Differential Proteomics Reveals Discrete Functions of Proteins Interacting with Hypo- versus Hyper-phosphorylated NS5A of the Hepatitis C Virus

• Published in: Journal of proteome research 2019-07, Vol.18 (7), p.2813-2825
• Main Authors: Pan, Ting-Chun, Lo, Chieh-Wen, Chong, Weng Man, Tsai, Chia-Ni, Lee, Kuan-Ying, Chen, Pin-Yin, Liao, Jung-Chi, Yu, Ming-Jiun
• Format: Article
• Language: English
• Subjects: DNA-Activated Protein Kinase - analysis; DNA-Activated Protein Kinase - metabolism; Hepacivirus - chemistry; Hepatitis C - complications; Hepatitis C - immunology; Hepatitis C - pathology; Humans; Liver Neoplasms - etiology; Phosphorylation; Protein Binding; Proteomics - methods; Viral Nonstructural Proteins - metabolism
• ISSN: 1535-3893; 1535-3907
• DOI: 10.1021/acs.jproteome.9b00130

Protein phosphorylation is a reversible post-translational modification that regulates many biological processes in almost all living forms. In the case of the hepatitis C virus (HCV), the nonstructural protein 5A (NS5A) is believed to transit between hypo- and hyper-phosphorylated forms that interact with host proteins to execute different functions; however, little was known about the proteins that bind either form of NS5A. Here, we generated two high-quality antibodies specific to serine 235 nonphosphorylated hypo- vs serine 235 phosphorylated (pS235) hyper-phosphorylated form of NS5A and for the first time segregated these two forms of NS5A plus their interacting proteins for dimethyl-labeling based proteomics. We identified 629 proteins, of which 238 were quantified in three replicates. Bioinformatics showed 46 proteins that preferentially bind hypo-phosphorylated NS5A are involved in antiviral response and another 46 proteins that bind pS235 hyper-phosphorylated NS5A are involved in liver cancer progression. We further identified a DNA-dependent kinase (DNA-PK) that binds hypo-phosphorylated NS5A. Inhibition of DNA-PK with an inhibitor or via gene-specific knockdown significantly reduced S232 phosphorylation and NS5A hyper-phosphorylation. Because S232 phosphorylation initiates sequential S232/S235/S238 phosphorylation leading to NS5A hyper-phosphorylation, we identified a new protein kinase that regulates a delicate balance of NS5A between hypo- and hyper-phosphorylation states, respectively, involved in host antiviral responses and liver cancer progression.