, F-actin staining of non-infected CESCs treated with 16 µg/mL of TAT-C3 transferase (bar, 20 µm). Lower panel, a picture of a representative BAC20-EGFP infection plaque obtained with 16 µg/mL of TAT-C3 transferase (bar, 200 µm). Right panel, the graph shows a quantitative analysis of plaques size in one of two to three representative experiments (***, p<0.001). The error bars represent the SEM of 50 plaques size. (B) Effect of LPA, an activator of Rho proteins, on F-actin (upper panel), plaques shape (lower panel) and plaques size (right panel), as in A (***, p<0.001). (C) Effect of Y-27632, an inhibitor of ROCKs, on F-actin (upper panel), plaques shape (lower panel) and plaques sizes (right panel), as in A (***, p<0.001). (D) Effect of Fasudil, a second inhibitor of ROCKs, on F-actin (upper panel), plaques shape (lower panel) and plaques size (right panel), as in A (*, 0.1</p

Inhibition of Rho-ROCK signaling results also in a rounding of MDV plaques.

<p>(A) Typical images of BAC20-EGFP infection plaques 4 days post-infection without treatment (lines a and b), or in the presence of 16 µg/mL of LPA (line c), 10 µM of IPA-3 (line d), 16 µg/mL of TAT-C3 transferase (line e), 20 µM of Y-27632 (line f) or 80 µM of Y-27632 (line g) are shown. For each condition, the cell monolayer was photographed in white light with phase contrast and infected cells in fluorescence with EGFPVP22. Cell monolayer (greyscale) and infection plaques (green) are shown merged (column 1). In some conditions, a semi-ordered cell arrangement is visible in uninfected region of the cell monolayer with phase contrast. This cell arrangement is indicated with white dashed lines on the pictures (column 2). Bars, 200 µm. (B) The graph shows a quantitative analysis of the ratio between plaque length and plaque width under the indicated growth conditions. A ratio of 1 corresponds to a perfect circle. The ratio will be increased by the degree to which the plaque is elongated. The median value for each experimental group is indicated by a horizontal bar. (***, p<0.001).</p

Actin cytoskeleton is rearranged in the center of MDV infection plaques but not in periphery.

<p>Immunofluorescence microscopy of non infected cells (column A) and BAC20-EGFP infected cells, after 4 days of growth on the CESCs monolayer are shown, in the center of a representative infection plaque (column B), or in the periphery (columns C–D). F-actin was stained with AlexaFluor594-Phalloidin (red) (line 2) and DNA with Hoechst 33342 (blue) (B1–D1). Infected cells express the EGFPVP22 protein (green) (line 3). Asterisks show infected cells still possessing stress fibers. Arrowheads show infected cells which present connections between their stress fibers and those of neighboring infected or uninfected cells. Note that pictures in columns B and C are a superposition of 6 z stacks of 0.250 µm each, whereas pictures A and D correspond to a unique cut. All scale bars, 20 µm.</p

ESCDL-1 stably expressing the YGFP Venus support GaHV-2 replication.

<p>(A) Venus expression by ESCDL-1-Venus at passage 8 post selection initiation (scale bar indicates 100 μm). Venus YGFP is equally distributed in the nucleus and cytoplasm. (B) Comparison of plaque areas of vBAC20UL17mRFP between the parental cell line and the ESCDL-1-Venus/Clone7. The plaque areas of virus produced on ESCDL-1-Venus/Clone7 (green Tuckey box and whiskers plots) and on ESCDL-1 (shaded Tuckey box and whiskers plot) were measured for the parental and the Venus expressing cell-lines at matching passages (2 independent experiments). Differences existing between the cells or viruses were non significant (Mann-Whitney test with P values over 0.5). (C) vBACRB1BUL17mRFP plaques on ESCDL-1-Venus/Clone7: note the strong up-regulation of Venus expression in infected cells (scale bar indicates 200 μm).</p

Constitutive expression of pUL49 (VP22) in ESCDL-1 complements the deletion of UL49 in GaHV2 genome.

<p>(A & B) Constitutive expression of pUL49 (VP22) in uncloned cell populations (A) and in ESCDL-1-UL49/clone 2 (B). VP22 staining by anti pUL49 Mabs and an Alexa Fluor^® 488 goat anti-mouse detects filamentous material between 2 strongly positive nuclei that appear to be still bound after cell division (white arrowhead). (C to H) Complementation of replication for BAC20ΔUL49 on ESCDL-1-UL49/clone 7 (C, E, G) and absence of viral dissemination in non-complementing ESCDL-1 (D, F, H). Viral replication was detected using a chicken hyper immune serum revealed by an Alexa Fluor^® 488 goat anti-chicken conjugate together with an anti-ICP4 Mab (C, D), a mixture of anti-gI and -gE Mabs (E, F), or a mixture of anti-pUL49 (VP22) Mabs (G, H) all revealed by an Alexa Fluor^® 594 goat anti-mouse conjugate. The restoration of pUL49 (VP22) expression is associated with the viral replication (G). Early-late (ICP4) and late (gE-gI) antigens are detected in isolated ESCDL-1 cells (D & F) and in panel H the arrow points to an isolated cell in which vBAC20ΔUL49 undergoes an aborted replication cycle as revealed by the polyclonal anti-MDV serum without detection of VP22. Scale bar represents 50μm.</p

TEM analysis of vBAC20 morphogenesis in ESCDL-1.

<p>(A) Overview of an infected cell with intranuclear A (black triangle) and B (black arrowheads) capsids and intracytoplasmic C capsids (white ellipses). The white arrow points to an image of capsid tegumentation in the cytoplasm. (B) Intranuclear accumulation of small particles (SP– 30 to 35 nm in diameter) arranged in a pseudo-crystalline structure in the vicinity of A capsids (black triangle). (C) Accumulation of primary enveloped virions in distended cisternae of the nuclear membrane (black arrows point to enveloped C capsids). (D) C capsid undergoing secondary envelopment: electron dense material, possibly of tegument origin, surrounding the capsid is surrounded by a membrane in close vicinity to the Golgi (bar represents 0.2 μm). (E) Multiple intracytoplasmic C capsids (white ellipses) close to an enveloped cytoplasmic particle (white arrowhead).</p