The prenylated pUS2 protein of pseudorabies virus contributes to phosphorylation of connexin 43 and suppression of gap junctional intercellular communication

Gap junctional intercellular communication (GJIC) enables the direct transfer of signaling molecules between neighboring cells and plays an important role in innate antiviral immunity by facilitating bystander defense responses. We previously showed that the alphaherpesvirus pseudorabies virus (PRV) suppresses GJIC through ERK1/2-mediated phosphorylation and degradation of the main gap junction (GJ) protein connexin 43 (Cx43), triggered by the viral tegument protein pUL46. Here, we identify the prenylated viral protein pUS2 as a second important regulator of this process during PRV infection. Using the attenuated PRV vaccine strain Bartha K61 and a panel of isogenic deletion mutants, we show that pUS2 is required for efficient Cx43 phosphorylation and GJIC suppression but is dispensable for ERK1/2 activation itself. We further demonstrate that this function of pUS2 depends on its C-terminal CAAX prenylation motif. A prenylation-deficient pUS2 mutant fails to localize ERK1/2 to the plasma membrane and, likely as a consequence, does not induce efficient Cx43 phosphorylation or GJIC downregulation. Our data support a model in which PRV employs a two-step strategy to suppress GJIC in infected cells: pUL46 triggers ERK1/2 activation, while newly expressed, prenylated pUS2 redirects the activated kinase to the plasma membrane to facilitate phosphorylation of Cx43. This work reveals how PRV exploits the spatial control of host kinase signaling to suppress gap junction intercellular communication.IMPORTANCEGap junctions (GJs) constitute key communication channels between cells in multicellular organisms. GJs allow the intercellular transfer of different small biomolecules, including messengers involved in the innate and adaptive antiviral response. We showed earlier that, during infection of epithelial cells with the porcine alphaherpesvirus pseudorabies virus (PRV), the viral tegument protein pUL46 triggers activation of ERK1/2, which in turn leads to phosphorylation of the major gap junction protein connexin 43 (Cx43) and closure of GJs. In the current report, we show that a second PRV protein, pUS2, contributes to Cx43 phosphorylation and GJ closure, likely by recruiting ERK1/2 to the plasma membrane. This function of pUS2 depends on its CAAX prenylation motif. The current data suggest that PRV-mediated Cx43 phosphorylation and GJ closure occur via a two-step process, involving the viral pUL46 and pUS2 proteins, and point to GJ modulation as a possible target for antiviral strategies.