RhoB supports rubella infection and impairs endothelial barrier integrity through downstream ROCK signaling.

backgroundDuring infection, alterations of the endothelium’s barrier function are multifaceted and virus-specific. Rubella virus (RuV) infection during pregnancy damages the endothelium in the placental and fetal vasculature, thereby contributing to the development of congenital rubella syndrome. However, the extent and mechanisms behind RuV-induced endothelial barrier disruption have not been previously documented.

methodsTo investigate if RuV directly impairs endothelial barrier integrity, we infected primary human umbilical vein endothelial cells and analyzed the barrier integrity using Electrical Cell-Substrate Impedance Sensing (ECIS). Furthermore, we applied fluorescence microscopy to analyze RuV-induced changes in cell morphology, the actin cytoskeleton, and cell–cell junctions. To dissect the molecular mechanisms behind the observed alterations induced by RuV infection in endothelial cells, we determined levels of RhoA and RhoB GTPases, followed by depletion of both proteins using siRNA transfection and application of pharmacological inhibitors of the Rho/ROCK signaling axis.

resultsRubella virus infection in endothelial cells induced a comparatively low level of cytopathogenicity, accompanied by an elongated morphology. Most notably, a reduction in endothelial barrier integrity occurred at later time points of infection, as measured by ECIS. While the overall expression levels of junctional proteins appeared unaffected, membrane localization of zonula occludens-1 (ZO-1) was significantly reduced. This was accompanied by a marked increase in actin stress fibers and enhanced RhoB GTPase levels. The siRNA-mediated downregulation combined with time-dependent inhibitor application confirmed a contributory role for RhoB to RuV infection at early post-entry steps. The elongated morphology of infected endothelial cells was associated with the chemokine CCL5. Notably, cell elongation induced by treatment with supernatant from infected cells was insufficient to impair the barrier integrity in the absence of productive RuV replication. In comparison, depletion of RhoB expression or ROCK inhibitor application revealed the involvement of the RhoB/ROCK signaling axis in the impairment of barrier integrity and induction of stress fibers by RuV infection. Additionally, altered localization of ZO-1 was restored after application of ROCK inhibitor Y-27632 to uninfected control levels.

conclusionsCollectively, our data highlight the involvement of Rho GTPase–ROCK signaling in alterations of endothelial cell actin dynamics and barrier integrity in rubella virus pathology.