Multiple receptor interactions trigger release of membrane and intracellular calcium stores critical for herpes simplex virus entry

Herpes simplex viruses (HSV) harness cellular calcium signaling pathways to facilitate viral entry. Confocal microscopy and small interfering RNA (siRNA) were used to identify the source of the calcium and to dissect the requisite viral-cell interactions. Binding of HSV to human epithelial cells induced no calcium response, but shifting the cells to temperatures permissive for penetration triggered increases in plasma membrane calcium followed by a global release of intracellular calcium. Transfection with siRNA targeting the proteoglycan syndecan-2 blocked viral binding and abrogated any calcium response. Transfection with siRNA targeting nectin-1, a glycoprotein D receptor, also prevented both membrane and intracellular calcium responses. In contrast, the membrane response was preserved after transfection with siRNA targeting integrinαv, a novel glycoprotein H receptor. The membrane response, however, was not sufficient for viral entry, which required interactions with integrinαv and release of inositol-triphosphate receptor-dependent intracellular calcium stores. Thus, calcium plays a critical, complex role in HSV entry.