Vue Yee, Mullon Patrick J, Leehangin Alexander, Maldonado-Luevano Emiliano, Hasselmann Tyler D, Mehta Kavi P M, Mohni Kareem N
Department of Molecular Medicine, Mayo Clinic, Rochester, Minnesota, USA.
Department of Comparative Biosciences, University of Wisconsin, Madison, Wisconsin, USA.
J Virol. 2025 Aug 18:e0089325. doi: 10.1128/jvi.00893-25.
Herpes simplex virus type 1 replicates in the nucleus of the host cell and utilizes many cellular proteins to facilitate DNA replication. HSV-1 DNA replication is closely linked with homologous recombination, and HSV-1 encodes a two-subunit recombinase consisting of an exonuclease (UL12) and a single-strand DNA-binding protein (ICP8). We identified the cellular deubiquitinating enzyme USP15 as a new UL12-binding partner. USP15 is one of the most enriched proteins on viral DNA and has recently been implicated in regulating cellular homologous recombination. Utilizing isolation of proteins on nascent DNA (iPOND) to compare the proteins on wild-type and UL12-deficient viruses, we observed that USP15 is not recruited to newly replicated DNA in the absence of UL12. We also observed that UL12 and USP15 interact directly using purified proteins. Together, these data suggest that UL12 directly recruits USP15 to viral DNA. UL12 stimulates a type of homology-directed repair called single-strand annealing (SSA). We generated a USP15 knockout cell line with an integrated GFP-based SSA reporter and observed that UL12 and USP15 both contribute to the maximal induction of SSA. In addition to the reporter cell line, there is a 10-fold reduction in the frequency of marker rescue, and individual replication forks move slower in the absence of USP15. These data support a role for USP15 in facilitating recombination during the virus life cycle and the maintenance of replication fork stability.IMPORTANCEHSV-1 is a ubiquitous pathogen in the global population that causes lifelong latent infection with sporadic reactivation in the host. It has long been appreciated that HSV-1 DNA replication exhibits high rates of recombination and is linked to the host DNA damage response. In this study, we show a novel interaction between UL12, a member of the HSV recombinase, and the deubiquitinating host enzyme USP15. We show that USP15 physically interacts with UL12 and is required for UL12 to maximally stimulate recombination. In the absence of USP15, we also observe an overall reduction in virus growth. This work demonstrated that host proteins facilitate viral-induced recombination. The interaction of USP15 with UL12 represents a potential target for intervention against HSV-1 infection and associated disease.
1型单纯疱疹病毒在宿主细胞核中复制，并利用多种细胞蛋白促进DNA复制。HSV-1 DNA复制与同源重组密切相关，HSV-1编码一种由核酸外切酶（UL12）和单链DNA结合蛋白（ICP8）组成的双亚基重组酶。我们鉴定出细胞去泛素化酶USP15是一种新的UL12结合伴侣。USP15是病毒DNA上最丰富的蛋白质之一，最近被认为参与调节细胞同源重组。利用新生DNA上蛋白质的分离技术（iPOND）比较野生型和UL12缺陷型病毒上的蛋白质，我们观察到在没有UL12的情况下，USP15不会被招募到新复制的DNA上。我们还观察到UL12和USP15使用纯化的蛋白质直接相互作用。总之，这些数据表明UL12直接将USP15招募到病毒DNA上。UL12刺激一种称为单链退火（SSA）的同源定向修复类型。我们用基于GFP的SSA报告基因构建了一个USP15基因敲除细胞系，并观察到UL12和USP15都有助于最大程度地诱导SSA。除了报告细胞系外，在没有USP15的情况下，标记拯救频率降低了10倍，单个复制叉移动得更慢。这些数据支持USP15在促进病毒生命周期中的重组和维持复制叉稳定性方面发挥作用。
重要性
HSV-1是全球人群中普遍存在的病原体，可导致宿主终身潜伏感染并偶发再激活。长期以来人们一直认识到HSV-1 DNA复制表现出高重组率，并与宿主DNA损伤反应有关。在这项研究中，我们展示了HSV重组酶成员UL12与宿主去泛素化酶USP15之间的新型相互作用。我们表明USP15与UL12发生物理相互作用，并且是UL12最大程度刺激重组所必需的。在没有USP15的情况下，我们还观察到病毒生长总体下降情况。这项工作证明宿主蛋白促进病毒诱导的重组。USP15与UL12的相互作用代表了针对HSV-1感染及相关疾病进行干预的潜在靶点。
