Department of Molecular and Cell Biology, University of California Berkeley, CA 94720, USA.
Department of Plant and Microbial Biology, University of California Berkeley, CA 94720, USA.
Nucleic Acids Res. 2023 Jan 11;51(1):182-197. doi: 10.1093/nar/gkac1190.
Alkaline exonucleases (AE) are present in several large DNA viruses including bacteriophage λ and herpesviruses, where they play roles in viral DNA processing during genome replication. Given the genetic conservation of AEs across viruses infecting different kingdoms of life, these enzymes likely assume central roles in the lifecycles of viruses where they have yet to be well characterized. Here, we applied a structure-guided functional analysis of the bifunctional AE in the oncogenic human gammaherpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV), called SOX. In addition to identifying a preferred DNA substrate preference for SOX, we define key residues important for DNA binding and DNA processing, and how SOX activity on DNA partially overlaps with its functionally separable cleavage of mRNA. By engineering these SOX mutants into KSHV, we reveal roles for its DNase activity in viral gene expression and infectious virion production. Our results provide mechanistic insight into gammaherpesviral AE activity as well as areas of functional conservation between this mammalian virus AE and its distant relative in phage λ.
碱性核酸外切酶（AE）存在于多种大型 DNA 病毒中，包括噬菌体 λ 和疱疹病毒，它们在基因组复制过程中发挥作用，参与病毒 DNA 的处理。鉴于感染不同生命领域的病毒之间 AE 的遗传保守性，这些酶可能在尚未得到充分描述的病毒生命周期中发挥核心作用。在这里，我们应用结构指导的功能分析方法，研究了致癌性人类γ疱疹病毒卡波济肉瘤相关疱疹病毒（KSHV）中的双功能 AE，称为 SOX。除了确定 SOX 的首选 DNA 底物偏好外，我们还定义了 DNA 结合和 DNA 处理的关键残基，以及 SOX 在 DNA 上的活性如何与其功能可分离的 mRNA 切割部分重叠。通过将这些 SOX 突变体工程化到 KSHV 中，我们揭示了其 DNase 活性在病毒基因表达和感染性病毒粒子产生中的作用。我们的研究结果为 γ 疱疹病毒 AE 活性提供了机制见解，以及哺乳动物病毒 AE 与其在噬菌体 λ 中的远亲之间的功能保守性领域。
