PUL21a-Cyclin A2 interaction is required to protect human cytomegalovirus-infected cells from the deleterious consequences of mitotic entry.
PUL21a-Cyclin A2 interaction is required to protect human cytomegalovirus-infected cells from the deleterious consequences of mitotic entry.
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DOI：
10.1371/journal.ppat.1004514
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发表时间：
2014-10
期刊：
影响因子：
6.7
通讯作者：
Wiebusch L
中科院分区：
文献类型：
--
作者：
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
进入有丝分裂伴随着细胞结构、新陈代谢和基因表达的巨大变化。许多病毒已经进化出阻止细胞周期停止的策略，以防止有丝分裂进入，可能是为了确保持续、不间断的病毒复制。在这里，我们展示了对于人类巨细胞病毒(HCMV)，如果病毒细胞周期停滞机制被禁用，参与病毒复制的细胞进入计划外有丝分裂，会发生什么。我们利用了一个巨细胞病毒突变体，由于其UL21a基因产物(PUL21a)中Cyclin A2结合基序的缺陷，已经失去了下调Cyclin A2的能力，从而使细胞停滞在G1/S的转变中。感染细胞中Cyclin A2的上调不仅触发了细胞DNA合成的启动，而且促进了Cyclin B1-CDK1的积聚和核转位，染色质过早凝聚和有丝分裂进入。核层解体和不规则的中期纺锤体形成表明，感染的细胞能够进入中期。然而，pUL21a仍然完整的后期促进复合体/环体(APC/C)抑制功能阻止了后期的启动。值得注意的是，必要的病毒IE2，而不是相关的染色体相关的IE1蛋白，在有丝分裂进入时消失，表明IE2在有丝分裂条件下固有的不稳定性。病毒DNA合成在有丝分裂中受损，表现为病毒复制区段的形态异常和BrdU掺入率显著降低。感染细胞的中期停滞时间延长，与性早熟姐妹染色单体分离和染色体物质的进行性碎裂相一致。我们得出结论，pUL21a的Cyclin A2结合功能有助于维持有利于完成HCMV复制周期的细胞周期状态。在HCMV复制过程中，非计划的有丝分裂进入会造成致命的后果，导致流产感染和细胞死亡。细胞周期蛋白A2是细胞分裂周期的关键调节因子。Cyclin A2的相互作用子通常包含短序列元件(RXL/Cy基序)，这些序列元件与Cyclin A2蛋白中的疏水斑块具有高亲和力。已知两种含有RXL/Cy的因子：i)CDK底物，由与Cyclin A2形成络合物的CDK亚单位处理；ii)CDK抑制剂，由于缺乏CDK磷酸化位点而稳定地与Cyclin A2-CDK结合。人巨细胞病毒(HCMV)已进化出一种新型的RXL/Cy蛋白。它的UL21a基因产物是一种高度不稳定的小蛋白，通过其N端的RXL/Cy基序与Cyclin A2结合，导致蛋白酶体有效地降解Cyclin A2。在这里，我们表明这一机制不仅对于病毒抑制细胞DNA合成是必不可少的，而且对于防止感染细胞进入有丝分裂也是必不可少的。非计划的有丝分裂进入后，伴随着异常的纺锤体形成，中期停滞，姐妹染色单体早熟分离，染色体断裂和细胞死亡。病毒的DNA复制和必需的病毒IE2蛋白的表达在有丝分裂中被取消。因此，pUL21a-Cyclin A2相互作用保护HCMV免受病毒和细胞功能在有丝分裂中崩溃的影响。
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. Cyclin A2 is a key regulator of the cell division cycle. Interactors of Cyclin A2 typically contain short sequence elements (RXL/Cy motifs) that bind with high affinity to a hydrophobic patch in the Cyclin A2 protein. Two types of RXL/Cy-containing factors are known: i) cyclin-dependent kinase (CDK) substrates, which are processed by the CDK subunit that complexes to Cyclin A2, and ii) CDK inhibitors, which stably associate to Cyclin A2-CDK due to the lack of CDK phosphorylation sites. Human cytomegalovirus (HCMV) has evolved a novel type of RXL/Cy-containing protein. Its UL21a gene product, a small and highly unstable protein, binds to Cyclin A2 via an RXL/Cy motif in its N-terminus, leading to efficient degradation of Cyclin A2 by the proteasome. Here, we show that this mechanism is not only essential for viral inhibition of cellular DNA synthesis, but also to prevent entry of infected cells into mitosis. Unscheduled mitotic entry is followed by aberrant spindle formation, metaphase arrest, precocious separation of sister chromatids, chromosomal fragmentation and cell death. Viral DNA replication and expression of the essential viral IE2 protein are abrogated in mitosis. Thus, pUL21a-Cyclin A2 interaction protects HCMV from a collapse of viral and cellular functions in mitosis.
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