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.

抽象的 爱泼斯坦-巴尔病毒 (EBV) 导致全球 95% 的成年人终身感染。 EB病毒 导致多种癌症，特别是在同时感染艾滋病毒的宿主中。 EBV 传播于 通过唾液作为宿主，从唾液穿过口腔和扁桃体上皮到达 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。 B 细胞感染后，会传递双链 DNA (dsDNA) EBV 基因组 到细胞核。关于感知传入 EBV 的宿主途径还有很多有待了解 基因组，以及正在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 与这些限制途径。我们使用 RNAseq 和多重串联质量标签 (TMT) 质谱法创建原发性人类 B 细胞感染和 EBV裂解复制。每个人都强调 EBV 强烈且特异性地下调 SMC5/6 粘连蛋白复合物在原代 B 细胞感染后 1 天内，在大多数表达之前 病毒基因，并在裂解重新激活后再次。我们发现 EBV 主要外皮蛋白 BNRF1 组装基于 Cullin-7 的泛素连接酶，针对蛋白酶体靶向 SMC5/6 降解。在没有 BNRF1 的情况下，SMC5/6 关闭 EBV 裂解复制室 并阻断 EBV 裂解性 DNA 合成。 CRISPR SMC6 敲除拯救复制区室 携带 BNRF1 敲除 EBV 的 Burkitt 细胞中形成。据我们所知，SMC5/6 因此 第一个可以限制 EBV 复制区室的宿主因素。然而，鲜为人知 关于 SMC5/6 如何将 EBV 基因组识别为外来基因。这个重要的问题仍然存在 跨双链 DNA 病毒基因组开放，重点是如何 病毒逃避 SMC5/6。我们的中心假设是 SMC5/6 粘连蛋白识别并 限制 EBV 潜伏期建立所需的病毒非 B 型 DNA 结构， 以 BNRF1 调节的方式进行裂解性 DNA 复制。我们的目标是 (1) 确定如何 SMC5/6 感知并限制新感染细胞中的潜在 EBV DNA，并 (2) 识别如何 SMC5/6 在重新激活时感知并限制裂解性 EBV DNA。总的来说，这些研究 解决有关 SMC5/6 如何发挥作用以区分病毒和病毒的关键悬而未决的问题 宿主 DNA，尽管人们越来越认识到 SMC5/6 在 双链病毒DNA的调控。