S-Like-Phase Cyclin-Dependent Kinases Stabilize the Epstein-Barr Virus BDLF4 Protein To Temporally Control Late Gene Transcription
S-Like-Phase Cyclin-Dependent Kinases Stabilize the Epstein-Barr Virus BDLF4 Protein To Temporally Control Late Gene Transcription
复制标题
S 样相细胞周期蛋白依赖性激酶稳定 Epstein-Barr 病毒 BDLF4 蛋白以暂时控制晚期基因转录
DOI：
10.1128/jvi.01707-18
复制
发表时间：
2019
影响因子：
5.4
通讯作者：
Kimura Hiroshi
中科院分区：
文献类型：
--
作者：
Sato Yoshitaka;Watanabe Takahiro;Suzuki Chihiro;Abe Yuichi;Masud H. M. Abdullah Al;Inagaki Tomoki;Yoshida Masahiro;Suzuki Takeshi;Goshima Fumi;Adachi Jun;Tomonaga Takeshi;Murata Takayuki;Kimura Hiroshi
时间控制的基因表达是疱疹病毒繁殖所必需的。为了实现这一点，疱疹病毒编码几种转录调节因子。在EB病毒中，BcRF 1与五种病毒蛋白（BDLF 4、BGLF 3、BFRF 2、BVLF 1和BDLF3.5）结合，在含TATT的启动子上形成病毒晚期（L）基因调控复合物，称为病毒前起始复合物（vPIC）。然而，vPIC的监管在很大程度上尚未探索。在这项研究中，我们使用激酶抑制剂文库进行了两次筛选，并确定了一系列细胞周期蛋白依赖性激酶（CDK）抑制剂，通过BDLF 4蛋白的不稳定性下调L基因的表达，而不会对病毒DNA复制产生任何影响。通过短发夹RNA（shRNA）和蛋白酶体抑制剂处理敲低CDK 2表明BDLF 4蛋白的磷酸化阻止了泛素介导的降解。此外，我们证明了细胞周期蛋白A和E相关的CDK 2复合物在体外磷酸化BDLF 4，我们确定了BDLF 4中的几个丝氨酸/苏氨酸磷酸化位点。磷酸化失活和拟磷酸化突变体揭示了在苏氨酸91处的磷酸化在稳定BDLF 4中起作用。因此，我们的研究结果表明，S-样期CDKs介导的调节L基因的表达，通过稳定的BDLF 4蛋白，这使得时间L基因表达系统更加robust.IMPORTANCELate（L）基因代表超过三分之一的疱疹病毒基因组，这表明，这些基因中的许多是不可缺少的病毒的生活周期。除BCRF 1、BDLF 2和BDLF 3外，EB病毒L基因由病毒调节因子转录，称为病毒前起始复合物（vPIC）和宿主RNA聚合酶II复合物。由于vPIC在β-和γ-疱疹病毒中是保守的，因此研究vPIC对病毒L基因表达的控制有助于开发特异性抑制β-和γ-疱疹病毒感染/疾病中这些过程的药物。在这项研究中，我们证明了CDK抑制剂诱导vPIC组分BDLF 4的不稳定，导致L基因表达和随后的后代生产减少。我们的研究结果表明，CDK抑制剂可能是一种治疗选择，对β和γ疱疹病毒与现有的抑制剂疱疹病毒裂解复制，如更昔洛韦。
Temporally controlled gene expression is necessary for the propagation of herpesviruses. To achieve this, herpesviruses encode several transcriptional regulators. In Epstein-Barr virus, BcRF1 associates with five viral proteins (BDLF4, BGLF3, BFRF2, BVLF1, and BDLF3.5) to form the viral late (L) gene regulatory complex, which is called the viral preinitiation complex (vPIC), on TATT-containing promoters. However, regulation of the vPIC has been largely unexplored. In this study, we performed two screens using a kinase inhibitor library and identified a series of cyclin-dependent kinase (CDK) inhibitors that downregulated the expression of L genes without any impact on viral DNA replication through destabilization of the BDLF4 protein. Knockdown of CDK2 by short hairpin RNA (shRNA) and proteasome inhibitor treatment showed that phosphorylation of the BDLF4 protein prevented ubiquitin-mediated degradation. Moreover, we demonstrated that cyclin A- and E-associated CDK2 complexes phosphorylated BDLF4in vitro, and we identified several serine/threonine phosphorylation sites in BDLF4. Phosphoinactive and phosphomimic mutants revealed that phosphorylation at threonine 91 plays a role in stabilizing BDLF4. Therefore, our findings indicate that S-like-phase CDKs mediate the regulation of L gene expression through stabilization of the BDLF4 protein, which makes the temporal L gene expression system more robust.IMPORTANCELate (L) genes represent more than one-third of the herpesvirus genome, suggesting that many of these genes are indispensable for the life cycle of the virus. With the exception of BCRF1, BDLF2, and BDLF3, Epstein-Barr virus L genes are transcribed by viral regulators, which are known as the viral preinitiation complex (vPIC) and the host RNA polymerase II complex. Because the vPIC is conserved in beta- and gammaherpesviruses, studying the control of viral L gene expression by the vPIC contributes to the development of drugs that specifically inhibit these processes in beta- and gammaherpesvirus infections/diseases. In this study, we demonstrated that CDK inhibitors induced destabilization of the vPIC component BDLF4, leading to a reduction in L gene expression and subsequent progeny production. Our findings suggest that CDK inhibitors may be a therapeutic option against beta- and gammaherpesviruses in combination with existing inhibitors of herpesvirus lytic replication, such as ganciclovir.