Chronic hepatitis caused by infection with the Hepatitis B virus is a life-threatening condition. In fact, 1 million people die annually due to liver cirrhosis or hepatocellular carcinoma. Recently, several studies demonstrated a molecular connection between the host DNA damage response (DDR) pathway and HBV replication and reactivation. Here, we investigated the role of Ataxia-telangiectasia-mutated (ATM) and Ataxia telangiectasia and Rad3-related (ATR) PI3-kinases in phosphorylation of the HBV core protein (HBc). We determined that treatment of HBc-expressing hepatocytes with genotoxic agents, e.g., etoposide or hydrogen peroxide, activated the host ATM-Chk2 pathway, as determined by increased phosphorylation of ATM at Ser1981 and Chk2 at Thr68. The activation of ATM led, in turn, to increased phosphorylation of cytoplasmic HBc at serine-glutamine (SQ) motifs located in its C-terminal domain. Conversely, down-regulation of ATM using ATM-specific siRNAs or inhibitor effectively reduced etoposide-induced HBc phosphorylation. Detailed mutation analysis of S-to-A HBc mutants revealed that S170 (S168 in a 183-aa HBc variant) is the primary site targeted by ATM-regulated phosphorylation. Interestingly, mutation of two major phosphorylation sites involving serines at positions 157 and 164 (S155 and S162 in a 183-aa HBc variant) resulted in decreased etoposide-induced phosphorylation, suggesting that the priming phosphorylation at these serine-proline (SP) sites is vital for efficient phosphorylation of SQ motifs. Notably, the mutation of S172 (S170 in a 183-aa HBc variant) had the opposite effect and resulted in massively up-regulated phosphorylation of HBc, particularly at S170. Etoposide treatment of HBV infected HepG2-NTCP cells led to increased levels of secreted HBe antigen and intracellular HBc protein. Together, our studies identified HBc as a substrate for ATM-mediated phosphorylation and mapped the phosphorylation sites. The increased expression of HBc and HBe antigens in response to genotoxic stress supports the idea that the ATM pathway may provide growth advantage to the replicating virus.

• MeSH
• Amino Acid Motifs MeSH
• Ataxia Telangiectasia Mutated Proteins metabolism   MeSH
• Hep G2 Cells MeSH
• Checkpoint Kinase 2 metabolism   MeSH
• Cytoplasm metabolism   virology   MeSH
• Etoposide pharmacology   MeSH
• Phosphorylation MeSH
• Hepatitis B e Antigens metabolism   MeSH
• Hepatocytes virology   MeSH
• Humans MeSH
• Hydrogen Peroxide pharmacology   MeSH
• DNA Damage * MeSH
• Viral Core Proteins chemistry   metabolism   MeSH
• Virus Replication drug effects   MeSH
• Serine metabolism   MeSH
• Trans-Activators genetics   metabolism   MeSH
• Viral Regulatory and Accessory Proteins genetics   metabolism   MeSH
• Hepatitis B virus drug effects   physiology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Infection with hepatitis B virus (HBV) often leads to development of chronic liver disease. In fact, 10% of infected adults and almost 90% of infected infants develop chronic hepatitis B associated with severe liver diseases, including acute liver failure, liver cirrhosis or hepatocellular carcinoma. At present there is no effective cure for chronic hepatitis B. The current treatment of chronically infected patients is long-term, expensive and relies on treatment with nucleos(t)ide analogs in combination with immune therapies, that frequently lead to adverse side effects. Recently, the National Institute of Health proposed strategic plan for Trans-NIH research to cure hepatitis B. The key priority is better understanding of HBV life cycle and its interactions with host cell. Due to the fact that HBV is a small double stranded DNA virus encoding only a limited number of proteins, HBV replication widely relies on host cell pathways and proteins. As demonstrated by numerous reports, HBV core protein (HBc) which is the main component of viral nucleocapsid, plays multiple roles in HBV life cycle and is engaged in many protein interaction networks of the host cell. Several recent studies have shown that HBV proteins can be modified by different types of posttranslational modifications (PTMs) that affect their protein-protein interactions, subcellular localization and function. In this review, we discuss diverse PTMs of HBc and their role in regulation of HBc function in the context of HBV replication and pathogenesis.

