Origin-independent assembly of Kaposi's sarcoma-associated herpesvirus DNA replication compartments in transient cotransfection assays and association with the ORF-K8 protein and cellular PML

Six predicted Kaposi's sarcoma virus herpesvirus (KSHV) proteins have homology with other well-characterized herpesvirus core DNA replication proteins and are expected to be essential for viral DNA synthesis, Intact Flag-tagged protein products from all six were produced from genomic expression vectors, although the ORF40/41 transcript encoding a primase helicase component proved to be spliced with a 127-bp intron. The intracellular localization of these six KSHV replication proteins and the mechanism of their nuclear translocation were investigated. SSB (single-stranded DNA binding protein, ORF6) and PPF (polymerase processivity factor, ORF59) were found to be intrinsic nuclear proteins, whereas POL (polymerase, ORF9), which localized in the cytoplasm on its own, was translocated to the nucleus when cotransfected with PPF. PAF (primase-associated factor, ORF40/41), a component of the primase-helicase tripartite subcomplex together with PRI (primase, ORF56) and HEL (helicase, ORF44), required the presence of all five other replication proteins for efficient nuclear translocation. Surprisingly, even in the absence of a lytic cycle replication origin (ori-Lyt) and any known initiator or origin binding protein, the protein products of all six KSHV core replication genes cooperated in a transient cotransfection assay to form large globular shaped pseudo-replication compartments (pseudo-RC), which excluded cellular DNA, These pseudo-RC structures were confirmed to include POL, SSB, PRI, and PAF but did not contain any newly synthesized DNA. Similar to the human cytomegalovirus system, the peripheries of these KSHV pre-RC were also found to be surrounded by punctate PML oncogenic domains (PODs). Furthermore, by transient cotransfection, the six KSHV core replication machinery proteins successfully replicated a plasmid containing EBV ori-Lyt in the presence of the Epstein-Barr virus-encoded DNA binding initiator protein, ZTA. The KSHV encoded K8 (ORF-K8) protein, which is a distant evolutionary homologue to ZTA, was incorporated into pseudo-RC structures formed by transient cotransfection with the six core KSHV replication genes. However, unlike ZTA, K8 displayed a punctate nuclear pattern both in transfected cells and at early stages of lytic infection and colocalized with the cellular PML proteins in PODs. Finally, K8 was also found to accumulate in functional viral RC, detected by incorporation of pulse-labeled bromodeoxyuridine into newly synthesized DNA in both tetadecanoyl phorbol acetate-induced JSC-1 primary effusion lymphoblasts and in KSHV lytically infected endothelial cells.