casein-kinase-ii and Hepatitis-C

Hepatitis C virus (HCV) chronically affects millions of individuals worldwide. The HCV nonstructural protein 5A (NS5A) plays a critical role in the viral assembly pathway. Domain 3 (D3) of NS5A is an unstructured polypeptide responsible for the interaction with the core particle assembly structure. Casein kinase 2 (CK2) phosphorylates NS5A-D3 at multiple sites that have mostly been predicted and only observed indirectly. In order to identify the CK2-dependent phosphorylation sites, we monitored the reaction between NS5A-D3 and CK2 in vitro by time-resolved NMR. We unambiguously identified four serine residues as substrates of CK2. The apparent rate constant for each site was determined from the reaction curves. Ser408 was quickly phosphorylated, whereas the three other serine residues reacted more slowly. These results provide a starting point from which to elucidate the role of phosphorylation in the mechanisms of viral assembly-and in the modulation of the viral activity-at the molecular level.

Topics: Amino Acid Sequence; Casein Kinase II; Hepacivirus; Hepatitis C; Humans; Molecular Sequence Data; Nuclear Magnetic Resonance, Biomolecular; Phosphorylation; Protein Structure, Tertiary; Serine; Viral Nonstructural Proteins

By sensing viral nucleic acids, host innate receptors elicit signaling pathways converging on TBK1-IFN regulatory factor (IRF)3 axis in mediating IFN-αβ induction and defense mechanisms. In contrast, viruses have evolved with diverse immune evasion/interference mechanisms to undermine innate receptor signaling and IFN response. In this regard, approaches enabling host to overcome such immune evasion/interference mechanisms are urgently needed to combat infections by epidemic/pandemic viruses. In this study, we report that protein kinase CK2 serves as a key component controlling TBK1 and IRF3 activation in IFN-inducing TLR, RIG-I-like receptors, and cGAS/STING signaling pathways. Accordingly, knocking down of CK2 expression or genetic ablation of its kinase activity resulted in elevated IFN-αβ response in response to infection by DNA and RNA viruses. Moreover, PP2A was identified as one of the intermediate phosphatases responsible for CK2-regulated IFN response, suggesting that CK2 may regulate TBK1 and IRF3 activation indirectly. Importantly, blockade of CK2 activity by small molecule inhibitor was able to activate TBK1, whereby eliciting effective host defense mechanisms against hepatitis C virus infection. Taken together, our results identify CK2 as a novel regulator of TBK1 and IRF3 and suggest that targeting CK2 by small molecular inhibitor may be a viable approach to prevent and treat viral infections.

Topics: Animals; Casein Kinase II; Cell Line; Disease Models, Animal; Hepatitis C; Herpes Simplex; Herpesvirus 1, Human; Humans; Interferon Regulatory Factor-3; Interferons; Membrane Proteins; Mice; Protein Serine-Threonine Kinases; Toll-Like Receptor 3; Toll-Like Receptor 4; Virus Diseases

Hepatitis C virus (HCV) is a significant pathogen, infecting some 170 million people worldwide. Persistent virus infection often leads to cirrhosis and liver cancer. In the infected cell many RNA directed processes must occur to maintain and spread infection. Viral genomic RNA is constantly replicating, serving as template for translation, and being packaged into new virus particles; processes that cannot occur simultaneously. Little is known about the regulation of these events. The viral NS5A phosphoprotein has been proposed as a regulator of events in the HCV life cycle for years, but the details have remained enigmatic. NS5A is a three-domain protein and the requirement of domains I and II for RNA replication is well documented. NS5A domain III is not required for RNA replication, and the function of this region in the HCV lifecycle is unknown. We have identified a small deletion in domain III that disrupts the production of infectious virus particles without altering the efficiency of HCV RNA replication. This deletion disrupts virus production at an early stage of assembly, as no intracellular virus is generated and no viral RNA and nucleocapsid protein are released from cells. Genetic mapping has indicated a single serine residue within the deletion is responsible for the observed phenotype. This serine residue lies within a casein kinase II consensus motif, and mutations that mimic phosphorylation suggest that phosphorylation at this position regulates the production of infectious virus. We have shown by genetic silencing and chemical inhibition experiments that NS5A requires casein kinase II phosphorylation at this position for virion production. A mutation that mimics phosphorylation at this position is insensitive to these manipulations of casein kinase II activity. These data provide the first evidence for a function of the domain III of NS5A and implicate NS5A as an important regulator of the RNA replication and virion assembly of HCV. The ability to uncouple virus production from RNA replication, as described herein, may be useful in understanding HCV assembly and may be therapeutically important.

Topics: Benzimidazoles; Casein Kinase II; Chromosome Mapping; Gene Deletion; Gene Expression Regulation, Viral; Gene Silencing; Hepacivirus; Hepatitis C; Mutagenesis, Site-Directed; Phosphorylation; Point Mutation; RNA-Dependent RNA Polymerase; RNA, Viral; Viral Core Proteins; Viral Nonstructural Proteins; Virion; Virus Replication