Separating late gene transcription from viral DNA replication in KSHV

将 KSHV 中的晚期基因转录与病毒 DNA 复制分离

• 批准号: 10739178
• 负责人: David W Morgens
• 金额: $ 11.93万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-18 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10739178
• 关键词: Acquired Immunodeficiency Syndrome; Affect; Automobile Driving; Binding; Biological; CRISPR/Cas technology; Cancer Etiology; Cells; DNA; DNA biosynthesis; DNA-Directed DNA Polymerase; DNA-Directed RNA Polymerase; Data; Dependence; Double Stranded DNA Virus; Elements; Essential Genes; Event; Gene Expression; Genes; Genetic; Genetic Transcription; Genome; Genomic Instability; Genomic approach; Herpesviridae; Human; Human Herpesvirus 8; Immunocompromised Host; Individual; Infection; Integration Host Factors; Knowledge; Late Gene Transcriptions; Life Cycle Stages; Link; Lytic; Malignant Neoplasms; Measures; Methods; Modeling; Molecular; Mutagenesis; Mutation; Nucleic Acid Regulatory Sequences; Oncogenic; Oncogenic Viruses; Oncornaviruses; Pathogenicity; Patients; Phenotype; Play; Postdoctoral Fellow; Process; Proteins; RNA; Regulator Genes; Regulatory Element; Replication Origin; Role; Testing; Therapeutic; Transcription Coactivator; Viral; Viral Genes; Viral Genome; Viral Proteins; Virion; Virus; Virus Diseases; Virus Replication; Work; ds-DNA; functional genomics; gammaherpesvirus; human DNA; lytic replication; mutant; particle; prevent; therapeutic target; transcription factor; viral DNA; virus genetics

Project Summary: Human oncogenic viruses are a major cause of cancer, with recent estimates that 15% of all cancers are associated with a viral infection. One such oncovirus is Kaposi’s sarcoma-associated herpesvirus (KSHV), which primarily affects untreated AIDS patients and other immunocompromised individuals. Like most viruses, KSHV relies both on host and unique viral processes for infection, each representing potential therapeutic vulnerabilities. One of these is an unusual link between the expression of an essential class of viral “late” genes and the replication of the viral dsDNA genome (vDNA). Though long recognized, the mechanism driving this link remains unknown. Here, I will use a multi-pronged functional genomics approach to identify and test new models for this process. The dependency of viral late gene transcription on vDNA replication depends on both the trans and cis viral components supporting vDNA replication. The trans component consists of the virally encoded vDNA replication factors. In Aim 1, I will use a method I have previously developed in my postdoc for high-throughput mutagenesis and phenotyping of viral mutants to identify mutations in these viral components that prevent late gene expression but still support vDNA replication. Preliminary work has already demonstrated that such mutations exist, and by expanding on this we can identify the role these viral proteins play in enabling late gene expression. In Aim 2, I will examine the viral origin of lytic replication, which is required in cis for both vDNA replication and late gene expression. While previous studies have been limited technically, new use of dCas9 will allow me to directly perturb the functional elements of this regulatory sequence and identify the distinct molecular events required to support vDNA replication and/or late gene expression. In my final aim, I will extend my work beyond the virus to identify and characterize the host components of this process. I propose to use genetic interactions to identify the host factors hijacked to support the viral life cycle. Specifically, by leveraging the mutants and functional elements discovered in my previous aims, I can further identify how the host supports or antagonizes this process both at the cis and trans levels. Each of these proposed aims will reveal new knowledge about vDNA replication and late gene expression, allow us to test models for how these processes are linked, and finally represent powerful functional genomic approaches that can be applied to many problems in KSHV and related dsDNA viruses.

项目概要： 人类致癌病毒是癌症的主要原因，最近估计所有癌症中有 15% 是由人类致癌病毒引起的。 与病毒感染有关的一种肿瘤病毒是卡波西肉瘤相关疱疹病毒（KSHV）。 与大多数病毒一样，KSHV 主要影响未经治疗的艾滋病患者和其他免疫功能低下的个体。 两者都依赖宿主和独特的病毒感染过程，每种都代表潜在的治疗方法 其中一个漏洞是一类重要的病毒“晚期”基因的表达之间存在不寻常的联系。 病毒双链 DNA 基因组 (vDNA) 的复制虽然早已被人们认识，但驱动这种联系的机制。 在这里，我将使用多管齐下的功能基因组学方法来识别和测试新模型。 对于这个过程。 病毒晚期基因转录对vDNA复制的依赖性取决于反式和顺式 支持 vDNA 复制的病毒成分 反式成分由病毒编码的 vDNA 组成。 在目标 1 中，我将使用我之前在博士后开发的高通量方法。 病毒突变体的诱变和表型分析，以识别这些病毒成分中预防晚期病毒的突变 基因表达但仍然支持 vDNA 复制。 突变是存在的，通过对此进行扩展，我们可以确定这些病毒蛋白在激活晚期基因中所起的作用 在目标 2 中，我将检查裂解复制的病毒起源，这对于两种 vDNA 都是顺式复制所必需的。 虽然之前的研究在技术上受到限制，但 dCas9 的新用途。 将使我能够直接干扰该调节序列的功能元件并识别不同的 支持 vDNA 复制和/或晚期基因表达所需的分子事件。 在我的最终目标中，我将把我的工作扩展到病毒之外，以识别和表征宿主成分 我建议利用遗传相互作用来识别被劫持以支持病毒生命的宿主因子。 具体来说，通过利用我之前目标中发现的突变体和功能元素，我可以 进一步确定宿主如何在顺式和反式水平上支持或拮抗这一过程。 拟议的目标将揭示有关 vDNA 复制和晚期基因表达的新知识，使我们能够测试 这些过程如何联系的模型，最终代表了强大的功能基因组方法 可应用于 KSHV 和相关 dsDNA 病毒的许多问题。