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

Epstein-Barr 病毒颠覆宿主 SMC5/6 限制途径的特征

• 批准号: 10679118
• 负责人: Benjamin Elison Gewurz
• 金额: $ 22.38万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-09 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10679118
• 关键词: 3-Dimensional; ATAC-seq; Acquired Immunodeficiency Syndrome; Address; Adult; B-Lymphocytes; Binding; Cell Compartmentation; Cell Nucleus; Cell Survival; Cells; ChIP-seq; Chromatin; Clustered Regularly Interspaced Short Palindromic Repeats; Co-Immunoprecipitations; Complex; Cullin Proteins; DNA; DNA Binding Domain; DNA Structure; DNA biosynthesis; Double Stranded DNA Virus; Epigenetic Process; Episome; Epithelium; Epstein-Barr Virus latency; Gene Expression; Genetic study; Genome; Genomic Segment; HIV; HIV/AIDS; Hairy Leukoplakia; Hour; Human; Human Herpesvirus 4; Infection; Infectious Mononucleosis; Integration Host Factors; Knock-out; Learning; Ligase; Lymphoma; Lytic; Lytic Virus; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Membrane; Molecular; Nasopharynx; Oral cavity; Pathway interactions; Patients; Persons; Phase; Proteins; Proteomics; Regulation; Role; Saliva; Series; Site; Stomach Carcinoma; Testing; Tonsil; Viral; Viral Genes; Viral Genome; Viral Load result; Virus; Virus Replication; cell growth; cell transformation; co-infection; cohesin; ds-DNA; insight; lytic replication; novel therapeutic intervention; programs; transcriptome sequencing; transmission process; ubiquitin ligase; viral DNA; viral genomics; virtual

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.

摘要 EB病毒（EBV）在全球95%的成年人中建立终身感染。EBV 导致多种癌症，特别是在合并感染艾滋病毒的宿主中。EB病毒是在 宿主通过唾液，从那里它易位通过口腔和扁桃体上皮， 到达B细胞室在艾滋病患者中，EBV裂解性复制导致口腔多毛 白斑病在B细胞感染后，双链DNA（dsDNA）EBV基因组被递送到 到细胞核。关于感知传入EBV的宿主途径还有很多东西有待了解 基因组，以及细胞中的病毒基因组进行裂解复制，以及EBV如何相互作用 与这些限制性途径。我们使用RNAseq和多重串联质量标签（TMT） 质谱法来创建原发性人类B细胞感染和 EBV裂解性复制。每一个都强调了EBV强烈而特异性地下调了 SMC 5/6粘附素复合物在原发性B细胞感染的1天内，在大多数表达之前， 病毒基因，并再次裂解再激活。我们发现，EBV主要被膜蛋白 BNRF 1组装一个基于Cullin-7的泛素连接酶，靶向SMC 5/6的蛋白酶体 降解在BNRF 1缺失的情况下，SMC 5/6关闭EBV裂解性复制区室 并阻断EBV裂解DNA合成。CRISPR SMC 6敲除拯救复制区 在携带BNRF 1敲除EBV的Burkitt细胞中形成。据我们所知，SMC 5/6因此 第一个宿主因素，可以限制EBV复制区室。然而， 关于SMC 5/6如何识别EBV基因组作为外来物。这个重要的问题一直存在 在双链DNA病毒基因组中开放，重点是如何 病毒逃避SMC 5/6。我们的中心假设是SMC 5/6粘附素识别并 限制EBV潜伏期建立所必需的病毒非B型DNA结构， 裂解DNA复制的方式调节BNRF 1。我们的目标是（1）确定如何 SMC 5/6在新感染的细胞中感知并限制潜伏的EBV DNA，并（2）确定如何 SMC 5/6在再活化时感测并限制裂解性EBV DNA。总的来说，这些研究 解决了关于SMC 5/6如何发挥功能的关键开放问题，以区分病毒和 尽管人们越来越认识到SMC 5/6在宿主DNA中的作用， 调节双链病毒DNA。