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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

The pUL21a-RXL/Cy motif is required for Cyclin A2 repression by HCMV.Density arrested human embryonic lung (HEL) fibroblasts were infected with HCMV reconstituted from TB40-BAC4-wt or derivatives carrying the indicated UL21a mutations. Cells were harvested at regular intervals and analyzed for viral and cellular gene expression by immunoblotting (A) and ribonuclease protection assay (B). Cell culture supernatants were analyzed for the number of IE protein-forming units (IU) by virus titration (C). Results are represented as mean values plus standard deviations of triplicate samples.
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ppat-1004514-g003: The pUL21a-RXL/Cy motif is required for Cyclin A2 repression by HCMV.Density arrested human embryonic lung (HEL) fibroblasts were infected with HCMV reconstituted from TB40-BAC4-wt or derivatives carrying the indicated UL21a mutations. Cells were harvested at regular intervals and analyzed for viral and cellular gene expression by immunoblotting (A) and ribonuclease protection assay (B). Cell culture supernatants were analyzed for the number of IE protein-forming units (IU) by virus titration (C). Results are represented as mean values plus standard deviations of triplicate samples.

Mentions: We then set out to investigate the functional consequences of pUL21a-Cyclin A2 interaction in the context of viral infection. To this end we introduced the RXL2 mutation into the HCMV genome by traceless BAC-mutagenesis and analyzed the recombinant virus in direct comparison to WT, UL21a deletion (ΔUL21a) and UL21a-PRAA mutant (APCmut) viruses. To ensure a synchronous start of lytic gene expression in infected cells, we used a high multiplicity of infection (MOI) and fibroblasts that were arrested in G0/G1 by contact inhibition. Accordingly, except the G1-specific Cyclin D1, cell cycle factors were barely detectable at the time of infection (Fig. 3A). Whereas Cyclin D1 expression was largely unaffected by HCMV infection and all four viruses led to a strong and sustained induction of Cyclin E1, they markedly differed with respect to Cyclin A2, Cyclin B1 and APC5 protein regulation. Both UL21a deletion and RXL2 point mutation abolished the characteristic block of Cyclin A2 expression observed in HCMV-WT infected cells. Whereas Cyclin A2 mRNA levels were only slightly elevated (see Fig. 3B), the effect was most pronounced at the level of protein expression, consistent with the observed proteasome dependency of Cyclin A2 down-regulation in pUL21a-transfected cells (Fig. 2). Remarkably, the RXL2 point mutation resulted in considerably higher peak levels of Cyclin A2 protein expression than the deletion of the whole UL21a open reading frame. This may be explained by the fact that the APC/C inhibitory function of UL21a is not affected by the loss of Cyclin A2 binding (see APC5 levels in Fig. 3A) and thus can contribute to Cyclin A2 stabilization. Cyclin A2 induction in HCMV-UL21a-RXL2mut-infected cells was followed by a strong up-regulation of Cyclin B1 mRNA and protein expression, most likely due to Cyclin A2-dependent transcriptional activation of the Cyclin B1 promoter [23]. Again, the effects of UL21a deletion were much weaker, hardly exceeding the moderate levels of Cyclin B1 induction in HCMV-WT-infected cells. Thus, the simultaneous loss of Cyclin A2 and APC/C-binding sites in pUL21a masks the full potential of its Cyclin A2 and B1-directed negative control function.


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)

The pUL21a-RXL/Cy motif is required for Cyclin A2 repression by HCMV.Density arrested human embryonic lung (HEL) fibroblasts were infected with HCMV reconstituted from TB40-BAC4-wt or derivatives carrying the indicated UL21a mutations. Cells were harvested at regular intervals and analyzed for viral and cellular gene expression by immunoblotting (A) and ribonuclease protection assay (B). Cell culture supernatants were analyzed for the number of IE protein-forming units (IU) by virus titration (C). Results are represented as mean values plus standard deviations of triplicate samples.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1004514-g003: The pUL21a-RXL/Cy motif is required for Cyclin A2 repression by HCMV.Density arrested human embryonic lung (HEL) fibroblasts were infected with HCMV reconstituted from TB40-BAC4-wt or derivatives carrying the indicated UL21a mutations. Cells were harvested at regular intervals and analyzed for viral and cellular gene expression by immunoblotting (A) and ribonuclease protection assay (B). Cell culture supernatants were analyzed for the number of IE protein-forming units (IU) by virus titration (C). Results are represented as mean values plus standard deviations of triplicate samples.
Mentions: We then set out to investigate the functional consequences of pUL21a-Cyclin A2 interaction in the context of viral infection. To this end we introduced the RXL2 mutation into the HCMV genome by traceless BAC-mutagenesis and analyzed the recombinant virus in direct comparison to WT, UL21a deletion (ΔUL21a) and UL21a-PRAA mutant (APCmut) viruses. To ensure a synchronous start of lytic gene expression in infected cells, we used a high multiplicity of infection (MOI) and fibroblasts that were arrested in G0/G1 by contact inhibition. Accordingly, except the G1-specific Cyclin D1, cell cycle factors were barely detectable at the time of infection (Fig. 3A). Whereas Cyclin D1 expression was largely unaffected by HCMV infection and all four viruses led to a strong and sustained induction of Cyclin E1, they markedly differed with respect to Cyclin A2, Cyclin B1 and APC5 protein regulation. Both UL21a deletion and RXL2 point mutation abolished the characteristic block of Cyclin A2 expression observed in HCMV-WT infected cells. Whereas Cyclin A2 mRNA levels were only slightly elevated (see Fig. 3B), the effect was most pronounced at the level of protein expression, consistent with the observed proteasome dependency of Cyclin A2 down-regulation in pUL21a-transfected cells (Fig. 2). Remarkably, the RXL2 point mutation resulted in considerably higher peak levels of Cyclin A2 protein expression than the deletion of the whole UL21a open reading frame. This may be explained by the fact that the APC/C inhibitory function of UL21a is not affected by the loss of Cyclin A2 binding (see APC5 levels in Fig. 3A) and thus can contribute to Cyclin A2 stabilization. Cyclin A2 induction in HCMV-UL21a-RXL2mut-infected cells was followed by a strong up-regulation of Cyclin B1 mRNA and protein expression, most likely due to Cyclin A2-dependent transcriptional activation of the Cyclin B1 promoter [23]. Again, the effects of UL21a deletion were much weaker, hardly exceeding the moderate levels of Cyclin B1 induction in HCMV-WT-infected cells. Thus, the simultaneous loss of Cyclin A2 and APC/C-binding sites in pUL21a masks the full potential of its Cyclin A2 and B1-directed negative control function.

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