Institute of Cancer & Genomic Sciences, College of Medical and Dental Sciences, The University of Birmingham, Birmingham, United Kingdom.
Institute of Metabolism & Systems Research, College of Medical and Dental Sciences, The University of Birmingham, Birmingham, United Kingdom.
J Virol. 2019 Jun 14;93(13). doi: 10.1128/JVI.00402-19. Print 2019 Jul 1.
Here, we show that the cellular DNA replication protein and ATR substrate SMARCAL1 is recruited to viral replication centers early during adenovirus infection and is then targeted in an E1B-55K/E4orf6- and cullin RING ligase-dependent manner for proteasomal degradation. In this regard, we have determined that SMARCAL1 is phosphorylated at S123, S129, and S173 early during infection in an ATR- and CDK-dependent manner, and that pharmacological inhibition of ATR and CDK activities attenuates SMARCAL1 degradation. SMARCAL1 recruitment to viral replication centers was shown to be largely dependent upon SMARCAL1 association with the RPA complex, while Ad-induced SMARCAL1 phosphorylation also contributed to SMARCAL1 recruitment to viral replication centers, albeit to a limited extent. SMARCAL1 was found associated with E1B-55K in adenovirus E1-transformed cells. Consistent with its ability to target SMARCAL1, we determined that E1B-55K modulates cellular DNA replication. As such, E1B-55K expression initially enhances cellular DNA replication fork speed but ultimately leads to increased replication fork stalling and the attenuation of cellular DNA replication. Therefore, we propose that adenovirus targets SMARCAL1 for degradation during infection to inhibit cellular DNA replication and promote viral replication. Viruses have evolved to inhibit cellular DNA damage response pathways that possess antiviral activities and utilize DNA damage response pathways that possess proviral activities. Adenovirus has evolved, primarily, to inhibit DNA damage response pathways by engaging with the ubiquitin-proteasome system and promoting the degradation of key cellular proteins. Adenovirus differentially regulates ATR DNA damage response signaling pathways during infection. The cellular adenovirus E1B-55K binding protein E1B-AP5 participates in ATR signaling pathways activated during infection, while adenovirus 12 E4orf6 negates Chk1 activation by promoting the proteasome-dependent degradation of the ATR activator TOPBP1. The studies detailed here indicate that adenovirus utilizes ATR kinase and CDKs during infection to promote the degradation of SMARCAL1 to attenuate normal cellular DNA replication. These studies further our understanding of the relationship between adenovirus and DNA damage and cell cycle signaling pathways during infection and establish new roles for E1B-55K in the modulation of cellular DNA replication.
在这里，我们表明细胞 DNA 复制蛋白和 ATR 底物 SMARCAL1 在腺病毒感染早期被招募到病毒复制中心，然后以 E1B-55K/E4orf6-和 cullin RING 连接酶依赖性方式靶向蛋白酶体降解。在这方面，我们已经确定 SMARCAL1 在感染早期以 ATR 和 CDK 依赖性方式在 S123、S129 和 S173 处被磷酸化，并且药理学抑制 ATR 和 CDK 活性会减弱 SMARCAL1 的降解。SMARCAL1 被招募到病毒复制中心在很大程度上取决于 SMARCAL1 与 RPA 复合物的关联，而 Ad 诱导的 SMARCAL1 磷酸化也有助于 SMARCAL1 招募到病毒复制中心，但程度有限。在腺病毒 E1 转化细胞中发现 SMARCAL1 与 E1B-55K 相关联。与其靶向 SMARCAL1 的能力一致，我们确定 E1B-55K 调节细胞 DNA 复制。因此，E1B-55K 的表达最初会增强细胞 DNA 复制叉速度，但最终会导致复制叉停滞增加和细胞 DNA 复制减弱。因此，我们提出腺病毒在感染期间将 SMARCAL1 靶向降解以抑制细胞 DNA 复制并促进病毒复制。病毒已经进化出抑制具有抗病毒活性的细胞 DNA 损伤反应途径，并利用具有促病毒活性的 DNA 损伤反应途径。腺病毒主要通过与泛素-蛋白酶体系统结合并促进关键细胞蛋白的降解来抑制 DNA 损伤反应途径。腺病毒在感染过程中差异调节 ATR DNA 损伤反应信号通路。细胞腺病毒 E1B-55K 结合蛋白 E1B-AP5 参与感染期间激活的 ATR 信号通路，而腺病毒 12 E4orf6 通过促进 ATR 激活剂 TOPBP1 的蛋白酶体依赖性降解来否定 Chk1 的激活。这里详述的研究表明，腺病毒在感染过程中利用 ATR 激酶和 CDK 促进 SMARCAL1 的降解，以减弱正常的细胞 DNA 复制。这些研究进一步加深了我们对感染期间腺病毒与 DNA 损伤和细胞周期信号通路之间关系的理解，并确立了 E1B-55K 在调节细胞 DNA 复制中的新作用。
