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，可在人类宿主中建立终身感染。我们工作的总体目标是定义对病毒复制和潜伏期重要的关键病毒-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靶点。HCMV编码单一DNA聚合酶（UL54）。由于疱疹病毒编码自己的DNA聚合酶，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，疱疹病毒基因组是复杂的，具有高gc含量和限制b家族DNA聚合酶的重复序列，如UL54。通过我们的合作努力，我们证明了特化宿主翻译聚合酶（TLS pol）在HCMV基因组复制和稳定性中的显著作用。TLS极点在复制叉的病变旁路或单链DNA间隙填充或同源定向修复中起作用，发生在合成后（叉后）。在未受干扰的DNA合成过程中，TLS极点还保持脆弱的位点稳定性。TLS poll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)。值得注意的是，我们发现y家族TLS位点（h、i、k和Rev1）和位点z对于维持HCMV基因组稳定性很重要。此外，我们的研究结果表明，极点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极点在确保病毒基因组完整性和产生病毒基因组多样性方面具有重要作用。我们还发现，TLS极点的耗尽对病毒基因组合成和复制的影响是不同的。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）途径在病毒生命周期中的重要性，对于理解病毒复制和延迟的机制具有重要意义。此外，令人兴奋的新数据表明，宿主TLS极点在对核苷类抗病毒疗法（如更昔洛韦）的耐药性进化中发挥了作用。我们假设HCMV积极招募TLS极点并选择相应的DDR通路来维持基因组完整性并调节病毒复制和延迟。目的1将确定HCMV将宿主TLS轮和其他DDR修复因子招募到病毒复制区及其发挥作用的子域的机制。目标2将定义宿主TLS poll和其他DDR修复因子作用于病毒序列以确保基因组稳定性并促进抗病毒抗性的机制。目的3将确定宿主DDR通路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 poll和DDR路径的病毒-宿主相互作用。我们的多pi合作工作建立了宿主TLS poll对病毒基因组稳定性和多样性的重要性，如果没有dr。古德鲁姆和博斯科。此外，该研究提供了独特的可能性，阐明了人类细胞中使用HCMV基因组作为模型系统的TLS极点生物学的新见解。