p53 and RPA Are Sequestered in Viral Replication Centers in the Nuclei of Cells Infected with Human Cytomegalovirus

Cells and virus.

Primary human foreskin fibroblasts were obtained from the University of California, San Diego, Medical Center and propagated in minimal essential medium with Earle’s salts (MEM). The cells were propagated in incubators maintained at 37°C and 5% CO₂, and the medium was supplemented with 10% heat-inactivated fetal bovine serum, l-glutamine (2 mM), penicillin (200 U/ml), streptomycin (200 μg/ml), amphotericin B (1.5 μg/ml), and gentamicin sulfate (50 μg/ml). The Towne strain of HCMV was obtained from the American Type Culture Collection (VR 977) and used at a multiplicity of infection of 5.

Cell cycle synchronization and infection conditions.

All experiments using fibroblasts were performed under G₀ synchronization conditions. Cells were seeded into flasks and allowed to become confluent. After 2 to 3 days at confluence, cells were reseeded onto 10-cm-diameter dishes at 10⁶ cells/dish. Approximately 1 h after plating, the medium was removed, and the infection inoculum (either virus or an equivalent amount of mock-conditioned medium, diluted 1:1 in fresh culture medium) was added to the cells. At 6 hpi, the inoculum was removed, fresh culture medium was added, and the cells were incubated for various times. All experiments were performed at least twice, and data presented in the present work are representative examples of those trials.

Western time course experiments.

Cells were synchronized, plated, and infected as described above. At the desired time points, cells were trypsinized, washed, and counted. Cells were then resuspended in Laemmli reducing sample buffer (LRSB) (2% sodium dodecyl sulfate [SDS], 10% glycerol, 100 mM dithiothreitol, 60 mM Tris pH 6.8, bromophenol blue dye, aprotinin and leupeptin [2 μg/ml each], and phenylmethylsulfonyl fluoride [1 mM]) at a concentration of 10⁴ cells/μl of buffer. Cells were then sonicated, boiled for 5 min, and spun at 16,000 × g for 10 min at 4°C to pellet any debris. Equal amounts of lysate (usually 25 to 30 μl) were then loaded onto an SDS-polyacrylamide gel electrophoresis minigel. Proteins were then transferred to Immobilon P (Millipore) and detected with mouse anti-p53 primary antibody (Ab) (DO-1) (Santa Cruz Biotechnology), followed by a goat anti-mouse secondary Ab conjugated with horseradish peroxidase (Amersham). Proteins were visualized with enhanced chemiluminescence reagents (Pierce), used according to the manufacturer’s instructions.

Metabolic labeling and pulse-chase immunoprecipitations.

Cells were synchronized, plated, and infected as described above. Prior to the radioactive pulse, normal culture medium was removed and cells were washed twice with phosphate-buffered saline (PBS) and refed with MEM lacking methionine but supplemented with 10% dialyzed fetal bovine serum. After 30 min, this starvation medium was removed and replaced with 3 ml of the same medium supplemented with 300 μCi of Tran³⁵S label (ICN) per plate. For metabolic labeling, cells were incubated in this labeling medium for a period of 2 h. The cells were then harvested as described below. For pulse-chase experiments, cells were incubated in labeling medium for 30 min and then one set of plates was harvested (pulse) and the other plates were washed twice with warm PBS, refed with normal culture medium supplemented with 1 mM cold methionine, and incubated for various lengths of time (chase). To harvest the cells, plates were washed twice in cold PBS and the cells were then scraped in PBS and transferred to 15-ml conical tubes. Cells were pelleted at 4°C, resuspended in 1 ml of cold PBS, and counted. Equal numbers of cells (approximately 5 × 10⁵) were then pelleted and resuspended in 100 μl of cold PBS. Three hundred microliters of RIPA buffer (50 mM Tris [pH 7.4], 120 mM NaCl, 0.1% deoxycholic acid, 2% Nonidet P-40, 0.2% SDS, aprotinin and leupeptin [2 μg/ml each], and dithiothreitol and phenylmethylsulfonyl fluoride [1 mM each]) was added, and the cells were sonicated for 2.5 min. After vortexing for an additional 10 min, the lysate was cleared with a 16,000 × g spin and transferred to a fresh tube containing an additional 600 μl of RIPA buffer. After gentle mixing, an aliquot was removed for analysis of total p53 concentration prior to incubation with Ab. The remaining lysate was mixed with 20 μl of agarose-coupled DO-1 Ab (Santa Cruz Biotechnology), an amount which we had determined would precipitate all of the p53 in the sample, and rocked for 2 h at 4°C. After incubation, 40 μl of a 50% protein A-Sepharose slurry was added, the beads were pelleted, and another aliquot was removed for determination of p53 concentration after incubation with Ab. The beads were washed three times in RIPA buffer, transferred to a fresh tube, and then washed once more. Proteins were eluted from the beads in LRSB and after boiling were electrophoresed through an SDS–10% polyacrylamide gel. Gels were stained and then subjected to fluorography, dried, and autoradiographed. Quantitation of bands was performed with a PhosphorImager (Molecular Dynamics).