• MeSH
• Phosphorylation MeSH
• Humans MeSH
• Protein Processing, Post-Translational MeSH
• Protein-Arginine N-Methyltransferases MeSH
• Viral Core Proteins genetics   MeSH
• Ubiquitination MeSH
• Hepatitis B virus * genetics   pathogenicity   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH

In mammals, protein arginine methyltransferase 5, PRMT5, is the main type II enzyme responsible for the majority of symmetric dimethylarginine formation in polypeptides. Recent study reported that PRMT5 restricts Hepatitis B virus (HBV) replication through epigenetic repression of HBV DNA transcription and interference with encapsidation of pregenomic RNA. Here we demonstrate that PRMT5 interacts with the HBV core (HBc) protein and dimethylates arginine residues within the arginine-rich domain (ARD) of the carboxyl-terminus. ARD consists of four arginine rich subdomains, ARDI, ARDII, ARDIII and ARDIV. Mutation analysis of ARDs revealed that arginine methylation of HBc required the wild-type status of both ARDI and ARDII. Mass spectrometry analysis of HBc identified multiple potential ubiquitination, methylation and phosphorylation sites, out of which lysine K7 and arginines R150 (within ARDI) and R156 (outside ARDs) were shown to be modified by ubiquitination and methylation, respectively. The HBc symmetric dimethylation appeared to be linked to serine phosphorylation and nuclear import of HBc protein. Conversely, the monomethylated HBc retained in the cytoplasm. Thus, overexpression of PRMT5 led to increased nuclear accumulation of HBc, and vice versa, down-regulation of PRMT5 resulted in reduced levels of HBc in nuclei of transfected cells. In summary, we identified PRMT5 as a potent controller of HBc cell trafficking and function and described two novel types of HBc post-translational modifications (PTMs), arginine methylation and ubiquitination.

• MeSH
• Phosphorylation MeSH
• Mass Spectrometry MeSH
• Humans MeSH
• Methylation MeSH
• Protein-Arginine N-Methyltransferases metabolism   physiology   MeSH
• Virus Replication physiology   MeSH
• Subcellular Fractions metabolism   MeSH
• Ubiquitination MeSH
• Hepatitis B virus enzymology   physiology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Kaposi sarcoma-associated herpesvirus is associated with two lymphoproliferative disorders, primary effusion lymphoma (PEL) and Castleman disease. In PEL, Kaposi sarcoma-associated herpesvirus is present in a latent form expressing only few viral genes. Among them is a viral homologue of cellular interferon regulatory factors, vIRF-3. To study the role of vIRF-3 in PEL lymphomagenesis, we analyzed the interaction of vIRF-3 with cellular proteins. Using yeast two-hybrid screen, we detected the association between vIRF-3 and c-Myc suppressor, MM-1alpha. The vIRF-3 and MM-1alpha interaction was also demonstrated by glutathione S-transferase pulldown assay and coimmunoprecipitation of endogenous vIRF-3 and MM-1alpha in PEL-derived cell lines. Overexpression of vIRF-3 enhanced the c-Myc-dependent transcription of the gene cdk4. Addressing the molecular mechanism of the vIRF-3-mediated stimulation, we demonstrated that the association between MM-1alpha and c-Myc was inhibited by vIRF-3. Furthermore, the recruitment of vIRF-3 to the cdk4 promoter and the elevated levels of the histone H3 acetylation suggest the direct involvement of vIRF-3 in the activation of c-Myc-mediated transcription. These findings indicate that vIRF-3 can effectively stimulate c-Myc function in PEL cells and consequently contribute to de-regulation of B-cell growth and differentiation.

• MeSH
• Financing, Organized MeSH
• Gene Library MeSH
• Interferon Regulatory Factors metabolism   MeSH
• Interferon Regulatory Factor-3 metabolism   MeSH
• Humans MeSH
• Herpesvirus 8, Human metabolism   MeSH
• Cell Line, Tumor MeSH
• Lymphoma, Primary Effusion metabolism   MeSH
• Proto-Oncogene Proteins c-myc metabolism   MeSH
• Two-Hybrid System Techniques MeSH
• Viral Proteins metabolism   MeSH
• Check Tag
• Humans MeSH