Departments of Biochemistry and Molecular Biology, Medical University of South Carolinagrid.259828.c, Charleston, South Carolina, USA.
Departments of Microbiology and Immunology, Medical University of South Carolinagrid.259828.c, Charleston, South Carolina, USA.
J Virol. 2022 May 11;96(9):e0213721. doi: 10.1128/jvi.02137-21. Epub 2022 Apr 11.
Vaccinia virus (VV), the prototypic poxvirus, encodes a repertoire of proteins responsible for the metabolism of its large dsDNA genome. Previous work has furthered our understanding of how poxviruses replicate and recombine their genomes, but little is known about whether the poxvirus genome undergoes DNA repair. Our studies here are aimed at understanding how VV responds to exogenous DNA damage introduced by UV irradiation. Irradiation of cells prior to infection decreased protein synthesis and led to an ∼12-fold reduction in viral yield. On top of these cell-specific insults, irradiation of VV infections at 4 h postinfection (hpi) introduced both cyclobutene pyrimidine dimer (CPD) and 6,4-photoproduct (6,4-PP) lesions into the viral genome led to a nearly complete halt to further DNA synthesis and to a further reduction in viral yield (∼35-fold). DNA lesions persisted throughout infection and were indeed present in the genomes encapsidated into nascent virions. Depletion of several cellular proteins that mediate nucleotide excision repair (XP-A, -F, and -G) did not render viral infections hypersensitive to UV. We next investigated whether viral proteins were involved in combatting DNA damage. Infections performed with a virus lacking the A50 DNA ligase were moderately hypersensitive to UV irradiation (∼3-fold). More strikingly, when the DNA polymerase inhibitor cytosine arabinoside (araC) was added to wild-type infections at the time of UV irradiation (4 hpi), an even greater hypersensitivity to UV irradiation was seen (∼11-fold). Virions produced under the latter condition contained elevated levels of CPD adducts, strongly suggesting that the viral polymerase contributes to the repair of UV lesions introduced into the viral genome. Poxviruses remain of significant interest because of their continuing clinical relevance, their utility for the development of vaccines and oncolytic therapies, and their illustration of fundamental principles of viral replication and virus/cell interactions. These viruses are unique in that they replicate exclusively in the cytoplasm of infected mammalian cells, providing novel challenges for DNA viruses. How poxviruses replicate, recombine, and possibly repair their genomes is still only partially understood. Using UV irradiation as a form of exogenous DNA damage, we have examined how vaccinia virus metabolizes its genome following insult. We show that even UV irradiation of cells prior to infection diminishes viral yield, while UV irradiation during infection damages the genome, causes a halt in DNA accumulation, and reduces the viral yield more severely. Furthermore, we show that viral proteins, but not the cellular machinery, contribute to a partial repair of the viral genome following UV irradiation.
痘病毒（VV）是典型的痘病毒，它编码了一系列负责其大 dsDNA 基因组代谢的蛋白质。以前的研究进一步加深了我们对痘病毒如何复制和重组其基因组的理解，但对于痘病毒基因组是否经历 DNA 修复知之甚少。我们在这里的研究旨在了解 VV 如何应对 UV 照射引入的外源性 DNA 损伤。在感染前对细胞进行照射会降低蛋白质合成，并导致病毒产量减少约 12 倍。除了这些细胞特异性损伤外，在感染后 4 小时（hpi）对 VV 感染进行照射会将环丁烷嘧啶二聚体（CPD）和 6,4-光产物（6,4-PP）损伤引入病毒基因组中，导致进一步 DNA 合成几乎完全停止，并进一步降低病毒产量（约 35 倍）。DNA 损伤持续存在于整个感染过程中，实际上存在于包裹在新生病毒粒子中的基因组中。几种介导核苷酸切除修复的细胞蛋白（XP-A、-F 和 -G）的耗竭并没有使病毒感染对 UV 更加敏感。我们接下来研究了病毒蛋白是否参与了对抗 DNA 损伤。用缺乏 A50 DNA 连接酶的病毒进行的感染对 UV 照射中度敏感（约 3 倍）。更引人注目的是，当在 UV 照射时（4 hpi）向野生型感染中添加 DNA 聚合酶抑制剂阿糖胞苷（araC）时，观察到对 UV 照射的敏感性更高（约 11 倍）。在后者条件下产生的病毒粒子含有更高水平的 CPD 加合物，强烈表明病毒聚合酶有助于修复引入病毒基因组的 UV 损伤。
痘病毒因其持续的临床相关性、在疫苗和溶瘤治疗开发方面的实用性以及对病毒复制和病毒/细胞相互作用的基本原理的说明而仍然具有重要意义。这些病毒的独特之处在于它们仅在感染的哺乳动物细胞的细胞质中复制，这为 DNA 病毒带来了新的挑战。痘病毒如何复制、重组和可能修复其基因组，我们仍只了解部分。我们使用 UV 照射作为一种外源性 DNA 损伤形式，研究了 VV 在受到损伤后如何代谢其基因组。我们表明，即使在感染前对细胞进行照射也会降低病毒产量，而在感染期间进行照射会损害基因组，导致 DNA 积累停止，并更严重地降低病毒产量。此外，我们表明，病毒蛋白而不是细胞机制有助于在 UV 照射后对病毒基因组进行部分修复。
