Virus assembly, which takes place during the late stage of viral replication, is essential for virus propagation. However, the underlying mechanisms remain poorly understood, especially for viruses with complicated structures. Here, we use correlative light and electron microscopy to examine the formation of cytoplasmic virion assembly compartments (cVACs) during infection by a γ-herpesvirus. These cVACs are membraneless organelles with liquid-like properties. Formation of cVACs during virus infection is mediated by ORF52, an abundant tegument protein. ORF52 undergoes liquid–liquid phase separation (LLPS), which is promoted by both DNA and RNA. Disrupting ORF52 phase separation blocks cVACs formation and virion production. These results demonstrate that phase separation of ORF52 is critical for cVACs formation. Our work defines herpesvirus cVACs as membraneless compartments that are generated through a process of LLPS mediated by a tegument protein and adds to the cellular processes that are facilitated by phase separation.
