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Significance Multiple families of DNA viruses including herpesviruses amplify their genomes in nuclear sites termed replication compartments. What benefits the viruses gain by this spatial and temporal control is unclear. We have analyzed the replication compartments induced by Epstein–Barr virus (EBV) and its DNA amplification in detail to elucidate their functions and regulation in EBV’s productive cycle. We found that EBV uses its replication compartments to coordinate the amplification of its genomes: Each compartment is seeded by single viral DNAs, each compartment supports similar levels of viral DNA synthesis, and each completes this synthesis as the replication machinery declines within it. Thus, replication compartments not only exclude cellular DNA synthesis but are hubs for the coordination of viral DNA amplification.

Four-dimensional analyses show that replication compartments are clonal factories in which Epstein–Barr viral DNA amplification is coordinated