Characterization of Epstein-Barr Virus Subversion of the Host SMC5/6 Restriction Pathway

摘要 爱泼斯坦-巴尔病毒(EBV)在全球95%的成年人中建立终身感染。EB病毒 会导致多种癌症，特别是在合并感染艾滋病毒的宿主中。EBV在两个人之间传播 通过唾液寄生，从唾液通过口腔和扁桃体上皮转移到 到达B细胞隔间。在艾滋病患者中，EBV裂解复制会导致口腔毛发 黏膜白斑。在B细胞感染后，双链DNA(DsDNA)EBV基因组被传递 到原子核。关于感知传入EBV的宿主途径，还有很多需要了解的地方 基因组，以及进行裂解复制的细胞中的病毒基因组，以及EBV如何相互作用 通过这些限制途径。我们使用了RNAseq和多路串联质量标签(TMT) 质谱学用于创建人类原发B细胞感染的时间蛋白质组图 EBV裂解复制。每个人都强调EBV强烈而具体地下调了 SMC5/6粘附素复合体在原发B细胞感染后1天内表达 病毒基因，并在裂解重新激活时再次激活。我们发现EB病毒的主要被膜蛋白 BNRF1组装一种基于cullin-7的泛素连接酶，针对蛋白酶体的SMC5/6 退化。在没有BNRF1的情况下，SMC5/6关闭EBV裂解复制区 并阻断EBV裂解DNA的合成。CRISPR SMC6基因敲除拯救了复制隔间 携带BNRF1基因敲除EBV的Burkitt细胞的形成。据我们所知，SMC5/6因此 可以限制EBV复制间隔的第一个主机因素。然而，人们对此知之甚少 关于SMC5/6如何识别EBV基因组是外来的。这个重要的问题一直存在 跨双链DNA病毒基因组开放，重点放在如何 病毒逃避SMC5/6。我们的中心假设是SMC5/6粘附素识别和 限制EBV潜伏期建立和建立所需的病毒非B型DNA结构 以BNRF1调节的方式裂解DNA复制。我们的目标是(1)确定如何 SMC5/6感知和限制新感染细胞中的潜伏EBV DNA，并(2)确定如何 SMC5/6在重新激活时识别和限制裂解的EBV DNA。总的来说，这些研究 回答有关SMC5/6如何工作以区分病毒和病毒的关键未决问题 宿主DNA，尽管人们越来越多地认识到SMC5/6在体内的作用，但它仍然几乎没有被研究 双链病毒DNA的调控。

Abstract Epstein-Barr virus (EBV) establishes lifelong infection in 95% of adults worldwide. EBV causes multiple cancers, particularly in hosts with HIV co-infection. EBV is transmitted between hosts through saliva, from which it translocates across the oral cavity and tonsillar epithelium to reach the B-cell compartment. In persons with AIDS, EBV lytic replication causes oral hairy leukoplakia. Upon B-cell infection, the double-stranded DNA (dsDNA) EBV genome is delivered to the nucleus. Much remains to be learned about host pathways that sense incoming EBV genomes, as well as viral genomes in cells undergoing lytic replication, and how EBV interacts with these restriction pathways. We used RNAseq and multiplexed tandem mass tag (TMT) mass spectrometry to create temporal proteomic maps of primary human B-cell infection and of EBV lytic replication. Each highlighted that EBV strongly and specifically downmodulates the SMC5/6 cohesin complex within 1 day of primary B-cell infection, prior to the expression of most viral genes, and again upon lytic reactivation. We identified that the EBV major tegument protein BNRF1 assembles a Cullin-7 based ubiquitin ligase that targets SMC5/6 for proteasomal degradation. In the absence of BNRF1, SMC5/6 shuts down EBV lytic replication compartments and blocks EBV lytic DNA synthesis. CRISPR SMC6 knockout rescues replication compartment formation in Burkitt cells carrying BNRF1 knockout EBV. To our knowledge, SMC5/6 is therefore the first host factor that can restrict EBV replication compartments. However, little is known about how SMC5/6 recognizes EBV genomes as foreign. This important question has remained open across double stranded DNA viral genomes, where focus has instead been on how viruses evade SMC5/6. Our central hypothesis is that the SMC5/6 cohesin recognizes and restricts viral non-B-form DNA structures necessary for EBV latency establishment and lytic DNA replication in a manner modulated by BNRF1. Our Aims are to (1) Identify how SMC5/6 senses and restricts latent EBV DNA in newly infected cells and to (2) Identify how SMC5/6 senses and restricts lytic EBV DNA upon reactivation. Collectively, these studies address the key open question about how SMC5/6 functions in order to discriminate viral from host DNA, which has remained nearly unstudied despite growing recognition of SMC5/6 roles in regulation of double stranded viral DNA.