During viral infection, pattern recognition receptors (PRRs) and their associated adaptors recruit TANK-binding kinase 1 (TBK1) to activate interferon regulatory factor 3 (IRF3), resulting in production of type I interferons (IFNs). ICP0 and ICP34.5 are among the proteins encoded by herpes simplex virus 1 (HSV-1) that modulate type I IFN signaling. We constructed a recombinant virus (ΔXX) that lacks amino acids 87 to 106, a portion of the previously described TBK1-binding domain of the γ34.5 gene (D. Verpooten, Y. Ma, S. Hou, Z. Yan, and B. He, J Biol Chem 284:1097–1105, 2009, https://doi.org/10.1074/JBC.M805905200). These 20 residues are outside the γ34.5 beclin1-binding domain (BBD) that interacts with beclin1 and regulates autophagy. Unexpectedly, ΔXX showed no deficit in replication in vivo in a variety of tissues and showed virulence comparable to that of wild-type and marker-rescued viruses following intracerebral infection. ΔXX was fully capable of mediating the dephosphorylation of eIF2α, and the virus was capable of controlling the phosphorylation of IRF3. In contrast, a null mutant in γ34.5 failed to control IRF3 phosphorylation due to an inability of the mutant to sustain expression of ICP0. Our data show that while γ34.5 regulates IRF3 phosphorylation, the TBK1-binding domain itself has no impact on IRF3 phosphorylation or on replication and pathogenesis in mice.

IMPORTANCE Interferons (IFNs) are potent activators of a variety of host responses that serve to control virus infections. The Herpesviridae have evolved countermeasures to IFN responses. Herpes simplex virus 1 (HSV-1) encodes the multifunctional neurovirulence protein ICP34.5. In this study, we investigated the biological relevance of the interaction between ICP34.5 and TANK-binding kinase 1 (TBK1), an activator of IFN responses. Here, we establish that although ICP34.5 binds TBK1 under certain conditions through a TBK1-binding domain (TBD), there was no direct impact of the TBD on viral replication or virulence in mice. Furthermore, we showed that activation of IRF3, a substrate of TBK1, was independent of the TBD. Instead, we provided evidence that the ability of ICP34.5 to control IRF3 activation is through its ability to reverse translational shutoff and sustain the expression of other IFN inhibitors encoded by the virus. This work provides new insights into the immunomodulatory functions of ICP34.5.

在病毒感染期间，模式识别受体（PRR）及其相关的衔接子募集TANK结合激酶1（TBK1）以激活干扰素调节因子3（IRF3），从而产生I型干扰素（IFN）。ICP0和ICP34.5是由单纯疱疹病毒1（HSV-1）编码的可调节I型IFN信号转导的蛋白。我们构建了重组病毒（ΔXX），该病毒缺少第87-106位氨基酸，即先前描述的γ34.5基因TBK1结合结构域的一部分（D. Verpooten，Y。Ma，S。Hou，Z。Yan和B. He，生物化学杂志284：1097-1105，2009，https：//doi.org/10.1074/JBC.M805905200 ）。这20个残基在与beclin1相互作用并调节自噬的γ34.5beclin1结合域（BBD）外部。出乎意料的是，ΔXX在体内复制没有显示出缺陷在脑内感染后，其毒力可与野生型和标记物拯救的病毒相媲美。ΔXX完全能够介导eIF2α的去磷酸化，并且病毒能够控制IRF3的磷酸化。相反，由于突变体无法维持ICP0的表达，γ34.5中的无效突变体无法控制IRF3磷酸化。我们的数据显示，虽然γ34.5调节IRF3磷酸化，但TBK1结合域本身对IRF3磷酸化或小鼠复制和发病机制没有影响。

重要信息干扰素（IFN）是多种宿主反应的有效激活剂，可控制病毒感染。在疱疹病毒已经发展出对IFN应答的对策。单纯疱疹病毒1（HSV-1）编码多功能神经毒力蛋白ICP34.5。在这项研究中，我们调查了ICP34.5与TANK结合激酶1（TBK1）（IFN应答的激活剂）之间相互作用的生物学相关性。在这里，我们确定，尽管ICP34.5在某些条件下通过TBK1结合域（TBD）结合TBK1，但是TBD对小鼠的病毒复制或毒力没有直接影响。此外，我们表明，IRF3（TBK1的底物）的激活独立于TBD。相反，我们提供的证据表明ICP34.5控制IRF3激活的能力是通过其逆转翻译关闭并维持该病毒编码的其他IFN抑制剂表达的能力来实现的。