Nebraska Center for Virology, University of Nebraska, Lincoln, Nebraska, USA.
School of Biological Sciences, University of Nebraska, Lincoln, Nebraska, USA.
J Virol. 2022 Jun 8;96(11):e0039822. doi: 10.1128/jvi.00398-22. Epub 2022 May 11.
Poxvirus proteins remodel signaling throughout the cell by targeting host enzymes for inhibition and redirection. Recently, it was discovered that early in infection the vaccinia virus (VACV) B12 pseudokinase copurifies with the cellular kinase VRK1, a proviral factor, in the nucleus. Although the formation of this complex correlates with inhibition of cytoplasmic VACV DNA replication and likely has other downstream signaling consequences, the molecular mechanisms involved are poorly understood. Here, we further characterize how B12 and VRK1 regulate one another during poxvirus infection. First, we demonstrate that B12 is stabilized in the presence of VRK1 and that VRK1 and B12 coinfluence their respective solubility and subcellular localization. In this regard, we find that B12 promotes VRK1 colocalization with cellular DNA during mitosis and that B12 and VRK1 may be tethered cooperatively to chromatin. Next, we observe that the C-terminal tail of VRK1 is unnecessary for B12-VRK1 complex formation or its proviral activity. Interestingly, we identify a point mutation of B12 capable of abrogating interaction with VRK1 and which renders B12 nonrepressive during infection. Lastly, we investigated the influence of B12 on the host factor BAF and antiviral signaling pathways and find that B12 triggers redistribution of BAF from the cytoplasm to the nucleus. In addition, B12 increases DNA-induced innate immune signaling, revealing a new functional consequence of the B12 pseudokinase. Together, this study characterizes the multifaceted roles B12 plays during poxvirus infection that impact VRK1, BAF, and innate immune signaling. Protein pseudokinases comprise a considerable fraction of the human kinome, as well as other forms of life. Recent studies have demonstrated that their lack of key catalytic residues compared to their kinase counterparts does not negate their ability to intersect with molecular signal transduction. While the multifaceted roles pseudokinases can play are known, their contribution to virus infection remains understudied. Here, we further characterize the mechanism of how the VACV B12 pseudokinase and human VRK1 kinase regulate one another in the nucleus during poxvirus infection and inhibit VACV DNA replication. We find that B12 disrupts regulation of VRK1 and its downstream target BAF, while also enhancing DNA-dependent innate immune signaling. Combined with previous data, these studies contribute to the growing field of nuclear pathways targeted by poxviruses and provide evidence of unexplored roles of B12 in the activation of antiviral immunity.
痘病毒蛋白通过靶向宿主酶进行抑制和重定向来重塑整个细胞的信号转导。最近发现，在感染早期，牛痘病毒 (VACV) B12 假激酶与细胞激酶 VRK1 共纯化，后者是一种前病毒因子，位于细胞核内。尽管这种复合物的形成与细胞质 VACV DNA 复制的抑制相关，并且可能具有其他下游信号转导后果，但涉及的分子机制仍知之甚少。在这里，我们进一步描述了 B12 和 VRK1 在痘病毒感染过程中如何相互调节。首先，我们证明 VRK1 的存在稳定了 B12，并且 VRK1 和 B12 共同影响它们各自的溶解度和亚细胞定位。在这方面，我们发现 B12 促进 VRK1 在有丝分裂期间与细胞 DNA 的共定位，并且 B12 和 VRK1 可能被共同连接到染色质上。接下来，我们观察到 VRK1 的 C 末端尾巴对于 B12-VRK1 复合物的形成或其前病毒活性不是必需的。有趣的是，我们鉴定出一种能够破坏 B12 与 VRK1 相互作用的 B12 点突变，并且该突变使 B12 在感染过程中不再具有抑制作用。最后，我们研究了 B12 对宿主因子 BAF 和抗病毒信号通路的影响，发现 B12 将 BAF 从细胞质重新分配到细胞核。此外，B12 增加了 DNA 诱导的先天免疫信号，揭示了 B12 假激酶的新功能后果。总的来说，这项研究描述了 B12 在痘病毒感染过程中发挥的多方面作用，这些作用影响 VRK1、BAF 和先天免疫信号。 蛋白质假激酶构成人类激酶组以及其他生命形式的相当大的一部分。最近的研究表明，与激酶对应物相比，它们缺乏关键的催化残基并不会否定它们与分子信号转导交叉的能力。虽然已知假激酶可以发挥多方面的作用，但它们对病毒感染的贡献仍未得到充分研究。在这里，我们进一步描述了在痘病毒感染过程中 VACV B12 假激酶和人类 VRK1 激酶如何在细胞核内相互调节以及如何抑制 VACV DNA 复制的机制。我们发现 B12 破坏了 VRK1 及其下游靶标 BAF 的调节，同时还增强了 DNA 依赖性先天免疫信号。结合以前的数据，这些研究为痘病毒靶向的核途径领域做出了贡献，并提供了 B12 在激活抗病毒免疫方面的未探索作用的证据。
