The formation of replication compartments, the subnuclear structures in which the
viral DNA genome is replicated, is a hallmark of
herpesvirus infections. The localization of
proteins and
viral DNA within human cytomegalovirus replication compartments is not well characterized. Immunofluorescence analysis demonstrated the accumulation of the
viral DNA polymerase subunit UL44 at the periphery of replication compartments and the presence of different populations of UL44 in infected cells. In contrast, the viral
single-stranded-DNA binding protein UL57 was distributed throughout replication compartments. Using "click chemistry" to detect
5-ethynyl-2'-deoxyuridine (EdU) incorporation into replicating
viral DNA and pulse-chase protocols, we found that
viral DNA synthesis occurs at the periphery of replication compartments and that replicated
viral DNA subsequently localizes to the interior of replication compartments. The interiors of replication compartments also contain regions in which UL44 and EdU-labeled
DNA are absent. The treatment of cells with a
viral DNA polymerase inhibitor reversibly caused the dispersal of both UL44 and EdU-labeled
viral DNA from replication compartments, indicating that ongoing
viral DNA synthesis is necessary to maintain the organization of replication compartments. Our results reveal a previously unappreciated complexity of the organization of human cytomegalovirus replication compartments.