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PUL21a-Cyclin A2 interaction is required to protect human cytomegalovirus-infected cells from the deleterious consequences of mitotic entry.

Eifler M, Uecker R, Weisbach H, Bogdanow B, Richter E, König L, Vetter B, Lenac-Rovis T, Jonjic S, Neitzel H, Hagemeier C, Wiebusch L - PLoS Pathog. (2014)

Bottom Line: Remarkably, the essential viral IE2, but not the related chromosome-associated IE1 protein, disappeared upon mitotic entry, suggesting an inherent instability of IE2 under mitotic conditions.Viral DNA synthesis was impaired in mitosis, as demonstrated by the abnormal morphology and strongly reduced BrdU incorporation rates of viral replication compartments.We conclude that the Cyclin A2-binding function of pUL21a contributes to the maintenance of a cell cycle state conducive for the completion of the HCMV replication cycle.

View Article: PubMed Central - PubMed

Affiliation: Labor für Pädiatrische Molekularbiologie, Charité Universitätsmedizin Berlin, Berlin, Germany.

ABSTRACT
Entry into mitosis is accompanied by dramatic changes in cellular architecture, metabolism and gene expression. Many viruses have evolved cell cycle arrest strategies to prevent mitotic entry, presumably to ensure sustained, uninterrupted viral replication. Here we show for human cytomegalovirus (HCMV) what happens if the viral cell cycle arrest mechanism is disabled and cells engaged in viral replication enter into unscheduled mitosis. We made use of an HCMV mutant that, due to a defective Cyclin A2 binding motif in its UL21a gene product (pUL21a), has lost its ability to down-regulate Cyclin A2 and, therefore, to arrest cells at the G1/S transition. Cyclin A2 up-regulation in infected cells not only triggered the onset of cellular DNA synthesis, but also promoted the accumulation and nuclear translocation of Cyclin B1-CDK1, premature chromatin condensation and mitotic entry. The infected cells were able to enter metaphase as shown by nuclear lamina disassembly and, often irregular, metaphase spindle formation. However, anaphase onset was blocked by the still intact anaphase promoting complex/cyclosome (APC/C) inhibitory function of pUL21a. Remarkably, the essential viral IE2, but not the related chromosome-associated IE1 protein, disappeared upon mitotic entry, suggesting an inherent instability of IE2 under mitotic conditions. Viral DNA synthesis was impaired in mitosis, as demonstrated by the abnormal morphology and strongly reduced BrdU incorporation rates of viral replication compartments. The prolonged metaphase arrest in infected cells coincided with precocious sister chromatid separation and progressive fragmentation of the chromosomal material. We conclude that the Cyclin A2-binding function of pUL21a contributes to the maintenance of a cell cycle state conducive for the completion of the HCMV replication cycle. Unscheduled mitotic entry during the course of the HCMV replication has fatal consequences, leading to abortive infection and cell death.

No MeSH data available.


Related in: MedlinePlus

Cyclin A2 up-regulation results in mitotic entry of infected cells.Cells were infected as described in the legend to figure 3. (A) At 72 hpi, their morphology was examined by phase-contrast microscopy to detect cell rounding. (B, C) Cells were further stained with propidium iodide, IE1/IE2-and phospho-histone H3(Ser10)-specific antibodies and analyzed by flow cytometry to determine the proportion of infected cells with (pre-)mitotic chromosome condensation. The results of one representative experiment (A, B) and of five independent experiments (C) are shown.
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ppat-1004514-g005: Cyclin A2 up-regulation results in mitotic entry of infected cells.Cells were infected as described in the legend to figure 3. (A) At 72 hpi, their morphology was examined by phase-contrast microscopy to detect cell rounding. (B, C) Cells were further stained with propidium iodide, IE1/IE2-and phospho-histone H3(Ser10)-specific antibodies and analyzed by flow cytometry to determine the proportion of infected cells with (pre-)mitotic chromosome condensation. The results of one representative experiment (A, B) and of five independent experiments (C) are shown.

