Unit of Molecular Genetics of RNA Viruses, Institut Pasteur, Paris, France; Interactomics, RNA and Immunity Laboratory, Institut Pasteur, Paris, France.
Institut Pasteur, Proteomics Core Facility, MSBio UtechS, UAR CNRS 2024, Université Paris Cité, Paris, France.
Mol Cell Proteomics. 2024 Nov;23(11):100856. doi: 10.1016/j.mcpro.2024.100856. Epub 2024 Oct 9.
Understanding the integrated regulation of cellular processes during viral infection is crucial for developing host-targeted approaches. We have previously reported that an optimal in vitro infection by influenza A virus (IAV) requires three components of Cullin 4-RING E3 ubiquitin ligases (CRL4) complexes, namely the DDB1 adaptor and two substrate recognition factors, DCAF11 and DCAF12L1, which mediate non-degradative poly-ubiquitination of the PB2 subunit of the viral polymerase. However, the impact of IAV infection on the CRL4 interactome remains elusive. Here, using Affinity Purification coupled with Mass Spectrometry (AP-MS) approaches, we identified cellular proteins interacting with these CRL4 components in IAV-infected and non-infected contexts. IAV infection induces significant modulations in protein interactions, resulting in a global loss of DDB1 and DCAF11 interactions, and an increase in DCAF12L1-associated proteins. The distinct rewiring of CRL4's associations upon infection impacted cellular proteins involved in protein folding, ubiquitination, translation, splicing, and stress responses. Using a split-nanoluciferase-based assay, we identified direct partners of CRL4 components and via siRNA-mediated silencing validated their role in IAV infection, representing potential substrates or regulators of CRL4 complexes. Our findings unravel the dynamic remodeling of the proteomic landscape of CRL4's E3 ubiquitin ligases during IAV infection, likely involved in shaping a cellular environment conducive to viral replication and offer potential for the exploration of future host-targeted antiviral therapeutic strategies.
了解病毒感染过程中细胞过程的综合调控对于开发针对宿主的方法至关重要。我们之前曾报道过，甲型流感病毒（IAV）的最佳体外感染需要三个 Cullin 4-RING E3 泛素连接酶（CRL4）复合物的组成部分，即 DDB1 接头和两个底物识别因子 DCAF11 和 DCAF12L1，它们介导病毒聚合酶 PB2 亚基的非降解性多泛素化。然而，IAV 感染对 CRL4 相互作用组的影响仍不清楚。在这里，我们使用亲和纯化结合质谱（AP-MS）方法，鉴定了 IAV 感染和未感染情况下与这些 CRL4 成分相互作用的细胞蛋白。IAV 感染会引起蛋白相互作用的显著调节，导致 DDB1 和 DCAF11 相互作用的全局丧失，以及 DCAF12L1 相关蛋白的增加。感染后 CRL4 关联的独特重排影响了涉及蛋白折叠、泛素化、翻译、剪接和应激反应的细胞蛋白。我们使用基于分裂纳米荧光素酶的测定法鉴定了 CRL4 成分的直接伙伴，并通过 siRNA 介导的沉默验证了它们在 IAV 感染中的作用，代表 CRL4 复合物的潜在底物或调节剂。我们的研究结果揭示了 IAV 感染期间 CRL4 E3 泛素连接酶的蛋白质组景观的动态重塑，可能参与塑造有利于病毒复制的细胞环境，并为探索未来针对宿主的抗病毒治疗策略提供了潜力。
