African swine fever virus is wrapped by the endoplasmic reticulum

African swine fever (ASF) virus is a large DNA virus that shares the striking icosahedral symmetry of iridoviruses and the genomic organization of poxviruses. Both groups of viruses have a complex envelope structure. In this study, the mechanism of formation of the inner envelope of ASF virus was investigated. Examination of thin cryosections by electron microscopy showed two internal membranes in mature intracellular virions and all structural intermediates. These membranes were in continuity with intracellular membrane compartments, suggesting that the virus gained two membranes from intracellular membrane cisternae. Immunogold electron microscopy showed the viral structural protein p17 and resident membrane proteins of the endoplasmic reticulum (ER) within virus assembly sites, virus assembly intermediates, and mature virions. Resident ER proteins were also detected by Western blotting of isolated virions. The data suggested the ASF virus was wrapped by the ER. Analysis of the published sequence of ASF virus (R. J. Yanez et al., Virology 208:249-278, 1995) revealed a reading frame, XP124L, that encoded a protein predicted to translocate into the lumen of the ER. Pulse-chase immunoprecipitation and glycosylation analysis of pXP124L, the product of the XP124L gene, showed that pXP124L was retained in the ER lumen after synthesis. When analyzed by immunogold electron microscopy, pXP124L localized to virus assembly intermediates and fully assembled virions. Western blot analysis detected pXP124L in virions isolated from Percoll gradients. The packaging of pXP124L from the lumen of the ER into the virion is consistent with ASF virus being wrapped by ER cisternae: a mechanism which explains the presence of two membranes in the viral envelope.