Yao F, Schaffer P A
Division of Molecular Genetics, Dana-Farber Cancer Institute, Boston, Massachusetts.
J Virol. 1994 Dec;68(12):8158-68. doi: 10.1128/JVI.68.12.8158-8168.1994.
The herpes simplex virus type 1 immediate-early protein ICP0 enhances expression of a spectrum of viral genes alone and synergistically with ICP4. To test whether ICP0 and ICP4 interact physically, we performed far-Western blotting analysis of proteins from mock-, wild-type-, and ICP4 mutant virus-infected cells with in vitro-synthesized [35S]Met-labeled ICP0 and ICP4 as probes. The ICP4 and ICP0 polypeptides synthesized in vitro exhibited molecular weights similar to those of their counterparts in herpes simplex virus type 1-infected cells, and the in vitro-synthesized ICP4 was able to bind to a probe containing the ICP4 consensus binding site. Far-Western blotting experiments demonstrated that ICP0 interacts directly and specifically with ICP4 and with itself. To further define the interaction between ICP0 and ICP4, we generated a set of glutathione S-transferase (GST)-ICP0 fusion proteins that contain GST and either ICP0 N-terminal amino acids 1 to 244 or 1 to 394 or C-terminal amino acids 395 to 616 or 395 to 775. Using GST-ICP0 fusion protein affinity chromatography and in vitro-synthesized [35S]Met-labeled ICP0 and ICP4, ICP4 was shown to interact preferentially with the fusion protein containing ICP0 C-terminal amino acids 395 to 775, whereas ICP0 interacted efficiently with both the N-terminal GST-ICP0 fusion proteins and the C-terminal GST-ICP0 fusion proteins containing amino acids 395 to 775. Fusion protein affinity chromatography also demonstrated that the C-terminal 235 amino acid residues of ICP4 are important for efficient interaction with ICP0. Collectively, these results reveal a direct and specific physical interaction between ICP0 and ICP4.
单纯疱疹病毒1型即刻早期蛋白ICP0可单独增强一系列病毒基因的表达，并与ICP4协同发挥作用。为了检测ICP0与ICP4是否存在物理相互作用，我们用体外合成的[35S]甲硫氨酸标记的ICP0和ICP4作为探针，对来自未感染、野生型和ICP4突变病毒感染细胞的蛋白质进行了远缘Western印迹分析。体外合成的ICP4和ICP0多肽的分子量与其在单纯疱疹病毒1型感染细胞中的对应物相似，且体外合成的ICP4能够与包含ICP4共有结合位点的探针结合。远缘Western印迹实验表明，ICP0可直接且特异性地与ICP4以及其自身相互作用。为了进一步明确ICP0与ICP4之间的相互作用，我们构建了一组谷胱甘肽S-转移酶（GST）-ICP0融合蛋白，这些融合蛋白包含GST以及ICP0的N端1至244个氨基酸或1至394个氨基酸，或者C端395至616个氨基酸或395至775个氨基酸。利用GST-ICP0融合蛋白亲和层析以及体外合成的[35S]甲硫氨酸标记的ICP0和ICP4，结果显示ICP4优先与包含ICP0 C端395至775个氨基酸的融合蛋白相互作用，而ICP0则能有效地与N端GST-ICP0融合蛋白以及包含395至775个氨基酸的C端GST-ICP0融合蛋白相互作用。融合蛋白亲和层析还表明，ICP4的C端235个氨基酸残基对于与ICP0的有效相互作用至关重要。总体而言，这些结果揭示了ICP0与ICP4之间直接且特异性的物理相互作用。
