ABSTRACTIntracellular signaling connected to integrin activation is known to induce cytoplasmic Ca2+ release, which in turn mediates a number of downstream signals. The cellular entry pathways of two closely related alphaherpesviruses, equine herpesviruses 1 and 4 (EHV-1 and EHV-4), are differentially regulated with respect to the requirement of interaction of glycoprotein H (gH) with α4β1-integrins. We show here that binding of EHV-1, but not EHV-4, to target cells resulted in a rapid and significant increase in cytosolic Ca2+ levels. EHV-1 expressing EHV-4 gH (gH4) in lieu of authentic gH1 failed to induce Ca2+ release, while EHV-4 with gH1 triggered significant Ca2+ release. Blocking the interaction between gH1 and α4β1-integrins, inhibiting phospholipase C (PLC) activation, or blocking binding of inositol 1,4,5-triphosphate (IP3) to its receptor on the endoplasmic reticulum (ER) abrogated Ca2+ release. Interestingly, phosphatidylserine (PS) was exposed on the plasma membrane in response to cytosolic calcium increase after EHV-1 binding through a scramblase-dependent mechanism. Inhibition of both Ca2+ release from the ER and scramblase activation blocked PS scrambling and redirected virus entry to the endocytic pathway, indicating that PS may play a role in facilitating virus entry directly at the plasma membrane.