Immunofluorescence.

Synchronized cells were seeded onto glass coverslips and infected as described above. At the desired time points, coverslips were washed twice in PBS and the cells were then fixed in a 3% paraformaldehyde solution in PBS. After 10 min, this was replaced with a 1% Triton X-100 solution in PBS. After a 5-min incubation, the cells were rinsed three times in PBS and then stored at 4°C until all samples were taken. Coverslips were then incubated for 10 min in blocking solution (1% normal goat serum in PBS–0.2% gelatin), washed in PBS, and incubated with one of several mouse primary Abs diluted in PBS–0.2% gelatin for 10 min. After extensive washing in PBS, the cells were then incubated with a fluorescein isothiocyanate-conjugated goat anti-mouse secondary Ab (Jackson Labs) (diluted in PBS–0.2% gelatin). Following more washes, the cells were finally incubated in Hoechst dye (Calbiochem) for 2 min to illuminate the DNA, washed and mounted onto slides using glycerol-paraphenylene diamine (an antiphotobleaching agent). For analysis of replication, cells were incubated for 30 min prior to harvesting at 48 hpi in bromodeoxyuridine (BrdU) labeling mix diluted in normal media. Coverslips were then harvested and fixed as described above. Prior to Ab detection, these coverslips were incubated in 4 M HCl diluted in PBS for 10 min to expose the BrdU residues. All slides were analyzed on a Nikon EFD-3 flourescence microscope equipped with Neofluor objectives and an MTT CCD2 camera, and images were captured by using NIH Image 1.59. Collected images were arranged and labeled by using Adobe Photoshop 3.0 and Adobe Illustrator 6.0.2. Monoclonal Abs used for fluorescence included commercially available anti-p53 (DO-1; Santa Cruz Biotechnology), anti-Rb (G3-245; Pharmingen), antitrinitrophenol (Pharmingen), and anti-BrdU (Boehringer Mannheim). Antibodies to HCMV UL44 and IE1 72 were kind gifts from L. Pereira and W. Britt, respectively. Anti-RPA was a kind gift from J. Wang. Note that attempts to colocalize proteins by double labeling using other commercially available Abs (raised in either rabbits or goats) proved unsuccessful due to their lack of avidity and/or their binding by the virally encoded immunoglobulin G Fc receptor-like protein.

RIPA buffer extractions.

Cells were harvested, counted, and pelleted in aliquots of 5 × 10⁵ cells/tube at 48 hpi. The pellets were then resuspended in 10 μl of PBS, to which was added 50 μl of RIPA buffer. Parallel sets of tubes were processed; one with and the other without a 2.5-min sonication before 10 min of vortexing. Thirty microliters of 3× LRSB was added to the cleared lysates, and the pelleted debris was resuspended in an equivalent amount of 1× LRSB. Samples were boiled, and one half of each tube was loaded and electrophoresed through an SDS–10% polyacrylamide gel and processed as described above for Western time course experiments.

Increased steady-state levels of p53 in infected fibroblasts are due to decreased degradation and not increased synthesis.

As we had previously reported (23), infection of fibroblasts with HCMV Towne strain induced elevated steady-state levels of p53 in infected cells relative to mock infected cells 24 to 36 hpi (Fig. 1A). These elevated steady-state levels could be due to an increase in the rate of synthesis and/or a decrease in the rate of degradation. In order to distinguish between these alternatives, we metabolically labeled cells every 2 h for the first 24 hpi and then again at 48 hpi. In our initial pilot experiments, we found that resuspension in RIPA buffer alone was not sufficient to extract all of the p53 from the infected cell nuclei (Fig. 1B). We therefore incorporated an additional sonication step in our lysis procedure to ensure complete extraction. Even under these extraction conditions, no burst of protein synthesis was found early in the infection that could account for the increased steady-state levels. A representative sample of these labelings is shown in Fig. 2A. In fact, when cells were pulse-labeled for 30 min at 48 hpi, we found that the synthesis levels of p53 in the infected cells were actually somewhat lower than in their mock-infected counterparts (approximately twofold) (Fig. 2B). Interestingly, analysis of the newly synthesized p53 during the chase period revealed a dramatic difference between the infected and uninfected cells; after an initial decrease to approximately half the incorporated label by 3 h, the labeled p53 in the infected cells became stabilized, with no further decrease observed for up to 6 h of chase. In contrast, the p53 in the mock-infected cells steadily decayed over the span of the experiment, going through approximately three half-lives in 6 h (half-life, ∼2 h). Preliminary experiments performed at 24 hpi revealed the same pattern of lower synthesis but extended stabilization as observed at 48 hpi (data not shown). Therefore, a stabilization rather than an increase in synthesis appeared to be the cause of the elevated steady-state levels of p53 observed in infected fibroblasts.

p53 is sequestered into discrete foci within the nuclei of infected cells.

