Biochemical requirements of virus wrapping by the endoplasmic reticulum: Involvement of ATP and endoplasmic reticulum calcium store during envelopment of African swine fever virus

Enwrapment by membrane cisternae has emerged recently as a mechanism of env elopment for large enveloped DNA viruses, such as herpesviruses, poxviruses , and African swine fever (ASF) virus. For both ASF virus and the poxviruse s, wrapping is a multistage process initiated by the recruitment of capsid proteins onto membrane cisternae of the endoplasmic reticulum (ER) or assoc iated ER-Golgi intermediate membrane compartments. Capsid assembly induces progressive bending of membrane cisternae into the characteristic shape of viral particles, and envelopment provides virions with two membranes in one step, We have used biochemical assays for ASF virus capsid recruitment, as sembly, and envelopment to define the cellular processes important for the enwrapment of viruses by membrane cisternae. Capsid assembly on the ER memb rane, and envelopment by ER cisternae, were inhibited when cells were deple ted of ATP or depleted of calcium by incubation with A23187 and EDTA or the ER calcium ATPase inhibitor, thapsigargin, Electron microscopy analysis sh owed that cells depleted of calcium were unable to assemble icosahedral par ticles. Instead, assembly sites contained crescent-shaped and bulbous struc tures and, in rare cases, empty closed five-sided particles. Interestingly, recruitment of the capsid protein from the cytosol onto the ER membrane di d not require ATP or an intact ER calcium store. The results show that foll owing recruitment of the virus capsid protein onto the ER membrane, subsequ ent stages of capsid assembly and enwrapment are dependent on ATP and are r egulated by the calcium gradients present across the ER membrane cisternae.