p53 and RPA are sequestered in viral replication centers in the nuclei of cells infected with human cytomegalovirus.
Previously, we reported that human cytomegalovirus (HCMV) infection of fibroblasts markedly affects p53 and other regulatory proteins and inhibits transit through the cell cycle (F. M. Jault, J.-M. Jault, F. Ruchti, E. A. Fortunato, C. Clark, J. Corbeil, D. D. Richman, and D. H. Spector, J. Virol. 69:6697-6704, 1995). Although the p53 steady-state levels are elevated throughout the infection, evidence suggests that the ability of p53 to transactivate some of its downstream targets is compromised. To elucidate the mechanisms governing the accumulation of p53, we examined the synthesis, stability, and localization of the protein in HCMV-infected fibroblasts. Synthesis of p53 was not increased in the infected cells during the first 24 h postinfection. In fact, pulse-chase experiments revealed that synthesis of p53 in infected fibroblasts was lower than in mock-infected cells. However, after an initial decay, the p53 was stabilized. In addition, beginning at approximately 30 h postinfection, p53 was localized to discrete foci within the nuclei of infected cells. The morphology of these foci suggested that they were replication centers. We confirmed that these are sites of DNA replication by demonstrating both incorporation of bromodeoxyuridine and localization of UL44 (the viral polymerase processivity factor) into these centers. The single-stranded DNA binding protein RPA was also sequestered. In contrast, Rb and HCMV IE1 72 remained distributed throughout the infected cell nuclei, indicating specific targeting of certain proteins. Taken together, our results provide two alternative mechanisms to account for the increased steady-state levels of p53 observed in HCMV-infected fibroblasts.
Fortunato
E A
EA
Department of Biology, University of California, San Diego, La Jolla 92093-0357, USA.
Spector
D H
DH
D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000378:metabolism*; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000032:analysis / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism* / Q000821:virology; D006801:Humans; D017874:Immediate-Early Proteins / Q000032:analysis; D051716:Replication Protein A; D016160:Retinoblastoma Protein / Q000032:analysis; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis / Q000378:metabolism*; D014764:Viral Proteins / Q000032:analysis; D014779:Virus Replication
D004268:DNA-Binding Proteins; C072217:ICP36 protein, Cytomegalovirus; C088645:IE1 protein, cytomegalovirus; D017874:Immediate-Early Proteins; C497167:RPA1 protein, human; D051716:Replication Protein A; D016160:Retinoblastoma Protein; D016159:Tumor Suppressor Protein p53; D014764:Viral Proteins