Interactions of Viral and Cellular Factors with Human Cytomegalovirus OriLyt
AuthorKagele, Dominique Ardena
AdvisorPari, Gregory S
Biochemistry and Molecular Biology
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HCMV requires both trans-acting factors and cis-acting elements to successfully replicate its genome during lytic infection. The activity of six "core" replication enzymes encoded by the virus: UL54 (polymerase), UL57 (single-stranded DNA binding protein), UL70 (primase), UL105 (helicase), UL105 (primase-associated factor), and UL44 (polymerase processivity factor) in addition to the initiator protein UL84 are required, as well as the major transactivator IE2. The 3 kb-long cis-acting element oriLyt, also required for lytic-phase DNA replication, is categorized into two essential regions. In addition to viral protein binding sites within oriLyt, many transcription factor-binding sites have been elucidated and characterized, suggesting a possible role for cellular proteins in the regulation of viral lytic DNA replication. In an effort to characterize the binding of the viral replication factors UL84, UL44, and IE2 across oriLyt, chromatin immunoprecipitation assays were performed using specific antibodies to immunoprecipitate each protein and associated DNA. Results from these experiments demonstrate that UL84 binds to regions where consensus C/EBP α and β transcription factor binding sequences are located. Mutation of these sites resulted in a loss of UL84 binding and abrogated oriLyt-dependent DNA replication, indicating that the interaction of UL84 with the C/EBP α sites within oriLyt is essential for lytic DNA replication. To identify other cellular factors that may be implicated in lytic DNA replication we used immobilized oriLyt DNA as bait to bind to viral and cellular factors present in infected cell nuclear extract. hnRNP K, IRS1, UL83, and several other proteins were identified by mass spectrometry analysis. We also show that UL84 interacts with hnRNP K in infected and transfected cells, and this interaction is enhanced by the addition of IE2 and UL44. These results reveal that viral proteins interact with cellular proteins to possibly regulate lytic DNA replication. UL84 has also been shown to interact with the polymerase processivity factor UL44. To further characterize this interaction, we performed co-immunoprecipitations and determined the interaction domain of UL44 lies between amino acids 355-400 of UL84, and amino acids 200-217 of UL44. Trans-dominant negative inhibition of oriLyt-dependent DNA replication was observed when fragments containing these regions of each protein were used in the transient cotransfection assay, demonstrating the interaction of UL84 with UL44 is essential for lytic DNA replication.