Olson Annabel T, Rico Amber B, Wang Zhigang, Delhon Gustavo, Wiebe Matthew S
Nebraska Center for Virology, University of Nebraska, Lincoln, Nebraska, USA.
School of Biological Sciences, University of Nebraska, Lincoln, Nebraska, USA.
J Virol. 2017 Jul 12;91(15). doi: 10.1128/JVI.00635-17. Print 2017 Aug 1.
The vaccinia virus B1 kinase is highly conserved among poxviruses and is essential for the viral life cycle. B1 exhibits a remarkable degree of similarity to vaccinia virus-related kinases (VRKs), a family of cellular kinases, suggesting that the viral enzyme has evolved to mimic VRK activity. Indeed, B1 and VRKs have been demonstrated to target a shared substrate, the DNA binding protein BAF, elucidating a signaling pathway important for both mitosis and the antiviral response. In this study, we further characterize the role of B1 during vaccinia infection to gain novel insights into its regulation and integration with cellular signaling pathways. We begin by describing the construction and characterization of the first B1 deletion virus (vvΔB1) produced using a complementing cell line expressing the viral kinase. Examination of vvΔB1 revealed that B1 is critical for the production of infectious virions in various cell types and is sufficient for BAF phosphorylation. Interestingly, the severity of the defect in DNA replication following the loss of B1 varied between cell types, leading us to posit that cellular VRKs partly complement for the absence of B1 in some cell lines. Using cell lines devoid of either VRK1 or VRK2, we tested this hypothesis and discovered that VRK2 expression facilitates DNA replication and allows later stages of the viral life cycle to proceed in the absence of B1. Finally, we present evidence that the impact of VRK2 on vaccinia virus is largely independent of BAF phosphorylation. These data support a model in which B1 and VRK2 share additional substrates important for the replication of cytoplasmic poxviruses. Viral mimicry of cellular signaling modulators provides clear evidence that the pathogen targets an important host pathway during infection. Poxviruses employ numerous viral homologs of cellular proteins, the study of which have yielded insights into signaling pathways used by both virus and cells alike. The vaccinia virus B1 protein is a homolog of cellular vaccinia virus-related kinases (VRKs) and is needed for viral DNA replication and likely other stages of the viral life cycle. However, much remains to be learned about how B1 and VRKs overlap functionally. This study utilizes new tools, including a B1 deletion virus and VRK knockout cells, to further characterize the functional links between the viral and cellular enzymes. As a result, we have discovered that B1 and VRK2 target a common set of substrates vital to productive infection of this large cytoplasmic DNA virus.
痘苗病毒B1激酶在痘病毒中高度保守，对病毒生命周期至关重要。B1与痘苗病毒相关激酶（VRK）家族（一类细胞激酶）具有显著的相似性，这表明该病毒酶已进化为模拟VRK活性。事实上，B1和VRK已被证明靶向共同的底物——DNA结合蛋白BAF，阐明了一条对有丝分裂和抗病毒反应都很重要的信号通路。在本研究中，我们进一步表征了B1在痘苗病毒感染过程中的作用，以深入了解其调控以及与细胞信号通路的整合。我们首先描述了使用表达病毒激酶的互补细胞系产生的首个B1缺失病毒（vvΔB1）的构建和表征。对vvΔB1的检测表明，B1对于多种细胞类型中感染性病毒粒子的产生至关重要，并且足以使BAF磷酸化。有趣的是，B1缺失后DNA复制缺陷的严重程度在不同细胞类型中有所不同，这使我们推测细胞VRK在某些细胞系中部分补偿了B1的缺失。使用缺乏VRK1或VRK2的细胞系，我们验证了这一假设，并发现VRK2的表达促进DNA复制，并使病毒生命周期的后期阶段在没有B1的情况下得以进行。最后，我们提供证据表明VRK2对痘苗病毒的影响在很大程度上独立于BAF磷酸化。这些数据支持了一个模型，即B1和VRK2共享对细胞质痘病毒复制至关重要的其他底物。病毒对细胞信号调节剂的模拟提供了明确的证据，表明病原体在感染过程中靶向重要的宿主途径。痘病毒利用众多细胞蛋白的病毒同源物，对其研究有助于深入了解病毒和细胞所使用的信号通路。痘苗病毒B1蛋白是细胞痘苗病毒相关激酶（VRK）的同源物，是病毒DNA复制以及可能的病毒生命周期其他阶段所必需的。然而，关于B1和VRK在功能上如何重叠仍有许多有待了解之处。本研究利用了新工具，包括B1缺失病毒和VRK基因敲除细胞，以进一步表征病毒和细胞酶之间的功能联系。结果，我们发现B1和VRK2靶向一组对这种大型细胞质DNA病毒的有效感染至关重要的共同底物。
