Abstract
<jats:p>Herpesvirus assembly requires coordination between the capsid, tegument, and viral envelope, but how the fragile interactions across these virion layers are integrated remains unclear. Using light-inducible seeding in living cells, we show that the intrinsically disordered region of the capsid-anchored tegument protein pp150 (UL32) of human cytomegalovirus (HCMV) undergoes liquid-liquid phase separation (LLPS) to organize this process. These condensates recruit the soluble tegument protein UL25 and the membrane-anchored protein UL71, linking viral proteins from spatially distinct virion sublayers. LLPS is mediated by distributed, multivalent contacts rather than defined sequence motifs, and is tunable by phosphorylation. Phosphomimetic substitution of CDK consensus sites suppresses condensation. In infected cells, this regulation is essential. A phosphomimetic virus yields no infectious progeny, whereas the phospho-ablated virus replicates poorly and accumulates aberrant cytoplasmic tegument-capsid condensates outside the assembly compartment. Thus, pp150 acts as a phosphorylation-regulated condensate scaffold coordinating HCMV tegument assembly and envelopment.</jats:p>