PRMT5: a Novel Regulator of Hepatitis B Virus Replication and an Arginine Methylase of HBV Core
In the study, we identified protein arginine methyltransferase 5 (PRMT5) as a novel restriction factor of HBV replication. We have also demonstrated that PRMT5 can interact with the HBV core and methylate its arginine residues within arginine-rich domain. We identified two types of HBc post-translational modifications: arginine methylation and ubiquitination. While monomethylated HBc retains cytoplasmic localization, symmetric dimethylation is linked to serine phosphorylation and nuclear transport. Thus arginine methylation and ubiquitination are novel types of HBc post-translational modifications which add another level of complexity to the understanding of HBc function and regulation of HBV replication
Sample Processing Protocol
Cells transfected with HA-tagged HBc-expression plasmid were lysed and incubated with anti-HA magnetic beads and extensively washed. The samples were then treated with sampling buffer and separated on SDS PAGE. The gel was separated into 14 bands, destained, reduced, alkylated and digested by trypsin. Peptides were extracted and analyzed by UltiMate 3000 RSLCnano system (Dionex) coupled to a TripleTOF 5600 mass spectrometer with a NanoSpray III source (Sciex).The peptides were first trapped, desalted and then separated on Acclaim PepMap100 analytical column (3 μm, 25 cm×75 μm ID) using 70 min gradient
Data Processing Protocol
The resulting raw files were searched against database consisting of human and HBV proteins (Uniprot as of Feb 15 2016) including HA tagged sequence of HBV Core protein. ProteinPilot search engine was set to select Iodoacetamide as alkylating agent, trypsin as protease, TripleTOF 5600 as instrument, PTM probabilitites included in the set called Biological modification, thorough search effort and running FDR analysis.
Lubyova B, Hodek J, Zabransky A, Prouzova H, Hubalek M, Hirsch I, Weber J. PRMT5: A novel regulator of Hepatitis B virus replication and an arginine methylase of HBV core. PLoS One. 2017 12(10):e0186982 PubMed: 29065155