The increased difficulty of extraction of p53 from the infected cell nuclei in the experiments described above led us to suspect that the subcellular localization of p53 might be altered in the infected fibroblasts. To investigate this possibility, cells were seeded onto glass coverslips and then infected or mock infected as described in Materials and Methods. At selected times postinfection, coverslips were removed, washed gently in PBS, fixed in paraformaldehyde, and then permeabilized with Triton X-100. We found this method of fixation coupled with the use of the DO-1 Ab to p53 to be the most reliable and reproducible combination for visualizing the staining pattern of p53 in both the mock-infected and infected cells. Surprisingly, we found that the p53 in infected cells was localized to discrete foci within the nuclei of virally infected cells beginning at approximately 30 hpi (Fig. 3a). These foci increased in size as the infection progressed and by 72 hpi almost completely covered the nucleus (data not shown). This pattern of staining was not apparent in mock-infected cells (Fig. 3b), where p53 was distributed uniformly throughout the entire nucleus. There was also no detectable staining observed when virally infected cells were incubated with a control Ab directed against trinitrophenol (Fig. 3c). These results support our hypothesis that a relocalization of p53 in the infected fibroblasts accounts for the increased difficulty of extraction and potentially for some of the stabilization of p53 observed in these cells.

p53 localizes to viral replication centers along with the HCMV protein UL44.

The staining pattern observed for p53 in the infected fibroblasts was reminiscent of that seen for proteins associated with viral replication. Closer examination of these foci in the virally infected cells by phase microscopy at 48 hpi revealed the distinct morphology of the viral replication centers produced by both HCMV and another herpesvirus, herpes simplex virus (HSV) (9) (arrows in Fig. 4b, f, and j). It was also apparent that these structures were not present within the mock-infected cell nuclei (Fig. 4d, h, and l).

To confirm that these structures were DNA replication centers, we stained for the HCMV protein UL44, an early protein that associates with the virally encoded DNA polymerase (48), is required for oriLyt dependent viral DNA replication (20, 37, 38), and localizes to these intranuclear inclusions during infection (22). Figure 4 shows that the UL44 protein also localized to these same structures within the virally infected nuclei (Fig. 4e and f [arrows]) and was not present in the mock-infected cells (Fig. 4g). These results confirmed earlier studies which colocalized UL44 and UL112-113, another class of viral proteins required for viral DNA replication (20, 37, 38), to these foci during HCMV infection (22, 51a). Moreover, by pulse-labeling the cells with BrdU, we were able to demonstrate that these foci were the primary sites of active DNA replication within the infected cells (Fig. 4i). We concluded from these results that the p53-containing foci in HCMV-infected cell nuclei were centers of viral replication.

Localization to replication centers is observed with some cellular and viral proteins but not others.

Previously, Wilcock and Lane (50) showed that HSV sequestered p53 as well as certain other host cell proteins, including Rb, PCNA, and RPA into viral replication centers. To determine whether HCMV also sequestered other cellular proteins into these centers and whether this might be a generalized phenomenon for cellular proteins normally present in the nucleus, we used indirect immunofluorescence staining to examine the viral and mock-infected fibroblasts at 30 hpi (Fig. 5); results obtained at 48 hpi were identical (data not shown). We focused on two cellular proteins: Rb, because we had previously observed modulation of both the steady-state levels and phosphorylation state of this protein upon infection, and RPA, because it is a protein involved in both cellular DNA replication and repair and is known to bind to p53. Figure 5 demonstrates that RPA but not Rb (Fig. 5e and a, respectively) localized to the replication centers. The viral immediate early protein IE1 72 (Fig. 5m) also did not localize to these foci, providing further evidence that there was specificity in the process. As noted above, the UL44 protein was clearly sequestered in these centers (Fig. 5i), and this localization could be observed as early as 24 hpi (data not shown). No foci were apparent in any of the mock-infected cells (Fig. 5c, g, k, and o). Furthermore, we observed the identical localization for each of these proteins in another permissive cell line, U373 (data not shown), providing evidence that this was not a cell type-specific phenomenon. Taken together, these results indicate that p53 and RPA are specifically sequestered into the viral replication centers of infected cells beginning at 30 hpi. This sequestering may in part contribute to the stabilization of p53 within infected fibroblasts.