HCMV DNA replication initiates at a distinct region called the lytic origin of DNA replication or oriLyt. Enzymes involved in DNA replication were elucidated using the transient replication assay. Based on this assay and subsequent experiments, it was demonstrated that the following open reading frames (ORFs) were required for oriLyt-dependent DNA replication: The """"""""core"""""""" replication proteins : UL54 (polymerase), UL44 (polymerase accessory protein), UL70 (primase), UL105 (helicase), UL102 (primase associated factor), UL57 (single-stranded DNA binding protein) and IE2-580aa (immediate early protein) and UL84 (early protein). We constructed an HCMV recombinant BAC with an insertion in the UL84 loci. This BAC was defective for both DNA replication and proper partitioning of UL44 and IE2 into nuclear replication compartments. We have now determined that UL84 is phosphorylated in transfected and infected cells and can form a stable physical association with itself in transfected cells. In addition, UL84 has UTPase activity which is likely part of an energy-generating system for helicase activity in DNA replication. Amino acid sequence analysis of UL84 reveals that it is a DExD/H box containing protein with RNA helicase consensus domains. Transient reporter assays, in which a region of oriLyt was ligated upstream of the luciferase gene, demonstrated the presence of strong promoter within oriLyt. In Vero cells, IE2 apparently represses a high basal level of activation from this promoter. However in permissive human fibroblasts (HFFs), the oriLyt promoter is quiescent and is activated by viral infection or the transfection of UL84 and IE2. Transient assays also show that further activation can be achieved by the cotransfection of the entire replication machinery. Our current studies demonstrate that a substantial portion of oriLyt facilitates DNA synthesis by transcriptional activation and the SV40 promoter can functionally substitute this activity. This proposal will seek to i) determine the precise phosphorylation sites within UL84; ii) identify the role of UL84 phosphorylation in DNA replication and the UL84-mediated regulation of IE2; and, iii) characterize any UL84-associated enzymatic activity.
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