Minute Virus of Mice Inhibits Transcription of the Cyclin B1 Gene during Infection
Minute Virus of Mice Inhibits Transcription of the Cyclin B1 Gene during Infection
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DOI：
10.1128/jvi.00428-17
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发表时间：
2017-07-01
影响因子：
5.4
通讯作者：
Pintel, David J.
中科院分区：
文献类型：
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作者：
Fuller, Matthew S.;Majumder, Kinjal;Pintel, David J.
小鼠微小病毒(MVM)的复制诱导持续的细胞DNA损伤反应(DDR)，然后病毒利用这一反应为细小病毒的有效接管准备核环境。MV诱导的DDR的一个重要方面是建立一个有效的有丝分裂前阻断，我们以前发现它不依赖于激活的p21和ATR/Chk1信号。与以前报道的其他研究不同，这种抑制依赖于细胞周期蛋白B1及其编码的RNA的显著、特异的缺失，从而阻止细胞周期蛋白B1/CDK1复合体的功能，从而阻止有丝分裂的进入。我们在这里表明，虽然细胞周期蛋白B1RNA的稳定性没有受到MVM感染的影响，但在感染后的后期，新生细胞周期蛋白B1RNA的产生显著减少。单独异位表达NS1并不能降低Cyclin B1的表达。MVM感染也降低了Cyclin B1蛋白的水平，而RNA水平通常会因DNA破坏试剂而增加。我们证明，在感染期间细胞周期蛋白B1表达降低的时候，RNA聚合酶II和必要的有丝分裂转录因子FOXM1在细胞周期蛋白B1基因启动子上的占有率显著减少。此外，虽然FOXM1的总水平保持不变，但磷酸化的、可能具有活性的FOXM1形式显著减少。通过成簇的规则间隔短回文重复序列(CRISPR)将具有固有活性的FOXM1结构或FOXM1的激活结构域靶向细胞周期蛋白B1基因启动子(CRISPR)-在MVM感染的细胞中，酶失活的Cas9增加了细胞周期蛋白B1的蛋白质和RNA水平，暗示FOXM1是MVM感染期间抑制细胞周期蛋白B1的关键靶点。小鼠细小病毒(MVM)的重要复制诱导持续的细胞DNA损伤反应(DDR)，病毒利用该反应为有效接管准备核环境。MVM诱导的DDR的一个重要方面是建立一个有效的有丝分裂前阻滞剂。这种阻断依赖于细胞周期蛋白B1及其编码RNA的显著、特异的缺失，从而排除了细胞周期蛋白B1/CDK1复合体进入有丝分裂所必需的功能。我们发现Cyclin B1的表达减少主要是在转录启动的水平上控制的。此外，在感染过程中，重要的有丝分裂转录因子FOXM1和RNA聚合酶II被发现以较低的水平占据了细胞周期蛋白B1基因的启动子。在MVM感染的细胞中，通过CRISPR催化失活的Cas9(DCas9)，在细胞周期蛋白B1基因启动子上引入具有固有活性的FOXM1或FOXM1的激活域，可增加细胞周期蛋白B1蛋白和RNA的表达，提示FOXM1是介导MVM诱导的细胞周期蛋白B1抑制的关键靶点。
Replication of minute virus of mice (MVM) induces a sustained cellular DNA damage response (DDR) which the virus then exploits to prepare the nuclear environment for effective parvovirus takeover. An essential aspect of the MVMinduced DDR is the establishment of a potent premitotic block, which we previously found to be independent of activated p21 and ATR/Chk1 signaling. This arrest, unlike others reported previously, depends upon a significant, specific depletion of cyclin B1 and its encoding RNA, which precludes cyclin B1/CDK1 complex function, thus preventing mitotic entry. We show here that while the stability of cyclin B1 RNA was not affected by MVM infection, the production of nascent cyclin B1 RNA was substantially diminished at late times postinfection. Ectopic expression of NS1 alone did not reduce cyclin B1 expression. MVM infection also reduced the levels of cyclin B1 protein, and RNA levels normally increased in response to DNA-damaging reagents. We demonstrated that at times of reduced cyclin B1 expression during infection, there was a significantly reduced occupancy of RNA polymerase II and the essential mitotic transcription factor FoxM1 on the cyclin B1 gene promoter. Additionally, while total FoxM1 levels remained constant, there was a significant decrease of the phosphorylated, likely active, forms of FoxM1. Targeting of a constitutively active FoxM1 construct or the activation domain of FoxM1 to the cyclin B1 gene promoter via clustered regularly interspaced short palindromic repeats (CRISPR)-enzymatically inactive Cas9 in MVM-infected cells increased both cyclin B1 protein and RNA levels, implicating FoxM1 as a critical target for cyclin B1 inhibition during MVM infection.IMPORTANCE Replication of the parvovirus minute virus of mice (MVM) induces a sustained cellular DNA damage response (DDR) which the virus exploits to prepare the nuclear environment for effective takeover. An essential aspect of the MVM-induced DDR is establishment of a potent premitotic block. This block depends upon a significant, specific depletion of cyclin B1 and its encoding RNA that precludes cyclin B1/CDK1 complex functions necessary for mitotic entry. We show that reduced cyclin B1 expression is controlled primarily at the level of transcription initiation. Additionally, the essential mitotic transcription factor FoxM1 and RNA polymerase II were found to occupy the cyclin B1 gene promoter at reduced levels during infection. Recruiting a constitutively active FoxM1 construct or the activation domain of FoxM1 to the cyclin B1 gene promoter via CRISPR-catalytically inactive Cas9 (dCas9) in MVM-infected cells increased expression of both cyclin B1 protein and RNA, implicating FoxM1 as a critical target mediating MVM-induced cyclin B1 inhibition.