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 Epstein-Barr病毒（EBV）在全球95％的成年人中建立了终身感染。 EBV 引起多种癌症，尤其是在HIV共感染的宿主中。 EBV在之间传播 通过唾液的宿主，它从口腔和扁桃体上皮转移到该唾液中 到达B细胞隔室。在艾滋病的人身上，ebv裂解复制会导致口腔毛茸茸 白细胞。 B细胞感染后，双链DNA（DSDNA）EBV基因组被传递 到核。关于接入EBV的宿主路径还有很多尚待了解 基因组以及经历裂解复制的细胞中的病毒基因组以及EBV的相互作用 使用这些限制途径。我们使用了RNASEQ和多路复用串联质量标签（TMT） 质谱法创建原代人B细胞感染的时间蛋白质组学图和 EBV裂解复制。每个人都强调EBV强烈，特别是下调了 在原发性B细胞感染后的1天内，SMC5/6粘蛋白复合物在大多数表达之前 病毒基因，再次在裂解重新激活后。我们确定了EBV主要的Tegument蛋白 BNRF1组装了基于Cullin-7的泛素连接酶，该连接酶靶向SMC5/6的蛋白酶体 降解。在没有BNRF1的情况下，SMC5/6关闭EBV裂解复制室 并阻止EBV裂解DNA合成。 CRISPR SMC6淘汰赛救援复制室 携带BNRF1基因敲除EBV的伯基特细胞中的形成。据我们所知，SMC5/6是 可以限制EBV复制室的第一个主机因子。但是，鲜为人知 关于SMC5/6如何将EBV基因组识别为外国。这个重要的问题仍然存在 跨双链DNA病毒基因组开放，而重点是如何 病毒逃避SMC5/6。我们的中心假设是SMC5/6粘着素识别和 限制EBV潜伏期建立所需的病毒非B形DNA结构 以BNRF1调制的方式进行裂解DNA复制。我们的目标是（1）确定如何 SMC5/6感应并限制新感染细胞中的潜在EBV DNA，并确定（2）如何确定如何 重新激活后SMC5/6感受并限制了裂解的EBV DNA。总的来说，这些研究 解决有关SMC5/6如何功能以区分病毒与病毒的关键问题 宿主DNA，尽管对SMC5/6角色的认识日益认识到，但仍未研究 双链病毒DNA的调节。