Intrinsic antiviral host factors confer cellular defence by limiting virus replication and are often counteracted by viral countermeasures. We reasoned that host factors that inhibit viral gene expression could be identified by determining proteins bound to viral DNA (vDNA) in the absence of key viral antagonists. Herpes simplex virus 1 (HSV-1) expresses E3 ubiquitin-protein ligase ICP0 (ICP0), which functions as an E3 ubiquitin ligase required to promote infection. Cellular substrates of ICP0 have been discovered as host barriers to infection but the mechanisms for inhibition of viral gene expression are not fully understood. To identify restriction factors antagonized by ICP0, we compared proteomes associated with vDNA during HSV-1 infection with wild-type virus and a mutant lacking functional ICP0 (ΔICP0). We identified the cellular protein Schlafen family member 5 (SLFN5) as an ICP0 target that binds vDNA during HSV-1 ΔICP0 infection. We demonstrated that ICP0 mediates ubiquitination of SLFN5, which leads to its proteasomal degradation. In the absence of ICP0, SLFN5 binds vDNA to repress HSV-1 transcription by limiting accessibility of RNA polymerase II to viral promoters. These results highlight how comparative proteomics of proteins associated with viral genomes can identify host restriction factors and reveal that viral countermeasures can overcome SLFN antiviral activity.

内在抗病毒宿主因子通过限制病毒复制赋予细胞防御能力，并且通常被病毒对策抵消。我们推断，在没有关键病毒拮抗剂的情况下，可以通过确定与病毒 DNA (vDNA) 结合的蛋白质来识别抑制病毒基因表达的宿主因素。单纯疱疹病毒 1 (HSV-1) 表达 E3 泛素蛋白连接酶 ICP0 (ICP0)，它作为促进感染所需的 E3 泛素连接酶发挥作用。已发现 ICP0 的细胞底物是宿主感染的屏障，但抑制病毒基因表达的机制尚不完全清楚。为了确定被 ICP0 拮抗的限制因素，我们比较了在 HSV-1 感染野生型病毒和缺乏功能性 ICP0 的突变体（ΔICP0）期间与 vDNA 相关的蛋白质组。我们将细胞蛋白 Schlafen 家族成员 5 (SLFN5) 鉴定为在 HSV-1 ΔICP0 感染期间结合 vDNA 的 ICP0 靶标。我们证明 ICP0 介导 SLFN5 的泛素化，从而导致其蛋白酶体降解。在没有 ICP0 的情况下，SLFN5 结合 vDNA 以通过限制 RNA 聚合酶 II 对病毒启动子的可及性来抑制 HSV-1 转录。这些结果强调了与病毒基因组相关的蛋白质的比较蛋白质组学如何识别宿主限制因素，并揭示病毒对策可以克服 SLFN 抗病毒活性。SLFN5 结合 vDNA 以通过限制 RNA 聚合酶 II 对病毒启动子的可及性来抑制 HSV-1 转录。这些结果强调了与病毒基因组相关的蛋白质的比较蛋白质组学如何识别宿主限制因素，并揭示病毒对策可以克服 SLFN 抗病毒活性。SLFN5 结合 vDNA 以通过限制 RNA 聚合酶 II 对病毒启动子的可及性来抑制 HSV-1 转录。这些结果强调了与病毒基因组相关的蛋白质的比较蛋白质组学如何识别宿主限制因素，并揭示病毒对策可以克服 SLFN 抗病毒活性。