Host DNA repair pathways in human cytomegalovirus replication

人类巨细胞病毒(HCMV)是一种双链DNA病毒，在人类宿主中建立终身感染。我们工作的首要目标是确定对病毒复制和潜伏期至关重要的关键病毒-宿主相互作用，这些相互作用为旨在限制病毒致病的抗病毒策略提供靶点。人巨细胞病毒编码一个DNA聚合酶(UL54)。由于疱疹病毒编码它们自己的DNA聚合酶，所以人们普遍认为它们不需要宿主聚合酶来复制它们的基因组。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，我们展示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著作用。TLS Pols在复制叉处的病变旁路或单链DNA缺口填充或发生在合成后(叉子后面)的同源定向修复中发挥作用。在不受干扰的DNA合成过程中，TLS Pols还保持了脆弱部位的稳定性。TLS Pols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 Pols(h，i，k和Rev1)和polz对于维持HCMV基因组的稳定性是重要的。此外，我们的结果表明Pols 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 Pols在确保病毒基因组完整性以及潜在地产生病毒基因组多样性方面发挥了重要作用。我们还发现，TLS Pols的耗尽对病毒基因组合成和复制产生了不同的影响。明确HCMV如何维持基因组的稳定性以及宿主TLS Pols和DNA损伤修复(DDR)通路在病毒生命周期中的重要性对于理解病毒复制和潜伏的机制非常重要。此外，令人兴奋的新数据表明，宿主TLS Pols在对更昔洛韦等核苷类抗病毒药物的耐药性演变中发挥了作用。我们假设HCMV主动招募TLS Pols并选择相应的DDR途径来维持基因组的完整性，并调节病毒的复制和潜伏时间。目的1将确定HCMV招募宿主TLS Pols和其他DDR修复因子到病毒复制区段和它们发挥作用的亚区的机制。AIM 2将确定宿主TLS Pols和其他DDR修复因子作用于病毒序列以确保基因组稳定和有助于抗病毒耐药性的机制。目的3将确定宿主DDR通路对病毒潜伏期的意义。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 Pols对于病毒基因组的稳定性和多样性的重要性，如果没有古德鲁姆博士和博斯科博士的共同专业知识，这是不可能的。此外，这项研究提供了独特的可能性，以人类巨细胞病毒基因组为模型系统，阐明人类细胞中TLS Pols的生物学特性。

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.