National Clinical Research Center for Geriatrics, and State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan, China.
MAX IV Laboratory, Lund University, PO Box 118, SE-22100 Lund, Sweden.
Int J Biol Macromol. 2024 Nov;280(Pt 4):136123. doi: 10.1016/j.ijbiomac.2024.136123. Epub 2024 Sep 28.
Non-structural protein 3 (Nsp3) is the largest protein encoded by the coronavirus (CoV) genome. It consists of multiple domains that perform critical functions during the viral life cycle. CoV-Y is the most C-terminal domain of Nsp3, and it exhibits evolutionary conservation across diverse CoVs; however, the exact biological function of CoV-Y remains unclear. Here, we determined the crystal structure of CoV-Y of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Nsp3 using the single-wavelength anomalous diffraction method. We revealed the interaction between CoV-Y and the host BRCA1-associated protein (BRAP) using immunoprecipitation-mass spectrometry experiments. This interaction was subsequently confirmed in cellular assays, and the precise binding-regions between these two proteins were clarified. We found that this interaction is conserved in SARS-CoV and Middle East respiratory syndrome coronavirus. Next, we demonstrated that CoV-Y enhances IκBα and IκBβ phosphorylation and promotes the nuclear translocation of the downstream NF-κB members p50 and p65 through binding to BRAP. The CoV-Y-BRAP interaction can upregulate the transcript levels of the host inflammatory cytokines. Overall, our findings illustrate the biological function of CoV-Y for the first time and provide novel insights into coronavirus regulation of host inflammatory responses, as well as a possible target for antiviral drug development.
非结构蛋白 3（Nsp3）是冠状病毒（CoV）基因组编码的最大蛋白。它由多个结构域组成，在病毒生命周期中发挥关键作用。CoV-Y 是 Nsp3 的最 C 末端结构域，在不同的 CoV 中表现出进化保守性；然而，CoV-Y 的确切生物学功能仍不清楚。在这里，我们使用单波长异常衍射法确定了严重急性呼吸综合征冠状病毒 2（SARS-CoV-2）Nsp3 的 CoV-Y 的晶体结构。我们通过免疫沉淀-质谱实验揭示了 CoV-Y 与宿主 BRCA1 相关蛋白（BRAP）之间的相互作用。这种相互作用随后在细胞测定中得到了证实，并阐明了这两种蛋白质之间的精确结合区域。我们发现这种相互作用在 SARS-CoV 和中东呼吸综合征冠状病毒中是保守的。接下来，我们证明 CoV-Y 通过与 BRAP 结合增强 IκBα 和 IκBβ 的磷酸化，并促进下游 NF-κB 成员 p50 和 p65 的核转位。CoV-Y-BRAP 相互作用可以上调宿主炎症细胞因子的转录水平。总的来说，我们的研究结果首次阐明了 CoV-Y 的生物学功能，为冠状病毒调节宿主炎症反应提供了新的见解，以及抗病毒药物开发的可能靶点。
