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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

Precocious separation of sister chromatids and progressive chromosome fragmentation predominate the chromosomal appearance of HCMV-UL21a-RXL2mut-infected cells.(A) Metaphase spreads from nocodazole-treated, non-infected cells were subjected to Giemsa staining and compared to equally prepared chromosomal material of HCMV-wt and HCMV-UL21a-RXL2mut-infected cells at 2 to 4 days post infection (dpi). Where indicated, magnified views of the encircled areas #1 and #2 in the adjacent image of RXL2mut-infected cells are shown. Scale bars: 5 µm. (B) Metaphase spreads were analyzed by fluorescence in situ hybridization (FISH) using whole chromosome painting probes for chromosomes 1 and 3. DNA was counterstained with DAPI. Typical examples of cells with intact metaphase chromosomes (IMC), fragmented chromosomes (FC), incomplete chromosome condensation (ICC) or precocious separation of sister chromatids (PSC) are shown and labeled accordingly. (C) Quantitative evaluation of FISH analysis based on at least 100 mitotic HCMV-UL21a-RXL2mut-infected cells per sample. Non-mitotic cells were not included in the analysis.
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ppat-1004514-g009: Precocious separation of sister chromatids and progressive chromosome fragmentation predominate the chromosomal appearance of HCMV-UL21a-RXL2mut-infected cells.(A) Metaphase spreads from nocodazole-treated, non-infected cells were subjected to Giemsa staining and compared to equally prepared chromosomal material of HCMV-wt and HCMV-UL21a-RXL2mut-infected cells at 2 to 4 days post infection (dpi). Where indicated, magnified views of the encircled areas #1 and #2 in the adjacent image of RXL2mut-infected cells are shown. Scale bars: 5 µm. (B) Metaphase spreads were analyzed by fluorescence in situ hybridization (FISH) using whole chromosome painting probes for chromosomes 1 and 3. DNA was counterstained with DAPI. Typical examples of cells with intact metaphase chromosomes (IMC), fragmented chromosomes (FC), incomplete chromosome condensation (ICC) or precocious separation of sister chromatids (PSC) are shown and labeled accordingly. (C) Quantitative evaluation of FISH analysis based on at least 100 mitotic HCMV-UL21a-RXL2mut-infected cells per sample. Non-mitotic cells were not included in the analysis.

Mentions: Giemsa staining revealed further chromosomal abnormalities, contrasting with the characteristic X-shaped chromosomal appearance of prometaphase-arrested control cells (Fig. 9A, upper left panel). Besides chromosomal fragmentation, HCMV-UL21a-RXL2mut-infected, mitotic cells displayed incompletely condensed chromosomes (Fig. 9A, magnified view #2) or showed signs of precocious separation of sister chromatids (Fig. 9A, magnified view #1). In addition, the extent of chromosomal fragmentation increased over time and “pulverized” chromosomes dominated the picture of mitotic cells at 3–4 days post infection (Fig. 9A, lower panel). Notably, the remaining non-mitotic HCMV-UL21a-RXL2mut-infected cells appeared at this time with fully developed viral replication compartments, visible as dark-stained intranuclear regions. This is in accordance with the observation of viral DNA replication in the G2-arrested, non-mitotic subpopulation (Fig. 5B, Fig. S1) and with the only moderate growth defect of pUL21a-Cyclin A2 binding-deficient viruses (Fig. 3C). It appears that not Cyclin A2 expression per se but mitotic entry had deleterious consequences for HCMV-infected cells.


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)

Precocious separation of sister chromatids and progressive chromosome fragmentation predominate the chromosomal appearance of HCMV-UL21a-RXL2mut-infected cells.(A) Metaphase spreads from nocodazole-treated, non-infected cells were subjected to Giemsa staining and compared to equally prepared chromosomal material of HCMV-wt and HCMV-UL21a-RXL2mut-infected cells at 2 to 4 days post infection (dpi). Where indicated, magnified views of the encircled areas #1 and #2 in the adjacent image of RXL2mut-infected cells are shown. Scale bars: 5 µm. (B) Metaphase spreads were analyzed by fluorescence in situ hybridization (FISH) using whole chromosome painting probes for chromosomes 1 and 3. DNA was counterstained with DAPI. Typical examples of cells with intact metaphase chromosomes (IMC), fragmented chromosomes (FC), incomplete chromosome condensation (ICC) or precocious separation of sister chromatids (PSC) are shown and labeled accordingly. (C) Quantitative evaluation of FISH analysis based on at least 100 mitotic HCMV-UL21a-RXL2mut-infected cells per sample. Non-mitotic cells were not included in the analysis.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4231158&req=5

ppat-1004514-g009: Precocious separation of sister chromatids and progressive chromosome fragmentation predominate the chromosomal appearance of HCMV-UL21a-RXL2mut-infected cells.(A) Metaphase spreads from nocodazole-treated, non-infected cells were subjected to Giemsa staining and compared to equally prepared chromosomal material of HCMV-wt and HCMV-UL21a-RXL2mut-infected cells at 2 to 4 days post infection (dpi). Where indicated, magnified views of the encircled areas #1 and #2 in the adjacent image of RXL2mut-infected cells are shown. Scale bars: 5 µm. (B) Metaphase spreads were analyzed by fluorescence in situ hybridization (FISH) using whole chromosome painting probes for chromosomes 1 and 3. DNA was counterstained with DAPI. Typical examples of cells with intact metaphase chromosomes (IMC), fragmented chromosomes (FC), incomplete chromosome condensation (ICC) or precocious separation of sister chromatids (PSC) are shown and labeled accordingly. (C) Quantitative evaluation of FISH analysis based on at least 100 mitotic HCMV-UL21a-RXL2mut-infected cells per sample. Non-mitotic cells were not included in the analysis.
Mentions: Giemsa staining revealed further chromosomal abnormalities, contrasting with the characteristic X-shaped chromosomal appearance of prometaphase-arrested control cells (Fig. 9A, upper left panel). Besides chromosomal fragmentation, HCMV-UL21a-RXL2mut-infected, mitotic cells displayed incompletely condensed chromosomes (Fig. 9A, magnified view #2) or showed signs of precocious separation of sister chromatids (Fig. 9A, magnified view #1). In addition, the extent of chromosomal fragmentation increased over time and “pulverized” chromosomes dominated the picture of mitotic cells at 3–4 days post infection (Fig. 9A, lower panel). Notably, the remaining non-mitotic HCMV-UL21a-RXL2mut-infected cells appeared at this time with fully developed viral replication compartments, visible as dark-stained intranuclear regions. This is in accordance with the observation of viral DNA replication in the G2-arrested, non-mitotic subpopulation (Fig. 5B, Fig. S1) and with the only moderate growth defect of pUL21a-Cyclin A2 binding-deficient viruses (Fig. 3C). It appears that not Cyclin A2 expression per se but mitotic entry had deleterious consequences for HCMV-infected cells.

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