Virus-Host Interactions Laboratory, Department of Biotechnology, College of Life Sciences and Biotechnology, Korea Universitygrid.222754.4, Seoul, Republic of Korea.
J Virol. 2022 Nov 9;96(21):e0037122. doi: 10.1128/jvi.00371-22. Epub 2022 Oct 26.
Gammaherpesviruses, including Epstein-Barr virus (EBV), are important human pathogens because they are associated with various tumors. Poly(ADP-ribose) polymerase 1 (PARP1) is a multifunctional host nuclear protein responsible for poly(ADP-ribosyl)ation (PARylation) of target proteins. While PARP1 acts as a negative regulator that suppresses the lytic replication of gammaherpesviruses, viruses are often equipped with various strategies to overcome PARP1 inhibition. However, the mechanisms of how EBV may modulate a repressive host protein, PARP1, are still elusive. In this study, we found that EBV reactivation induced PARP1 downregulation in EBV-infected cells. EBV DNA polymerase processivity factor EA-D, encoded by the gene, directly interacted with the central automodification domain (AD) of PARP1 and was necessary and sufficient to downregulate PARP1 via K29-linked polyubiquitination. Moreover, knockdown of EA-D in B95.8 cells restored PARP1 levels and abrogated the expression of ZTA (also known as ZEBRA), a switch molecule of the EBV life cycle during reactivation. Interestingly, PARP1 PARylated RTA, another key switch molecule, and decreased RTA transactivation on the promoters of the , and genes. EA-D alleviated the PARylation of RTA and further enhanced RTA-mediated transactivation of these lytic promoters in reporter assays. Taken together, our results suggest that EBV viral processivity factor plays a key role in facilitating lytic replication by inducing PARP1 degradation via its interaction with the PARP1 AD, which is a highly conserved mechanism among gammaherpesviruses to counteract host repressive activity of PARP1 against viral lytic replication. PARP1 acts as a negative regulator of lytic replication in EBV. To successfully enter the reactivation cycle, EBV has developed multiple strategies to counteract the host's repressive mechanisms. In this study, we investigated how EBV manipulated the host repressive factor PARP1 to facilitate lytic replication. The EBV processivity factor EA-D downregulated PARP1 in a proteasome-dependent manner via its direct binding with PARP1 AD. The knockdown of EA-D restored the PARP1 level and inhibited ZTA expression during reactivation. Interestingly, PARP1 PARylated RTA and EA-D reduced the PARylation of RTA, thereby promoting the promoter activity. These results suggest that EA-D plays a key role in EBV lytic replication by inducing PARP1 degradation in addition to supporting DNA replication as a viral processivity factor. Given that the KSHV processivity factor also induces PARP1 degradation and enhances RTA function, gammaherpesviruses share a conserved molecular mechanism to overcome the inhibitory effects of PARP1, promoting lytic replication.
γ疱疹病毒，包括 EBV（Epstein-Barr virus），是重要的人类病原体，因为它们与各种肿瘤有关。多聚（ADP-核糖）聚合酶 1（PARP1）是一种多功能的宿主核蛋白，负责靶蛋白的聚（ADP-核糖）化（PARylation）。虽然 PARP1 作为一种负调控因子，抑制γ疱疹病毒的裂解复制，但病毒通常具有各种策略来克服 PARP1 抑制。然而，EBV 如何调节抑制性宿主蛋白 PARP1 的机制仍不清楚。在这项研究中，我们发现 EBV 再激活诱导 EBV 感染细胞中 PARP1 的下调。由 基因编码的 EBV DNA 聚合酶进程因子 EA-D 直接与 PARP1 的中央自动修饰结构域（AD）相互作用，通过 K29 连接的多泛素化，足以下调 PARP1。此外，在 B95.8 细胞中敲低 EA-D 可恢复 PARP1 水平并消除 ZTA（也称为 ZEBRA）的表达，ZTA 是 EBV 生活周期再激活时的开关分子。有趣的是，PARP1 对另一个关键开关分子 RTA 进行 PARylation，并降低 RTA 在启动子上的转录激活作用。 ，和 基因。EA-D 减轻 RTA 的 PARylation，并在报告基因实验中进一步增强 RTA 介导的这些裂解启动子的转录激活。总之，我们的研究结果表明，EBV 病毒进程因子通过其与 PARP1 AD 的相互作用诱导 PARP1 降解，在促进裂解复制中发挥关键作用，这是γ疱疹病毒中一种高度保守的机制，可对抗宿主对 PARP1 抑制病毒裂解复制的抑制活性。PARP1 是 EBV 裂解复制的负调控因子。为了成功进入再激活周期，EBV 已开发出多种策略来对抗宿主的抑制机制。在这项研究中，我们研究了 EBV 如何操纵宿主抑制因子 PARP1 以促进裂解复制。EBV 进程因子 EA-D 通过其与 PARP1 AD 的直接结合，以蛋白酶体依赖的方式下调 PARP1。EA-D 的敲低恢复了再激活过程中的 PARP1 水平并抑制了 ZTA 的表达。有趣的是，PARP1 对 RTA 进行 PARylation，而 EA-D 减少了 RTA 的 PARylation，从而促进了 启动子活性。这些结果表明，EA-D 除了作为病毒进程因子支持 DNA 复制外，还通过诱导 PARP1 降解在 EBV 裂解复制中发挥关键作用。鉴于 KSHV 进程因子也诱导 PARP1 降解并增强 RTA 功能，γ疱疹病毒具有一种保守的分子机制，可克服 PARP1 的抑制作用，促进裂解复制。
