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. 1998 Mar;72(3):2033-9.
doi: 10.1128/JVI.72.3.2033-2039.1998.

p53 and RPA are sequestered in viral replication centers in the nuclei of cells infected with human cytomegalovirus

E A Fortunato  1 , D H Spector
Affiliations

p53 and RPA are sequestered in viral replication centers in the nuclei of cells infected with human cytomegalovirus

E A Fortunato et al. J Virol. 1998 Mar.

Abstract

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.

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Figures

FIG. 1
FIG. 1
The p53 protein is stabilized in HCMV-infected fibroblasts but is not easily extracted from their nuclei. (A) Confluence-synchronized fibroblasts were collected at the indicated times by trypsinization, counted, and lysed in LRSB as described in Materials and Methods. Each lane of the SDS–15% polyacrylamide gel was loaded with 3 × 105 cell equivalents. Proteins were transferred to Immobilon and detected with the monoclonal Ab DO-1 directed against p53. (B) At 48 hpi, cells were harvested and aliquots of 5 × 105 cells were lysed in RIPA buffer with or without an additional sonication step (+Son and −Son, respectively) to help improve extraction as indicated. Lysates were cleared, and the pelleted debris was resuspended in an equivalent amount of LRSB. One half of each aliquot, either pellet (P) or supernatant (S) was then electrophoresed through an SDS–10% polyacrylamide gel, transferred to Immobilon P, and probed with DO-1 Ab.
FIG. 2
FIG. 2
Stabilization of p53 is due to decreased degradation, not increased synthesis. (A) Cells were metabolically labeled for 2-h periods at the indicated times postinfection as described in Materials and Methods. Approximately 5 × 105 cells were sonicated in RIPA buffer as described in the legend to Fig. 1B to ensure complete extraction of p53, and cleared lysates were incubated with agarose-coupled DO-1 Ab for 2 h at 4°C with gentle rocking. The beads were then washed several times in RIPA buffer and resuspended in LRSB, and the eluted proteins were run on an 10% SDS–polyacrylamide gel, fluorographed, dried, and exposed to film. M, mock-infected cells; V, virally infected cells. (B) Cells were pulse-labeled at 48 hpi, chased for the indicated times as described in Materials and Methods, and then processed as described for panel A. An agarose-coupled Ab to SV40 large-T antigen was used as a control for specificity. (C) Quantitation of data from panel B by using PhosphorImager software. For each set of data, the pulse was taken as 100% incorporation of label. m48, mock-infected cells pulse-labeled at 48 hpi; v48, virally infected cells pulse-labeled at 48 hpi.
FIG. 3
FIG. 3
p53 is sequestered into discrete foci in the nucleus of infected fibroblasts. Cells were confluence synchronized, seeded onto glass coverslips, infected, and at 30 hpi were processed as described in Materials and Methods. Viral (a) and mock (b) cells were stained with DO-1 Ab. Viral cells were also stained with a control Ab to trinitrophenol to show specificity of staining (c). Hoechst dye was used to illuminate the DNA of the imaged cells (d through f). Magnification ≈×410. FITC, fluorescein isothiocyanate.
FIG. 4
FIG. 4
p53 localizes to viral replication centers in infected fibroblasts as delineated by staining of the HCMV DNA polymerase processivity factor UL44 and BrdU incorporation. At 48 hpi, coverslips were fixed and permeabilized as described in Materials and Methods. (a and c) Staining with DO-1 (anti p53); (e and g) staining with CH16.1 (anti UL44); (i and k) staining with anti BrdU. Arrows in the phase-contrast micrographs point to the electron-dense structures that are viral replication centers. BrdU images are taken from a separate experiment in which, 30 min prior to harvesting at 48 hpi, cells were incubated in media containing BrdU. Magnification ≈ ×370. FITC, fluorescein isothiocyanate.
FIG. 5
FIG. 5
Localization to replication centers in infected fibroblasts is observed with some cellular and viral proteins, but not others. Coverslips were processed for immunofluorescence at 30 hpi. Cells were stained with PMG-345 (anti-Rb) (a and c), monoclonal Ab to RPA (e and g), CH16.1 (anti-UL44) (i and k), and monoclonal Ab to IE1 72 (m and o). Hoechst dye was used to illuminate the DNA in all imaged cells. Magnification ≈ ×290. FITC, fluorescein isothiocyanate.
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