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. 2010 Mar 19;6(3):e1000814.
doi: 10.1371/journal.ppat.1000814.

Human cytomegalovirus protein pUL117 targets the mini-chromosome maintenance complex and suppresses cellular DNA synthesis

Zhikang Qian  1 , Van Leung-Pineda, Baoqin Xuan, Helen Piwnica-Worms, Dong Yu
Affiliations

Human cytomegalovirus protein pUL117 targets the mini-chromosome maintenance complex and suppresses cellular DNA synthesis

Zhikang Qian et al. PLoS Pathog. .

Abstract

Modulation of host DNA synthesis is essential for many viruses to establish productive infections and contributes to viral diseases. Human cytomegalovirus (HCMV), a large DNA virus, blocks host DNA synthesis and deregulates cell cycle progression. We report that pUL117, a viral protein that we recently identified, is required for HCMV to block host DNA synthesis. Mutant viruses in which pUL117 was disrupted, either by frame-shift mutation or by a protein destabilization-based approach, failed to block host DNA synthesis at times after 24 hours post infection in human foreskin fibroblasts. Furthermore, pUL117-deficient virus stimulated quiescent fibroblasts to enter S-phase, demonstrating the intrinsic ability of HCMV to promote host DNA synthesis, which was suppressed by pUL117. We examined key proteins known to be involved in inhibition of host DNA synthesis in HCMV infection, and found that many were unlikely involved in the inhibitory activity of pUL117, including geminin, cyclin A, and viral protein IE2, based on their expression patterns. However, the ability of HCMV to delay the accumulation of the mini-chromosome maintenance (MCM) complex proteins, represented by MCM2 and MCM4, and prevent their loading onto chromatin, was compromised in the absence of pUL117. When expressed alone, pUL117 slowed cell proliferation, delayed DNA synthesis, and inhibited MCM accumulation. Knockdown of MCM proteins by siRNA restored the ability of pUL117-deficient virus to block cellular DNA synthesis. Thus, targeting MCM complex is one mechanism pUL117 employs to help block cellular DNA synthesis during HCMV infection. Our finding substantiates an emerging picture that deregulation of MCM is a conserved strategy for many viruses to prevent host DNA synthesis and helps to elucidate the complex strategy used by a large DNA virus to modulate cellular processes to promote infection and pathogenesis.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. UL117-deficient virus failed to block host DNA synthesis during infection of actively growing human foreskin fibroblasts (HFFs).
Actively growing HFFs were mock-infected, or infected with wild type HCMV (wt), or pUL117-deficient virus (dlpUL117), and nocodozale was added 8 hours post infection (hpi). At 48 hpi, cells were stained with propidium iodide (PI) and their DNA content was determined by flow cytometry based cell cycle analysis. (A) HFFs that were mock-infected or infected with recombinant HCMV without gancyclovir (GCV) treatment. (B) HFFs that were infected in the presence of GCV at indicated concentrations.
Figure 2
Figure 2. pUL117 was required to block host DNA synthesis of HCMV-infected cells at late times during infection.
Actively growing HFFs were mock-infected, or infected with wild type HCMV (ADwt), or pUL117-deficient virus (ADdlpUL117), and nocodozale was added 8 hpi. (A) Cells were sorted by HCMV-driven GFP expression at 48 hpi, stained with PI, and GFP+ cells were analyzed for their DNA content by flow cytometry. (B) Cells were fixed and labeled with the antibody to viral protein pUL44 (α-pUL44) at 48 hpi. Cellular distributions of GFP and pUL44 signals were analyzed by two-color flow cytometry. Specificity of GFP or pUL44 signal was demonstrated by analyzing infection of recombinant HCMV ADwt-cre that did not express GFP or ADwt without pUL44 staining, respectively. (C) Cells were infected in the presence of GCV (30µg/ml), double-stained with PI and the antibody to viral proteins IE1/IE2 at 24 and 48 hpi, and IE1/IE2+ cells were determined for their DNA content.
Figure 3
Figure 3. pUL117 was required to block host DNA synthesis of G0-synchronized HFFs during HCMV infection.
Subconfluent HFFs were synchronized at G0 by serum starvation and then infected with recombinant HCMV in the presence of serum. Nocodazole was added at 8 hpi. (A and B) Cells were infected in the presence of GCV (30µg/ml), double-stained with PI and α-pUL44 at 24 and 48 hpi, and analyzed by flow cytometry for their DNA content and pUL44 expression. Shown is a representative of three reproducible, independent experiments. (A) Signal profiles of pUL44 (x-axis) and PI (y-axis) of mock- or virus- infected cells at 48 hpi. Cells within gate R3 or R4 represents pUL44-negative or pUL44-positive cells, respectively. (B) Mock-infected cells in R3 and virus-infected cells in R4 were analyzed for their DNA content. Percentages of cells in S-phase or G2/M-phases were shown as solid or open bars, respectively. (C) The rate of DNA synthesis of infected cells with or without GCV (30µg/ml) was measured by [3H] thymidine incorporation at 48 hpi. Shown are relative amounts of incorporated [3H] thymidine in each sample with thymidine incorporation in mock-infected cells in the absence of GCV set at 100%. (D) Accumulation of viral DNA in infected cells with or without GCV (30µg/ml) was measured by realtime-quantitative PCR at 48 hpi.
