INRA, UMR1282, Infectious Diseases and Public Health, ISP, BIOVA team, Nouzilly, France.
PLoS One. 2012;7(8):e44072. doi: 10.1371/journal.pone.0044072. Epub 2012 Aug 27.
Marek's Disease Virus (MDV) is an avian alpha-herpesvirus that only spreads from cell-to-cell in cell culture. While its cell-to-cell spread has been shown to be dependent on actin filament dynamics, the mechanisms regulating this spread remain largely unknown. Using a recombinant BAC20 virus expressing an EGFPVP22 tegument protein, we found that the actin cytoskeleton arrangements and cell-cell contacts differ in the center and periphery of MDV infection plaques, with cells in the latter areas showing stress fibers and rare cellular projections. Using specific inhibitors and activators, we determined that Rho-ROCK pathway, known to regulate stress fiber formation, and Rac-PAK, known to promote lamellipodia formation and destabilize stress fibers, had strong contrasting effects on MDV cell-to-cell spread in primary chicken embryo skin cells (CESCs). Inhibition of Rho and its ROCKs effectors led to reduced plaque sizes whereas inhibition of Rac or its group I-PAKs effectors had the adverse effect. Importantly, we observed that the shape of MDV plaques is related to the semi-ordered arrangement of the elongated cells, at the monolayer level in the vicinity of the plaques. Inhibition of Rho-ROCK signaling also resulted in a perturbation of the cell arrangement and a rounding of plaques. These opposing effects of Rho and Rac pathways in MDV cell-to-cell spread were validated for two parental MDV recombinant viruses with different ex vivo spread efficiencies. Finally, we demonstrated that Rho/Rac pathways have opposing effects on the accumulation of N-cadherin at cell-cell contact regions between CESCs, and defined these contacts as adherens junctions. Considering the importance of adherens junctions in HSV-1 cell-to-cell spread in some cell types, this result makes of adherens junctions maintenance one potential and attractive hypothesis to explain the Rho/Rac effects on MDV cell-to-cell spread. Our study provides the first evidence that MDV cell-to-cell spread is regulated by Rho/Rac signaling.
马立克氏病病毒（MDV）是一种仅在细胞培养物中通过细胞间传播的禽类α疱疹病毒。虽然已经表明其细胞间传播依赖于肌动蛋白丝动力学，但调节这种传播的机制在很大程度上仍然未知。使用表达 EGFPVP22 被膜蛋白的重组 BAC20 病毒，我们发现 MDV 感染斑的中心和外围的肌动蛋白细胞骨架排列和细胞-细胞接触不同，后一区域的细胞显示出应力纤维和罕见的细胞突起。使用特定的抑制剂和激活剂，我们确定 Rho-ROCK 途径，已知调节应力纤维形成，以及 Rac-PAK，已知促进片状伪足形成并使应力纤维不稳定，对鸡胚皮肤细胞（CESC）中的 MDV 细胞间传播具有强烈的对比作用。Rho 的抑制及其 ROCKs 效应物导致斑块大小减小，而 Rac 或其 I 组-PAKs 效应物的抑制则产生相反的效果。重要的是，我们观察到 MDV 斑块的形状与在斑块附近单层水平伸长细胞的半有序排列有关。Rho-ROCK 信号的抑制也导致细胞排列的扰动和斑块的变圆。这两种 Rho 和 Rac 途径在 MDV 细胞间传播中的相反作用在两种具有不同体外传播效率的亲本 MDV 重组病毒中得到了验证。最后，我们证明 Rho/Rac 途径对 CESC 之间细胞-细胞接触区域 N-钙粘蛋白的积累有相反的影响，并将这些接触定义为黏着连接。考虑到黏着连接在某些细胞类型中的 HSV-1 细胞间传播中的重要性，这一结果使黏着连接的维持成为一个潜在的有吸引力的假说，以解释 Rho/Rac 对 MDV 细胞间传播的影响。我们的研究首次证明 MDV 细胞间传播受 Rho/Rac 信号调节。
