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. 2017 Jan 27;13(1):e1006193.
doi: 10.1371/journal.ppat.1006193. eCollection 2017 Jan.

Synthetic lethal mutations in the cyclin A interface of human cytomegalovirus

Affiliations

Synthetic lethal mutations in the cyclin A interface of human cytomegalovirus

Henry Weisbach et al. PLoS Pathog. .

Abstract

Generally, the antagonism between host restriction factors and viral countermeasures decides on cellular permissiveness or resistance to virus infection. Human cytomegalovirus (HCMV) has evolved an additional level of self-imposed restriction by the viral tegument protein pp150. Depending on a cyclin A-binding motif, pp150 prevents the onset of viral gene expression in the S/G2 cell cycle phase of otherwise fully permissive cells. Here we address the physiological relevance of this restriction during productive HCMV infection by employing a cyclin A-binding deficient pp150 mutant virus. One consequence of unrestricted viral gene expression in S/G2 was the induction of a G2/M arrest. G2-arrested but not mitotic cells supported viral replication. Cyclin A destabilization by the viral gene product pUL21a was required to maintain the virus-permissive G2-arrest. An HCMV double-point mutant where both pp150 and pUL21a are disabled in cyclin A interaction forced mitotic entry of the majority of infected cells, with a severe negative impact on cell viability and virus growth. Thus, pp150 and pUL21a functionally cooperate, together building a cell cycle synchronization strategy of cyclin A targeting and avoidance that is essential for productive HCMV infection.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. The essential function of pp150 in the late phase of HCMV infection is not compromised by lack of cylin A binding.
Density-arrested fibroblasts were infected with the indicated recombinant viruses. (A) The cellular DNA content was analyzed at regular intervals by propidium iodide staining and flow cytometry. Shown are DNA histograms where a 2n (n = haploid number of chromosomes) DNA content is indicative of G0/G1 and a 4n DNA content of G2/M cells. Where indicated, ganciclovir (GCV) was added to the cells at day 1 post infection (dpi) to discriminate between viral and cellular DNA replication. (B) The expression of selected IE, early and late gene products was monitored by immunoblot analysis of whole cell lysates; loading control: GAPDH; hpi: hours post infection. (C) To obtain virus growth curves, cell culture supernatants were collected on a daily basis and analyzed for infectious titers. Data points are displayed on a logarithmic scale and represent means and standard deviations of biological triplicates.
Fig 2
Fig 2. Loss of pp150-cyclin A interaction leads to an HCMV-permissive G2 arrest.
Fibroblast cultures were partially synchronized in early S phase and infected with pp150-WT or pp150-RXL mutant viruses. Where indicated, cells were pulse-labeled with 5-ethynyl-2’-deoxyuridine (EdU) 60 min before infection to distinguish S phase from G1 cells. (A) Cell cycle progression of infected and non-infected control cells was analyzed by flow cytometry. (B) Distribution over G0/1, S and G2/M cell cycle phases was calculated and presented as means and standard deviations of biological triplicates. (C) To assess cell division, cell numbers were monitored over time. (D) At 24 h, HCMV-pp150-RXLmut-infected cells were either treated with ganciclovir to inhibit viral DNA synthesis or left untreated. DNA histograms were obtained by flow cytometry. (E) EdU incorporation and the expression of selected viral immediate early (IE1/2), early (gB) and late (pp28) gene products were determined by flow cytometry at different time points post infection. EdU-positive cells are displayed in red, EdU-negative cells in gray. (F) IE-positive cells were analyzed by flow cytometry for phosphorylation of histone H3 at serine 10 (pH3(ser10)), a marker of mitotic chromatin condensation. The relative proportion of mitotic cells (mitotic index) is given in percent of total cells. (G) The averages and standard deviations of mitotic indices were calculated from six independent experiments. Statistically significant differences, based on a two-tailed, paired Student’s t test, are marked with asterisks; ** (p < .01); * (p < .05).
Fig 3
Fig 3. Pp150 and pUL21a cooperate to prevent HCMV-infected cells from entering a non-productive and unstable mitotic state.
Fibroblasts were first synchronized in early S phase and then (at time point 0 h) infected with the indicated recombinant viruses. Cells and cell culture supernatants were harvested at regular intervals and analyzed for cell numbers, cell cycle distribution and virus growth. (A) DNA histograms of propidium iodide stained cells. (B) To determine mitotic indices of infected cells, the relative numbers of pH3(ser10) and IE1/2-positive cells were quantified by flow cytometry. Data represent the means and standard deviations of biological triplicates. (C) For virus growth curves, titers of infectious virus progeny were determined in biological triplicates. (D) The numbers of viable, propidium iodide excluding cells were determined by flow cytometry. (E) The observations were summarized into a model that visualizes the chain of events after S phase infection, leading to either a virus-permissive G1/G2 cell cycle arrest or a non-permissive mitotic state and cell death. Different numbers of asterisks, symbolizing virus progeny, indicate how efficient each virus can replicate in the particular cell cycle phases. In the case of the pUL21a-RXL mutant, it is currently unclear to what extent S phase infected cells contribute to G1, G2 and M phase arrested populations.

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This work was supported by grant WI2043/3-1 from the Deutsche Forschungsgemeinschaft (URL: http://www.dfg.de) to LW and CH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.