Mentions: We then examined whether the Cyclin A2-induced progression through S-phase is followed by mitotic entry of infected cells. Entry into mitosis is marked by cell rounding and chromatin condensation and therefore can be readily visualized by phase contrast microscopy and DAPI staining. Both methods revealed a high number of mitotic cells at 48 h after infection with the UL21a-RXL2 mutant virus (Fig. 4C, 5A). To quantitatively assess the number of mitotic cells, we performed flow cytometry of histone H3-serine 10 phosphorylation (pH3ser10), a marker of condensed chromatin. Parallel analysis of DNA content and IE1/2 protein expression in combination with an appropriate gating strategy ensured that only single, IE-positive cells were evaluated (Fig. S2). We found that the percentage of HCMV-UL21a-RXL2mut-infected cells displaying H3(ser10) phosphorylation increased from about 20% at day 2 to almost 30% at day 3 before it declined again to just under 10% at day 5 (Fig. 5B–C). Some cells had undergone chromatin condensation prematurely, i.e. before completion of DNA replication (Fig. 5B). In case of UL21a deletion, the proportion of mitotic cells was also increased compared to HCMV-WT, but remained constantly below 3% and showed no signs of premature mitotic entry. This low rate of mitosis is well supported by the only moderate strength of Cyclin B1 induction in these cells (Fig. 3A). As it is rather unusual that a point mutation has a stronger phenotype than a whole gene knockout, it was important to ensure that cyclin up-regulation and mitotic entry were not caused by unwanted secondary mutations in the genome of the UL21a-RXL2-mutant virus. We therefore repaired the RXL2 mutation and analyzed the cell cycle effects of the UL21a-RXL2-revertant virus. Cyclin A2 and B1 protein expression as well as mitotic cell numbers were reverted to HCMV-WT levels (Fig. S3), demonstrating that the observed changes are specifically linked to the pUL21a-RXL2 motif. We concluded that pUL21a-Cyclin A2 interaction is not only required for the inhibition DNA synthesis in HCMV-infected cells but also for the inhibition of mitotic entry.


PUL21a-Cyclin A2 interaction is required to protect human cytomegalovirus-infected cells from the deleterious consequences of mitotic entry.

Eifler M, Uecker R, Weisbach H, Bogdanow B, Richter E, König L, Vetter B, Lenac-Rovis T, Jonjic S, Neitzel H, Hagemeier C, Wiebusch L - PLoS Pathog. (2014)

Cyclin A2 up-regulation results in mitotic entry of infected cells.Cells were infected as described in the legend to figure 3. (A) At 72 hpi, their morphology was examined by phase-contrast microscopy to detect cell rounding. (B, C) Cells were further stained with propidium iodide, IE1/IE2-and phospho-histone H3(Ser10)-specific antibodies and analyzed by flow cytometry to determine the proportion of infected cells with (pre-)mitotic chromosome condensation. The results of one representative experiment (A, B) and of five independent experiments (C) are shown.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4231158&req=5

