Deng Wentao, Lin Biing Yuan, Jin Ge, Wheeler Crystal G, Ma Tianlin, Harper J Wade, Broker Thomas R, Chow Louise T
Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, 510 McCallum Basic Health Sciences Building, 1918 University Blvd., Birmingham, AL 35294-0005, USA.
J Virol. 2004 Dec;78(24):13954-65. doi: 10.1128/JVI.78.24.13954-13965.2004.
Cyclin-dependent kinases (CDKs) play key roles in eukaryotic DNA replication and cell cycle progression. Phosphorylation of components of the preinitiation complex activates replication and prevents reinitiation. One mechanism is mediated by nuclear export of critical proteins. Human papillomavirus (HPV) DNA replication requires cellular machinery in addition to the viral replicative DNA helicase E1 and origin recognition protein E2. E1 phosphorylation by cyclin/CDK is critical for efficient viral DNA replication. We now show that E1 is phosphorylated by CDKs in vivo and that phosphorylation regulates its nucleocytoplasmic localization. We identified a conserved regulatory region for localization which contains a dominant leucine-rich nuclear export sequence (NES), the previously defined cyclin binding motif, three serine residues that are CDK substrates, and a putative bipartite nuclear localization sequence. We show that E1 is exported from the nucleus by a CRM1-dependent mechanism unless the NES is inactivated by CDK phosphorylation. Replication activities of E1 phosphorylation site mutations are reduced and correlate inversely with their increased cytoplasmic localization. Nuclear localization and replication activities of most of these mutations are enhanced or restored by mutations in the NES. Collectively, our data demonstrate that CDK phosphorylation controls E1 nuclear localization to support viral DNA amplification. Thus, HPV adopts and adapts the cellular regulatory mechanism to complete its reproductive program.
细胞周期蛋白依赖性激酶（CDKs）在真核生物DNA复制和细胞周期进程中发挥关键作用。起始前复合物组分的磷酸化激活复制并防止再次起始。一种机制是由关键蛋白的核输出介导的。人乳头瘤病毒（HPV）DNA复制除了需要病毒复制性DNA解旋酶E1和起始识别蛋白E2外，还需要细胞机制。细胞周期蛋白/CDK对E1的磷酸化对于有效的病毒DNA复制至关重要。我们现在表明，E1在体内被CDKs磷酸化，并且磷酸化调节其核质定位。我们鉴定了一个保守的定位调节区域，其包含一个显性富含亮氨酸的核输出序列（NES）、先前定义的细胞周期蛋白结合基序、三个作为CDK底物的丝氨酸残基以及一个推定的双分型核定位序列。我们表明，除非NES被CDK磷酸化失活，否则E1通过一种依赖CRM1的机制从细胞核输出。E1磷酸化位点突变的复制活性降低，并且与其增加的细胞质定位呈负相关。NES中的突变增强或恢复了大多数这些突变的核定位和复制活性。总体而言，我们的数据表明CDK磷酸化控制E1的核定位以支持病毒DNA扩增。因此，HPV采用并适应细胞调节机制以完成其繁殖程序。