Figure 4
Figure 4. pUL117-deficient virus stimulated host DNA synthesis of quiescent HFFs in the absence of serum.
HFFs were synchronized at G0 by serum starvation and contact inhibition, and then infected with recombinant HCMV with GCV (30µg/ml) but without serum. (A) Nocodazole was added at 8 hpi and the percentage of IE1/IE2+ cells in S-phase was determined by flow cytometry at indicated times post infection. Shown is a representative of three reproducible, independent experiments. (B) The rate of DNA synthesis of infected HFFs at 72 or 96 hpi was measured by [3H] thymidine incorporation. *, P<0.05; **, P<0.01 by Student's t test.
Figure 5
Figure 5. Lack of newly synthesized pUL117 was directly responsible for the failure of pUL117-deficient virus to block host DNA synthesis.
Recombinant HCMV virus ADpFKBP-UL117 carried a functional pUL117 variant that was tagged with a FKBP destabilization domain at its N terminus (pFKBP-UL117). HFFs were infected with ADpFKBP-UL117 with or without stabilizing ligand Shld1 (1µM). Nocodozale was added at 8 hpi, infected cells were harvested at 48 hpi, the accumulation of pUL117 or pFKBP-UL117 was determined by immunoblotting analysis (A), and the DNA content by flow cytometry (B). Percentages of cells in S-phase or G2/M-phases were shown as solid or open bars, respectively.
Figure 6
Figure 6. pUL117-deficient virus failed to block accumulation and chromatin loading of MCM components.
Subconfluent HFFs were synchronized at G0 by serum starvation, and then infected with recombinant HCMV in the presence of serum. (A) At indicated times, total lysate or chromatin fractions from equal number of cells were analyzed by immunoblotting for viral and cellular factors known to modulate the cell cycle and DNA replication. Also shown were pUL117-related proteins and actin control. (B) Amounts of MCM2 and MCM4 in total cell lysates or chromatin fractions from equal number of infected cells at 24 and 30 hpi were quantified as described in Materials and Methods. The ratio of MCM in mutant virus infection to that in wild type virus infection was calculated. Shown are the averages of the results from three independent sets of infections. *, P<0.05; **, P<0.01 by Student's t test. (C) The completion of fractionation was determined by immunoblotting of viral protein IE1 and cellular protein Lamin A in soluble and chromatin fractions. (D) At indicated times post infection, RNA from infected cells was extracted, treated with TURBO DNA-free reagent (Ambion) to remove contaminating DNA, and the amounts of MCM4 and GAPDH control transcripts were determined by reverse transcription-couple realtime-quantitative PCR analysis. All data sets were within the linear ranges of standard curves shown in Figure S5. The amount of MCM4 transcript was then normalized to that of GAPDH transcript. For each time point, the normalized amount of MCM4 transcript in mock infection was set at 1.
Figure 7
Figure 7. pUL117 was not required for premature accumulation of APC substrates in HCMV infection.
Subconfluent HFFs were synchronized at G0 by serum starvation, and then infected with recombinant HCMV in the presence of serum. At indicated times, total lysate from equal number of cells were analyzed by immunoblotting for APC substrates cdc6, cyclin B1, and geminin. Also shown were pUL117-related proteins, IE1 and actin control.
Figure 8
Figure 8. pUL117 was sufficient to inhibit proliferation and cellular DNA replication.
HFFs were transduced with retro-viral vector either expressing GFP (HF-GFP) or pUL117 (HF-GFP/UL117) as previously described . (A) Cells were synchronized at G0 by serum starvation, stimulated into the cell cycle by sub-confluent re-seeding with serum, and total cell lysate or chromatin fractions from equal number of cells were examined by immunoblot analysis at indicated times. (B) MCM2 and MCM4 accumulation and their chromatin loading were quantified as described in the legend to Fig. 6B. The ratio of MCM in GFP-transduced cells to that in GFP/UL117-transduced cells was calculated. Shown are the averages of the results from three independent experiments. *, P<0.05; **, P<0.01; n.s. (not significant), P>0.05 by Student's t test. (C) At 24 hpi, the rate of DNA synthesis was measured by [3H] thymidine incorporation. (D) In a parallel experiment, equal numbers of HF-GFP and HF-GFP/UL117 were re-seeded at sub-confluency with serum and their proliferations were determined by scoring cell numbers during 24–120 h.
Figure 9
Figure 9. Knockdown of MCM2 and MCM4 restores the ability of pUL117-deficient virus to block host DNA synthesis.
HFFs were transfected with a negative control siRNA or siRNAs targeting MCM2 and MCM4. (A) Immunoblot analysis of MCM 2 and MCM4 at 48 hours after siRNA transfection. (B) Transfected cells were starved and infected in the presence of GCV (30µg/ml) as described in Materials and Methods, double-stained with PI and α-pUL44 at 48 hpi, and pUL44+ cells were determined for their DNA content. Percentages of cells in S-phase or G2/M-phases were shown as solid or open bars, respectively. Shown is a representative from three reproducible, independent experiments.
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