Quantitative Proteomics Reveals a Novel Role of the E3 Ubiquitin-Protein Ligase FANCL in the Activation of the Innate Immune Response through Regulation of TBK1 Phosphorylation during Peste des Petits Ruminants Virus Infection

Peste
des petits ruminants virus (PPRV) infection causes considerable
innate immunosuppression in its host, which promotes viral replication.
However, how the host rescues the innate immune response to counteract
this immunosuppression during viral replication remains largely unknown.
To explore the mechanisms of how a host counteracts PPRV-mediated
innate immunosuppression, a high-throughput quantitation proteomic
approach (isobaric tags for relative and absolute quantitation in
conjunction with LC–MS/MS) was used to investigate the proteome
landscape of goat fetal fibroblasts (GFFs) in response to PPRV infection.
Eventually, 497 upregulated proteins and 358 downregulated proteins
were identified. Many of the differentially expressed proteins were
enriched in immune-related pathways. Blocking the activation of the
innate immune response with a specific inhibitor BX795 in GFFs remarkably
promoted PPRV replication, suggesting the significant antiviral role
of the enriched immune-related pathways. The GO enrichment analysis
showed that the host protein FANCL revealed a similar expression pattern
to these innate immune-related proteins. In addition, the analysis
of protein–protein interaction networks reveals a potential
relationship between FANCL and the innate immune pathway. We determined
that FANCL inhibited PPRV infection by enhancing type I interferon
(IFN) and IFN-stimulated gene expression. Further investigation determined
that FANCL induced type I IFN production by promoting TBK1 phosphorylation,
thus impairing PPRV-mediated immunosuppression.

小反刍兽疫病毒（Peste des petits ruminants virus, PPRV）感染会导致宿主出现显著的先天免疫抑制，进而促进病毒复制。然而，在病毒复制期间，宿主如何恢复先天免疫应答以对抗这种免疫抑制，目前仍未明确。
为探究宿主拮抗PPRV介导的先天免疫抑制的机制，本研究采用高通量定量蛋白质组学方法——即同位素相对与绝对定量标记技术（isobaric tags for relative and absolute quantitation）联合液相色谱-串联质谱（LC–MS/MS）——分析山羊胎成纤维细胞（goat fetal fibroblasts, GFFs）感染PPRV后的蛋白质组图谱。最终共鉴定得到497个上调蛋白与358个下调蛋白。大量差异表达蛋白富集于免疫相关通路。在GFFs中使用特异性抑制剂BX795阻断先天免疫应答的激活，可显著促进PPRV复制，这表明上述富集的免疫相关通路发挥着重要的抗病毒作用。基因本体（Gene Ontology, GO）富集分析显示，宿主蛋白FANCL的表达模式与这些先天免疫相关蛋白高度相似。此外，蛋白质相互作用网络分析揭示了FANCL与先天免疫通路之间存在潜在关联。我们证实FANCL通过增强I型干扰素（type I interferon, IFN）及其刺激基因（IFN-stimulated gene）的表达来抑制PPRV感染。进一步研究表明，FANCL通过促进TANK结合激酶1（TBK1）的磷酸化诱导I型干扰素产生，从而削弱PPRV介导的先天免疫抑制。