casein-kinase-ii and Hepatitis-B

To investigate mechanisms that TMEM2 activation inhibits hepatitis B virus (HBV) infection in hepatocarcinoma (HCC) cells, co-immunoprecipitation (Co-IP) and mass spectrometry were used in screening interacting proteins for TMEM2. Levels of casein kinase 2 subunit α3 (CSNK2A3) in HCC cells were found to be inhibited or overexpressed using siRNAs and pcDNA3.1-CSNK2A3, respectively. Effect of CSNK2A3 expression on cell proliferation was analyzed using MTS, while its effect on HBV infection was measured using ddPCR and IHC. Western blotting and JAK inhibitor ruxolitinib were also used to determine whether TMEM2-regulated CSNK2A3 expression and HBV infection were affected by JAK-STAT signaling. Co-IP and mass spectrometry results showed that CSNK2A3 interacts with TMEM2. Moreover, overexpression of CSNK2A3 significantly inhibited cell proliferation, while inhibition of CSNK2A3 promoted proliferation of HCC cells. In addition, overexpression of CSNK2A3 was observed to significantly enhance HBV infection, while siRNA knockdown of CSNK2A3 inhibited HBV infection. Notably, effect of CSNK2A3 overexpression on HBV infection was suppressed by TMEM2 overexpression. Further mechanistic analyses have revealed that TMEM2 could antagonize the effects of CSNK2A3 on cell proliferation and HBV infection via JAK-STAT pathway activation. In conclusion, TMEM2 has been determined to bind to CSNK2A3 to inhibit HBV infection via activation of the JAK-STAT pathway.

Topics: Brain; Carcinoma, Hepatocellular; Case-Control Studies; Casein Kinase II; Epilepsy, Temporal Lobe; Hepatitis B; Hepatitis B virus; Humans; Janus Kinase 1; Liver Neoplasms; Membrane Proteins; PPAR gamma; STAT Transcription Factors; Tumor Cells, Cultured

Human La protein (hLa) is a multifunctional RNA-binding protein involved in the regulation of hepatitis B virus (HBV) expression. Casein kinase II (CK2), a protein kinase, is known to activate hLa by phosphorylating Ser(366). Tetrabromobenzimidazole (TBBz) has been shown to be a specific inhibitor of CK2 activity, which suggests that TBBz may be useful for reducing HBV gene expression. The aim of our study was to determine whether inhibition of CK2 by TBBz and decreased phosphorylation of hLa Ser(366) (pLa) would reduce HBV gene expression. pLa and total La expression levels were evaluated by immunohistochemistry in human liver tissues with or without HBV infection. HepG2.2.15 cells (an HBV-expressing cell line) were treated with TBBz, and cell viability and pLa levels were evaluated. Knockdown of hLa and CK2 levels by specific siRNA and mutant hLa Ala(366) were utilized to establish the roles of pLa and CK2 in HBV gene expression. HBV DNA replication and HBsAg and HBeAg levels were analysed in HepG2.2.15 cell supernatants by standard methods. pLa was significantly overexpressed in HBV-infected human liver samples. TBBz decreased the phosphorylation of hLa, which coincided with decreased HBV expression. Mutant hLa Ala(366) had reduced viral expression compared with hLa Ser(366) treatment in hLa siRNA knockdown cells. Knockdown of CK2 also decreased the HBV parameters. hLa plays a key role in the regulation of HBV gene expression in a CK2-dependent mechanism via phosphorylation of hLa at Ser(366).

Topics: Adolescent; Adult; Aged; Benzimidazoles; Casein Kinase II; Cell Line, Tumor; Dose-Response Relationship, Drug; Down-Regulation; Female; Gene Expression Regulation, Viral; Hepatitis B; Hepatitis B Antigens; Hepatitis B virus; Humans; Liver; Male; Middle Aged; Mutation, Missense; Phosphoproteins; Phosphorylation; Serine; Virus Replication; Young Adult

The human La (hLa) protein functions in RNA metabolism and is activated by casein kinase 2 (CK2) phosphorylation. Hepatitis B virus (HBV) can exploit hLa to stabilize its RNA and promote its pathogenesis. To enhance our knowledge of host molecular pathways involved in HBV pathogenesis, a bioinformatic approach was used to generate an expression profile of all predicted target genes of CK2-activated hLa in HBV-infected cells. A computerized literature search was performed to identify English language studies of HBV-, hLa- and CK2-related molecules. The data were pooled and the genes were classified in three functional groups by gene ontology (GO) analysis. HBV, hLa and CK2 targets were predicted, respectively, by a computational method, followed by screening for matching gene symbols in the NCBI human sequences, GO, pathway and network analyses. hLa targets and respective networks in the viral mechanisms of HBV were obtained by the final integrative analysis. Thirty-seven hub genes were identified by overlap calculation, suggesting that hLa may play an important role in the development and progression of HBV through cytokine-cytokine receptor interaction, hematopoietic cell lineage, cell adhesion molecules (CAMs), antigen processing and presentation, Jak-STAT signalling pathway, natural killer cell-mediated cytotoxicity, apoptosis, T-cell receptor signalling pathway, complement and coagulation cascades, protein export and other pathways. Our data may help researchers to predict the molecular mechanisms of hLa in the development and progression of HBV through CK2 comprehensively. Moreover, the present data indicate that hLa targets may be a series of promising candidates for HBV.

Topics: Casein Kinase II; Computational Biology; Disease Progression; Gene Expression Regulation, Viral; Gene Regulatory Networks; Genes, Regulator; Hepatitis B; Hepatitis B virus; Humans; Phosphoproteins; Phosphorylation; RNA, Viral; Signal Transduction; Transcriptome; Virulence

Two alpha-type CK2-activated PKAs (CK2-aPKAIalpha and CK2-aPKAIIalpha) were biochemically characterized in vitro using GST-HBV core fusion protein (GST-Hcore) and GST-Hcore157B as phosphate acceptors. It was found that (i), in the absence of cAMP, these two CK2-aPKAs phosphorylated both Ser-170 and Ser-178 on GST-Hcore and Hcore157B; (ii) this phosphorylation was approx. 4-fold higher than their phosphorylation by cAMP-activated PKAs; and (iii) suramin effectively inhibited the phosphorylation of Hcore157B by CK2-aPKAIIalpha through its direct binding to Hcore157B in vitro. These results suggest that high phosphorylation of HBV-CP by two CK2-aPKAs, in the absence of cAMP, may be involved in the pregenomic RNA (pgRNA) encapsidation and DNA-replication in HBV-infected cells.

Topics: Animals; Antineoplastic Agents; Casein Kinase II; Cattle; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Enzyme Activation; Genome, Viral; Hepatitis B; Hepatitis B virus; Humans; Phosphorylation; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; RNA, Viral; Serine; Suramin; Viral Core Proteins; Virus Replication