Pastor Florentin, Charles Emilie, Belmudes Lucid, Chabrolles Hélène, Cescato Marion, Rivoire Michel, Burger Thomas, Passot Guillaume, Durantel David, Lucifora Julie, Couté Yohann, Salvetti Anna
International Center for Research in Infectiology (CIRI), INSERM U1111, Université Claude Bernard Lyon, CNRS, UMR5308, ENS, Lyon, France.
Université Grenoble Alpes, CEA, INSERM, UA13 BGE, CEA, CNRS, FR2048, Grenoble, France.
Front Microbiol. 2024 May 22;15:1415449. doi: 10.3389/fmicb.2024.1415449. eCollection 2024.
Phosphorylation is a major post-translation modification (PTM) of proteins which is finely tuned by the activity of several hundred kinases and phosphatases. It controls most if not all cellular pathways including anti-viral responses. Accordingly, viruses often induce important changes in the phosphorylation of host factors that can either promote or counteract viral replication. Among more than 500 kinases constituting the human kinome only few have been described as important for the hepatitis B virus (HBV) infectious cycle, and most of them intervene during early or late infectious steps by phosphorylating the viral Core (HBc) protein. In addition, little is known on the consequences of HBV infection on the activity of cellular kinases. The objective of this study was to investigate the global impact of HBV infection on the cellular phosphorylation landscape early after infection. For this, primary human hepatocytes (PHHs) were challenged or not with HBV, and a mass spectrometry (MS)-based quantitative phosphoproteomic analysis was conducted 2- and 7-days post-infection. The results indicated that while, as expected, HBV infection only minimally modified the cell proteome, significant changes were observed in the phosphorylation state of several host proteins at both time points. Gene enrichment and ontology analyses of up- and down-phosphorylated proteins revealed common and distinct signatures induced by infection. In particular, HBV infection resulted in up-phosphorylation of proteins involved in DNA damage signaling and repair, RNA metabolism, in particular splicing, and cytoplasmic cell-signaling. Down-phosphorylated proteins were mostly involved in cell signaling and communication. Validation studies carried out on selected up-phosphorylated proteins, revealed that HBV infection induced a DNA damage response characterized by the appearance of 53BP1 foci, the inactivation of which by siRNA increased cccDNA levels. In addition, among up-phosphorylated RNA binding proteins (RBPs), SRRM2, a major scaffold of nuclear speckles behaved as an antiviral factor. In accordance with these findings, kinase prediction analysis indicated that HBV infection upregulates the activity of major kinases involved in DNA repair. These results strongly suggest that HBV infection triggers an intrinsic anti-viral response involving DNA repair factors and RBPs that contribute to reduce HBV replication in cell culture models.
磷酸化是蛋白质的一种主要翻译后修饰（PTM），由数百种激酶和磷酸酶的活性精细调节。它控制着大多数（如果不是全部的话）细胞通路，包括抗病毒反应。因此，病毒常常会引起宿主因子磷酸化的重要变化，这些变化既可以促进也可以对抗病毒复制。在构成人类激酶组的500多种激酶中，只有少数几种被描述为对乙型肝炎病毒（HBV）感染周期很重要，其中大多数通过磷酸化病毒核心（HBc）蛋白在感染的早期或晚期阶段发挥作用。此外，关于HBV感染对细胞激酶活性的影响知之甚少。本研究的目的是调查感染后早期HBV感染对细胞磷酸化格局的整体影响。为此，用HBV对原代人肝细胞（PHH）进行或不进行攻击，并在感染后2天和7天进行基于质谱（MS）的定量磷酸化蛋白质组分析。结果表明，正如预期的那样，HBV感染仅对细胞蛋白质组产生最小程度的修饰，但在两个时间点均观察到几种宿主蛋白的磷酸化状态发生了显著变化。对上调和下调磷酸化蛋白的基因富集和本体分析揭示了感染诱导的共同和不同特征。特别是，HBV感染导致参与DNA损伤信号传导和修复、RNA代谢（尤其是剪接）以及细胞质细胞信号传导的蛋白质磷酸化上调。下调磷酸化的蛋白质大多参与细胞信号传导和通讯。对选定的上调磷酸化蛋白进行的验证研究表明，HBV感染诱导了一种DNA损伤反应，其特征是出现53BP1病灶，通过siRNA使其失活会增加cccDNA水平。此外，在上调磷酸化的RNA结合蛋白（RBP）中，核斑点的主要支架SRRM2表现为一种抗病毒因子。与这些发现一致，激酶预测分析表明HBV感染会上调参与DNA修复的主要激酶的活性。这些结果强烈表明，HBV感染触发了一种内在的抗病毒反应，涉及DNA修复因子和RBP，它们有助于在细胞培养模型中减少HBV复制。
