The human cytomegalovirus IE2 and UL112-113 proteins accumulate in viral DNA replication compartments that initiate from the periphery of promyelocytic leukemia protein-associated nuclear bodies (PODs or ND10)

During human cytomegalovirus (HCMV) infection, the periphery of promyelocyt ic leukemia protein (PML)associated nuclear bodies (also known as PML oncog enic domains [PODs] or ND10) are sites for both input viral genome depositi on and immediate-early (IE) gene transcription. At very early times after i nfection, the IE1 protein localizes to and subsequently disrupts PODs, wher eas the IE2 protein Localizes within or adjacent to PODs. This process appe ars to be required for efficient viral gene expression and DNA replication. We have investigated the initiation of viral DNA replication compartment f ormation by studying the localization of viral IE proteins, DNA replication proteins, and the PML protein during productive infection. Localization of IE2 adjacent to PODs between 2 and 6 h after infection was confirmed by co nfocal microscopy of human fibroblasts (HF cells) infected with both wild-t ype HCMV(Towne) and with an IE1-deletion mutant HCMV(CR208) that fails to d isrupt PODs. In HCMV(Towne) -infected HE cells at 24 to 48 h, IE2 also accu mulated in newly formed viral DNA replication compartments containing the p olymerase processivity factor (UL44), the single-stranded DNA binding prote in (SSB; UL57), the UL112-113 accessory protein, and newly incorporated bro modeoxyuridine (BrdU). Double labeling of the HCMV(CR208)-infected HF cells demonstrated that formation of viral DNA replication compartments initiate s within granular structures that bud from the periphery of some of the POD s and subsequently coalesce into larger structures that are flanked by PODs . In transient DNA transfection assays, both the N terminus (codons 136 to 290) and the C terminus (codons 379 to 579) of IE2 exon 5, but not the cent ral region between them, were found to be necessary for both the punctate d istribution of IE2 and its association with PODs. Like IE2, the UL112-113 a ccessory replication protein was also distributed in a POD-associated patte rn in both DNA-transfected and virus-infected cells beginning at 6 h. Furth ermore, when all six replication core machinery proteins (polymerase comple x, SSB, and helicase-primase complex) were expressed together in the presen ce of UL112-113, they also accumulated at POD-associated sites, suggesting that the UL112-113 protein (but not IE2) may play a role in recruitment of viral replication fork proteins into the periphery of PODs. These results s how that (i) subsequent to accumulating at the periphery of PODs, IE2 is in corporated together with the core proteins into viral DNA replication compa rtments that initiate from the periphery of PODs and then grow to fill the space between groups of PODs, and (ii) the UL112-113 protein appears to hav e a key role in assembling and recruiting the tore replication machinery pr oteins in the initial stages of viral replication compartment formation.