Host DNA repair pathways in human cytomegalovirus replication

人类巨细胞病毒复制中的宿主DNA修复途径

• 批准号: 10715597
• 负责人: GIOVANNI BOSCO
• 金额: $ 61.69万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-16 至 2028-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10715597
• 关键词: Antiviral Therapy; Antiviral resistance; Architecture; Biological Models; Biology; Bypass; Cells; Chemicals; Chromatin; Chromosome Fragile Sites; Complex; Complication; Cytomegalovirus; DNA; DNA Damage; DNA Repair; DNA Repair Pathway; DNA Structure; DNA biosynthesis; DNA lesion; DNA replication fork; DNA-Directed DNA Polymerase; Data; Diagnostic; Double Stranded DNA Virus; Elements; Ensure; Event; Evolution; FANCD2 protein; Family; Ganciclovir; Genetic Recombination; Genome; Genome Stability; Genomic Instability; Goals; Guanine + Cytosine Composition; Hematopoietic; Herpesviridae; Human; Immunocompromised Host; Infection; Integration Host Factors; Lesion; Life; Life Cycle Stages; Mammalian Cell; Mediating; Molecular; Nuclear; Nucleosides; Organ; Pathway interactions; Polymerase; Process; Proteins; Publishing; Recruitment Activity; Repair Complex; Resistance; Risk; Role; Single Nucleotide Polymorphism; Single-Stranded DNA; Site; Solid; Stem cell transplant; Therapeutic; Transplant Recipients; Viral; Viral Drug Resistance; Viral Genome; Viral Pathogenesis; Virus; Virus Latency; Virus Replication; Work; antiviral nucleoside analog; genome integrity; inhibitor; insight; knock-down; novel; programs; reactivation from latency; recruit; repaired; response; ubiquitin isopeptidase; viral genomics; virus host interaction

Human Cytomegalovirus (HCMV) is a double-stranded DNA virus that establishes life-long infection in the human host. The overarching objective of our work is to define critical virus-host interactions important for virus replication and latency, which provide targets for antiviral strategies aimed at limiting viral pathogenesis. HCMV encodes a single DNA polymerase (UL54). As herpesviruses encode their own DNA polymerase, it has been broadly presumed that they do not require host polymerases for the replication of their genomes. However, herpesvirus genomes are complex with high-GC content and repeat sequences that constrain the B-family DNA polymerases, such as UL54. Through our collaborative effort, we demonstrated a striking role for specialized host translesion polymerases (TLS pols) in HCMV genome replication and stability. TLS pols function in lesion bypass at the replication fork or in single-stranded DNA gap filling or homology-directed repair that occurs post-synthesis (behind the fork). TLS pols also maintain fragile site stability during unperturbed DNA synthesis. TLS pols include the Y-family polymerases eta (h), iota (i), kappa (k) and Rev 1, as well as the error-prone, B-family polymerase zeta (z). Strikingly, we found that Y-family TLS pols (h,i,k, and Rev1) and pol z are important to maintain HCMV genome stability. Further, our results indicate that pols h, i, and k generate single nucleotide variants across the viral genome. These findings indicate important roles for host TLS pols in ensuring viral genomic integrity and potentially in generating viral genome diversity. We also found that depletion of TLS pols differentially impacts viral genome synthesis and replication. Defining how HCMV maintains genomic stability and the significance of host TLS pols and DNA damage repair (DDR) pathways on the viral lifecycle is important for understanding mechanisms of virus replication and latency. Further, exciting new data indicates a role for host TLS pols in the evolution of resistance to nucleoside antiviral therapies, such as ganciclovir. We hypothesize that HCMV actively recruits TLS pols and coopts corresponding DDR pathways to maintain genome integrity and regulate viral replication and latency. Aim 1 will determine the mechanisms by which HCMV recruits host TLS pols and other DDR repair factors to viral replication compartments and the subdomains in which they function. Aim 2 will define the mechanisms by which host TLS pols and other DDR repair factors act on viral sequences to ensure genome stability and contribute to antiviral resistance. Aim 3 will determine the significance of host DDR pathways to viral latency. These aims are driven by our published work and exciting preliminary data identifying virus-host interactions that control host TLS pols and DDR pathways. Our multi-PI collaborative work establishes the importance of host TLS pols for the stability and diversity of viral genomes and would not be possible without the combined expertise of Drs. Goodrum and Bosco. Further, this study offers the unique possibility of illuminating new insights into the biology of TLS pols in human cells using the HCMV genome as a model system.

人类巨细胞病毒 (HCMV) 是一种双链 DNA 病毒，可在人类宿主体内形成终身感染。我们工作的首要目标是定义对病毒复制和潜伏期很重要的关键病毒-宿主相互作用，这为旨在限制病毒发病机制的抗病毒策略提供了目标。 HCMV 编码单个 DNA 聚合酶 (UL54)。由于疱疹病毒编码自己的 DNA 聚合酶，因此人们普遍认为它们不需要宿主聚合酶来复制其基因组。然而，疱疹病毒基因组非常复杂，具有高 GC 含量和限制 B 家族 DNA 聚合酶（例如 UL54）的重复序列。通过我们的合作，我们证明了专门的宿主跨损伤聚合酶 (TLS pols) 在 HCMV 基因组复制和稳定性中的显着作用。 TLS pols 在复制叉处的病变旁路或单链 DNA 间隙填充或合成后（复制叉后面）发生的同源定向修复中发挥作用。 TLS pol 还在不受干扰的 DNA 合成过程中保持脆弱的位点稳定性。 TLS pol 包括 Y 家族聚合酶 eta (h)、iota (i)、kappa (k) 和 Rev 1，以及容易出错的 B 家族聚合酶 zeta (z)。引人注目的是，我们发现 Y 家族 TLS pol（h、i、k 和 Rev1）和 pol z 对于维持 HCMV 基因组稳定性很重要。此外，我们的结果表明 pol h、i 和 k 在病毒基因组中产生单核苷酸变异。这些发现表明宿主 TLS pol 在确保病毒基因组完整性和潜在产生病毒基因组多样性方面发挥着重要作用。我们还发现 TLS pol 的消耗对病毒基因组合成和复制有不同的影响。定义 HCMV 如何维持基因组稳定性以及宿主 TLS pol 和 DNA 损伤修复 (DDR) 途径对病毒生命周期的重要性对于理解病毒复制和潜伏机制非常重要。此外，令人兴奋的新数据表明宿主 TLS pols 在核苷抗病毒疗法（如更昔洛韦）耐药性进化中发挥着作用。我们假设 HCMV 主动招募 TLS pol 并选择相应的 DDR 途径来维持基因组完整性并调节病毒复制和潜伏期。目标 1 将确定 HCMV 将宿主 TLS pol 和其他 DDR 修复因子招募到病毒复制区室及其发挥作用的子域的机制。目标 2 将定义宿主 TLS pol 和其他 DDR 修复因子作用于病毒序列的机制，以确保基因组稳定性并有助于抗病毒耐药性。目标 3 将确定宿主 DDR 途径对病毒潜伏期的重要性。这些目标是由我们发表的工作和令人兴奋的初步数据驱动的，这些数据确定了控制宿主 TLS pol 和 DDR 途径的病毒-宿主相互作用。我们的多 PI 协作工作确立了宿主 TLS pol 对病毒基因组稳定性和多样性的重要性，如果没有 Drs. 的综合专业知识，这是不可能实现的。古德鲁姆和博斯科。此外，这项研究提供了独特的可能性，可以使用 HCMV 基因组作为模型系统，阐明人类细胞中 TLS pols 生物学的新见解。