Haller Sherry L, Park Chorong, Bruneau Ryan C, Megawati Dewi, Zhang Chi, Vipat Sameera, Peng Chen, Senkevich Tatiana G, Brennan Greg, Tazi Loubna, Rothenburg Stefan
bioRxiv. 2024 May 16:2024.05.16.594589. doi: 10.1101/2024.05.16.594589.
The antiviral protein kinase R (PKR) is activated by viral double-stranded RNA and phosphorylates translation initiation factor eIF2α, thereby inhibiting translation and virus replication. Most poxviruses contain two PKR inhibitors, called E3 and K3 in vaccinia virus (VACV), which are determinants of viral host range. The prevailing model for E3 function is that it inhibits PKR through the non-specific sequestration of double-stranded (ds) RNA. Our data revealed that Syrian hamster PKR was resistant to E3, which is at odds with the sequestration model. However, Syrian hamster PKR was still sensitive to K3 inhibition. In contrast, Armenian hamster PKR showed opposite sensitivities, being sensitive to E3 and resistant to K3 inhibition. Mutational analyses of hamster PKRs showed that sensitivity to E3 inhibition was largely determined by the region linking the dsRNA-binding domains and the kinase domain of PKR, whereas two amino acid residues in the kinase domain (helix αG) determined sensitivity to K3. Expression of PKRs in congenic cells showed that Syrian hamster PKR containing the two Armenian hamster PKR residues in helix-αG was resistant to wild type VACV infection, and that cells expressing either hamster PKR recapitulated the phenotypes observed in species-derived cell lines. The observed resistance of Syrian hamster PKR to E3 explains its host range function and challenges the paradigm that dsRNA-binding PKR inhibitors mainly act by the sequestration of dsRNA.
The molecular mechanisms that govern the host range of viruses are incompletely understood. A small number of poxvirus genes have been identified that influence the host range of poxviruses. We show that the host range functions of E3 and K3, two host range factors from vaccinia virus, are a result of species-specific interactions with the antiviral protein kinase R (PKR) and that PKR from closely related species displayed dramatic differences in their sensitivities to these viral inhibitors. While there is a substantial body of work demonstrating host-specific interactions with K3, the current model for E3-mediated PKR inhibition is that E3 non-specifically sequesters dsRNA to prevent PKR activation. This model does not predict species-specific sensitivity to E3; therefore, our data suggest that the current model is incomplete, and that dsRNA sequestration is not the primary mechanism for E3 activity.
抗病毒蛋白激酶R（PKR）被病毒双链RNA激活，并使翻译起始因子eIF2α磷酸化，从而抑制翻译和病毒复制。大多数痘病毒含有两种PKR抑制剂，在痘苗病毒（VACV）中称为E3和K3，它们是病毒宿主范围的决定因素。关于E3功能的普遍模型是，它通过非特异性隔离双链（ds）RNA来抑制PKR。我们的数据显示，叙利亚仓鼠的PKR对E3具有抗性，这与隔离模型不一致。然而，叙利亚仓鼠的PKR对K3抑制仍敏感。相比之下，亚美尼亚仓鼠的PKR表现出相反的敏感性，对E3敏感而对K3抑制具有抗性。对仓鼠PKR的突变分析表明，对E3抑制的敏感性很大程度上由连接PKR的dsRNA结合结构域和激酶结构域的区域决定，而激酶结构域中的两个氨基酸残基（αG螺旋）决定了对K3的敏感性。在同基因细胞中PKR的表达表明，在αG螺旋中含有两个亚美尼亚仓鼠PKR残基的叙利亚仓鼠PKR对野生型VACV感染具有抗性，并且表达任一仓鼠PKR的细胞都重现了在物种来源的细胞系中观察到的表型。观察到的叙利亚仓鼠PKR对E3的抗性解释了其宿主范围功能，并挑战了dsRNA结合型PKR抑制剂主要通过隔离dsRNA起作用的范式。
控制病毒宿主范围的分子机制尚未完全了解。已经鉴定出少数影响痘病毒宿主范围的痘病毒基因。我们表明，来自痘苗病毒的两个宿主范围因子E3和K3的宿主范围功能是与抗病毒蛋白激酶R（PKR）进行物种特异性相互作用的结果，并且来自密切相关物种的PKR对这些病毒抑制剂的敏感性存在显著差异。虽然有大量工作证明了与K3的宿主特异性相互作用，但目前关于E3介导的PKR抑制的模型是，E3非特异性隔离dsRNA以防止PKR激活。该模型无法预测对E3的物种特异性敏感性；因此，我们的数据表明当前模型不完整，并且dsRNA隔离不是E3活性的主要机制。
