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

Abstract

Phosphorylation is a major post-translation modification (PTM) of proteins, and small molecules, 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 help 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, scarce information is available 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 at early after infection. For this, primary human hepatocytes (PHH) 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, important changes were observed on the phosphorylation level 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 following 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. Several down-phosphorylated factors, mostly involved in cell signaling and communication, were also detected. Validation studies performed on some selected up-phosphorylated proteins, revealed that HBV infection induced a DNA damage response characterized by the appearance of 53BP1 foci, whose siRNA-mediated knock-down increased cccDNA levels. In addition, among up-phosphorylated RBPs, SRRM2, a major scaffold of nuclear speckles behaved as 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 composed by DNA repair factors and RBPs that contribute to reduce HBV replication in cell culture models.

The Dual-Specificity Kinase Dyrk1a Interacts With the Hepatitis B Virus Genome and Regulates the Production of Viral Rna

“…Therefore, it is possible that a low and transient stimulation of HBx mRNA transcription early upon cccDNA formation may lead to the synthesis of a basal level of HBx sufficient to induce Smc5/6 degradation (72). In this regard, it is interesting to note that in a recent phospho-proteomic analysis we found that DYRK1A was among the top list of kinases whose activity is predicted to be up-regulated upon HBV infection (73).…”