If you have any problems related to the accessibility of any content (or if you want to request that a specific publication be accessible), please contact (email@example.com). We will work to respond to each request in as timely a manner as possible.
Characterization of Human Cytomegalovirus UL84
AdvisorPari, Gregory S
Biochemistry and Molecular Biology
StatisticsView Usage Statistics
ABSTRACT: HCMV oriLyt-dependent DNA synthesis requires six core replication proteins, UL44 (DNA processivity factor), UL54 (DNA polymerase), UL70 (primase), UL105 (helicase), UL102 (primase-associated factor) and UL57 (single-stranded DNA-binding protein), and two non-core proteins, UL84 and IE2. UL84 is a non-core multifuctional protein that shuttles from the nucleus to the cytoplasm and is required for oriLyt-dependent DNA replication and viral growth. UL84 was previously shown to interact with IE2 (IE86) in infected cells, and this interaction down-regulates IE2-mediated transcriptional activation in transient assays. UL84 and IE2 cooperatively activate a promoter within HCMV oriLyt and UL84 alone can interact with an RNA stem-loop within oriLyt and is bound to this structure within the virion. In an effort to investigate the binding partners for UL84 in infected cells, we immunoprecipitated UL84 from protein lysates prepared from HCMV-infected human fibroblasts by using a UL84-specific antibody and resolved the immunoprecipitated protein complexes by two-dimensional gel electrophoresis. We subsequently identified individual proteins by matrix-assisted laser desorption ionization-tandem time of flight analysis (MALDITOF). This analysis revealed that UL84 interacts with viral proteins UL44, pp65, and IE2. In addition, a number of cell-encoded proteins were identified, including ubiquitin-conjugating enzyme E2, casein kinase II (CKII), and the multifunctional protein p32. We also confirmed these interactions in infected and cotransfected cells by coimmunoprecipitation assays followed by Western blotting. Ubiquitination of UL84 occurred in the presence and absence of the proteasome activity inhibitor MG132 in infected cells and was consistant with monoubiquitination. The identification of UL84 binding partners is a significant step toward the understanding of the function of this significant replication protein. This dissertation also defines the roles of CK2 and nucleocytoplasmic shuttling of UL84. We demonstrate that pUL84 is a substrate for CK2 in vitro and determine that two putative CK2 phosphorylation sites within pUL84 mediate binding to CK2. Mutation of a threonine residue at amino acid (aa) 148 and a serine residue at aa 157 within the pUL84 protein resulted in the inability of the protein to interact with the CK2 subunit in transfected cells. Interaction of pUL84 with CK2 was essential for complementation of oriLyt-dependent DNA replication, suggesting that phosphorylation is an essential modification.To address the role of nucleocytoplasmic shuttling of UL84 in the context of the viral genome, we generated an HCMV bacterial artificial chromosome (BAC) with mutation in UL84 protein, LLL228/230/359AAA. This non-shuttling UL84 BAC mutant was unable to produce infectious virus after transfection into human fibroblasts, whereas a revertant virus readily produced viral plaques and infectious virus. Real-time quantitative PCR showed that non-shuttling UL84 BAC was DNA replication deficient in that no increase in the accumulation of viral DNA was observed in either the intracellular or supernatant. This suggests that the nucleocytoplasmic shuttling of UL84 may have an important role in the viral DNA replication. Immunofluorescence assay was performed to investigate the subcellular localization of HCMV viral encoded proteins: UL84, UL44, IE2, UL83 and IRS1 in wild type verses UL84 recombinant non-shuttling BAC. The distribution of IE2 protein in non-shuttling UL84 BAC was noticeably different from that observed in wt BAC transfected cells. Small punctate structures within the nucleus were observed implicating the role of nucleocytoplasmic shuttling of UL84 in the formation of IE2 replicaion compartments. An analysis of viral mRNA transcriptions by real-time PCR showed that mRNA transcripts from all representative kinetic classes of viral genes were accumulated to the same degree as wt BAC up to 96hs pt in cells transfected with non-shuttling UL84 BAC. These data show that nucleocytoplasmic shuttling of UL84 provides an essential DNA replication function and possibly influences the distribution of IE2 in the nucleus.