ppat-1004514-g005: Cyclin A2 up-regulation results in mitotic entry of infected cells.Cells were infected as described in the legend to figure 3. (A) At 72 hpi, their morphology was examined by phase-contrast microscopy to detect cell rounding. (B, C) Cells were further stained with propidium iodide, IE1/IE2-and phospho-histone H3(Ser10)-specific antibodies and analyzed by flow cytometry to determine the proportion of infected cells with (pre-)mitotic chromosome condensation. The results of one representative experiment (A, B) and of five independent experiments (C) are shown.
Mentions: We then examined whether the Cyclin A2-induced progression through S-phase is followed by mitotic entry of infected cells. Entry into mitosis is marked by cell rounding and chromatin condensation and therefore can be readily visualized by phase contrast microscopy and DAPI staining. Both methods revealed a high number of mitotic cells at 48 h after infection with the UL21a-RXL2 mutant virus (Fig. 4C, 5A). To quantitatively assess the number of mitotic cells, we performed flow cytometry of histone H3-serine 10 phosphorylation (pH3ser10), a marker of condensed chromatin. Parallel analysis of DNA content and IE1/2 protein expression in combination with an appropriate gating strategy ensured that only single, IE-positive cells were evaluated (Fig. S2). We found that the percentage of HCMV-UL21a-RXL2mut-infected cells displaying H3(ser10) phosphorylation increased from about 20% at day 2 to almost 30% at day 3 before it declined again to just under 10% at day 5 (Fig. 5B–C). Some cells had undergone chromatin condensation prematurely, i.e. before completion of DNA replication (Fig. 5B). In case of UL21a deletion, the proportion of mitotic cells was also increased compared to HCMV-WT, but remained constantly below 3% and showed no signs of premature mitotic entry. This low rate of mitosis is well supported by the only moderate strength of Cyclin B1 induction in these cells (Fig. 3A). As it is rather unusual that a point mutation has a stronger phenotype than a whole gene knockout, it was important to ensure that cyclin up-regulation and mitotic entry were not caused by unwanted secondary mutations in the genome of the UL21a-RXL2-mutant virus. We therefore repaired the RXL2 mutation and analyzed the cell cycle effects of the UL21a-RXL2-revertant virus. Cyclin A2 and B1 protein expression as well as mitotic cell numbers were reverted to HCMV-WT levels (Fig. S3), demonstrating that the observed changes are specifically linked to the pUL21a-RXL2 motif. We concluded that pUL21a-Cyclin A2 interaction is not only required for the inhibition DNA synthesis in HCMV-infected cells but also for the inhibition of mitotic entry.

Bottom Line: Remarkably, the essential viral IE2, but not the related chromosome-associated IE1 protein, disappeared upon mitotic entry, suggesting an inherent instability of IE2 under mitotic conditions.Viral DNA synthesis was impaired in mitosis, as demonstrated by the abnormal morphology and strongly reduced BrdU incorporation rates of viral replication compartments.We conclude that the Cyclin A2-binding function of pUL21a contributes to the maintenance of a cell cycle state conducive for the completion of the HCMV replication cycle.

View Article: PubMed Central - PubMed

Affiliation: Labor für Pädiatrische Molekularbiologie, Charité Universitätsmedizin Berlin, Berlin, Germany.

ABSTRACT
Entry into mitosis is accompanied by dramatic changes in cellular architecture, metabolism and gene expression. Many viruses have evolved cell cycle arrest strategies to prevent mitotic entry, presumably to ensure sustained, uninterrupted viral replication. Here we show for human cytomegalovirus (HCMV) what happens if the viral cell cycle arrest mechanism is disabled and cells engaged in viral replication enter into unscheduled mitosis. We made use of an HCMV mutant that, due to a defective Cyclin A2 binding motif in its UL21a gene product (pUL21a), has lost its ability to down-regulate Cyclin A2 and, therefore, to arrest cells at the G1/S transition. Cyclin A2 up-regulation in infected cells not only triggered the onset of cellular DNA synthesis, but also promoted the accumulation and nuclear translocation of Cyclin B1-CDK1, premature chromatin condensation and mitotic entry. The infected cells were able to enter metaphase as shown by nuclear lamina disassembly and, often irregular, metaphase spindle formation. However, anaphase onset was blocked by the still intact anaphase promoting complex/cyclosome (APC/C) inhibitory function of pUL21a. Remarkably, the essential viral IE2, but not the related chromosome-associated IE1 protein, disappeared upon mitotic entry, suggesting an inherent instability of IE2 under mitotic conditions. Viral DNA synthesis was impaired in mitosis, as demonstrated by the abnormal morphology and strongly reduced BrdU incorporation rates of viral replication compartments. The prolonged metaphase arrest in infected cells coincided with precocious sister chromatid separation and progressive fragmentation of the chromosomal material. We conclude that the Cyclin A2-binding function of pUL21a contributes to the maintenance of a cell cycle state conducive for the completion of the HCMV replication cycle. Unscheduled mitotic entry during the course of the HCMV replication has fatal consequences, leading to abortive infection and cell death.

No MeSH data available.


Related in: MedlinePlus