Deciphering the phospho-signature induced by hepatitis B virus in primary human hepatocytes

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 protein (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 two- and seven-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 times 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. ### Competing Interest Statement The authors have declared no competing interest. * caRNAs : chromatin-associated RNAs cccDNA : covalently-closed circular DNA DDR : DNA damage response dGepaRG : differentiated HepaRG dpi : days post-infection DSBs : DNA double-stranded breaks HBV : Hepatitis B Virus HIV-1 : human immunodeficiency virus 1 IF : ummunofluorescence IU/ml : International Units/ml KD : knock-down MOI : multiplicity of infection MS : mass spectrometry NHEJ : non-homologous end-joining PEIU/ml : Paul Erlich Institute Units/ml pgRNA : pregenomic RNA PHHs : primary human hepatocytes PPIs : protein-protein interactions RBPs : RNA-binding proteins rcDNA : relaxed circular DNA S/MAR : scaffold/matrix attachment region SARS-Cov2 : severe acute respiratory syndrome coronavirus 2 Vge/ml : viral genome equivalents/ml Wt : wild type