Hepatitis B virus uses e antigen (HBe), which is dispensable for virus infectivity, to modulate host immune responses and achieve viral persistence in human hepatocytes. The HBe precursor (p25) is directed to the endoplasmic reticulum (ER), where cleavage of the signal peptide (sp) gives rise to the first processing product, p22. P22 can be retro-translocated back to the cytosol or enter the secretory pathway and undergo a second cleavage event, resulting in secreted p17 (HBe). Here, we report that translocation of p25 to the ER is promoted by translocon-associated protein complex. We have found that p25 is not completely translocated into the ER; a fraction of p25 is phosphorylated and remains in the cytoplasm and nucleus. Within the p25 sp sequence, we have identified three cysteine residues that control the efficiency of sp cleavage and contribute to proper subcellular distribution of the precore pool.

• MeSH
• Cysteine metabolism   MeSH
• Endoplasmic Reticulum metabolism   MeSH
• Hepatitis B e Antigens * metabolism   MeSH
• Hepatitis B * metabolism   MeSH
• Humans MeSH
• Membrane Glycoproteins MeSH
• Protein Sorting Signals genetics   MeSH
• Calcium-Binding Proteins MeSH
• Receptors, Cytoplasmic and Nuclear MeSH
• Receptors, Peptide MeSH
• Hepatitis B virus metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

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

Hepatitis B virus (HBV) core protein (HBc) plays many roles in the HBV life cycle, such as regulation of transcription, RNA encapsidation, reverse transcription, and viral release. To accomplish these functions, HBc interacts with many host proteins and undergoes different post-translational modifications (PTMs). One of the most common PTMs is ubiquitination, which was shown to change the function, stability, and intracellular localization of different viral proteins, but the role of HBc ubiquitination in the HBV life cycle remains unknown. Here, we found that HBc protein is post-translationally modified through K29-linked ubiquitination. We performed a series of co-immunoprecipitation experiments with wild-type HBc, lysine to arginine HBc mutants and wild-type ubiquitin, single lysine to arginine ubiquitin mutants, or single ubiquitin-accepting lysine constructs. We observed that HBc protein could be modified by ubiquitination in transfected as well as infected hepatoma cells. In addition, ubiquitination predominantly occurred on HBc lysine 7 and the preferred ubiquitin chain linkage was through ubiquitin-K29. Mass spectrometry (MS) analyses detected ubiquitin protein ligase E3 component N-recognin 5 (UBR5) as a potential E3 ubiquitin ligase involved in K29-linked ubiquitination. These findings emphasize that ubiquitination of HBc may play an important role in HBV life cycle.

• MeSH
• Arginine genetics   MeSH
• Cell Line MeSH
• Hep G2 Cells MeSH
• HEK293 Cells MeSH
• Hepatitis B genetics   MeSH
• Carcinoma, Hepatocellular genetics   MeSH
• Humans MeSH
• Lysine genetics   MeSH
• Cell Line, Tumor MeSH
• Protein Processing, Post-Translational genetics   MeSH
• Ubiquitin genetics   MeSH
• Ubiquitination genetics   MeSH
• Ubiquitin-Protein Ligases genetics   MeSH
• Viral Proteins genetics   MeSH
• Hepatitis B virus genetics   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

Recombinant interferon-α (IFN-α) treatment functionally cures chronic hepatitis B virus (HBV) infection in some individuals and suppresses virus replication in hepatocytes infected in vitro. We studied the antiviral effect of conditioned media (CM) from peripheral blood mononuclear cells (PBMCs) stimulated with agonists of Toll-like receptors (TLRs) 2, 7, 8 and 9. We found that CM from PBMCs stimulated with dual-acting TLR7/8 (R848) and TLR2/7 (CL413) agonists were more potent drivers of inhibition of HBe and HBs antigen secretion from HBV-infected primary human hepatocytes (PHH) than CM from PBMCs stimulated with single-acting TLR7 (CL264) or TLR9 (CpG-B) agonists. Inhibition of HBV in PHH did not correlate with the quantity of PBMC-produced IFN-α, but it was a complex function of multiple secreted cytokines. More importantly, we found that the CM that efficiently inhibited HBV production in freshly isolated PHH via various cytokine repertoires and mechanisms did not reduce covalently closed circular (ccc)DNA levels. We confirmed our data with a cell culture model based on HepG2-NTCP cells and the plasmacytoid dendritic cell line GEN2.2. Collectively, our data show the importance of dual-acting TLR agonists inducing broad cytokine repertoires. The development of poly-specific TLR agonists provides novel opportunities towards functional HBV cure.

• MeSH
• Hep G2 Cells MeSH
• Hepatitis B, Chronic virology   MeSH
• Cytokines metabolism   MeSH
• Hepatocytes virology   MeSH
• Interferon-alpha metabolism   MeSH
• DNA, Circular metabolism   MeSH
• Culture Media, Conditioned pharmacology   MeSH
• Drug Delivery Systems MeSH
• Leukocytes, Mononuclear metabolism   MeSH
• Humans MeSH
• Immunity, Innate drug effects   MeSH
• Virus Replication drug effects   MeSH
• Toll-Like Receptors agonists   metabolism   MeSH
• Hepatitis B virus physiology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't 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

Viruses have developed numerous strategies to counteract the host cell defense. Kaposi's sarcoma-associated herpesvirus (KSHV) is a DNA tumor virus linked to the development of Kaposi's sarcoma, Castleman's disease, and primary effusion lymphoma (PEL). The virus-encoded viral interferon regulatory factor 3 (vIRF-3) gene is a latent gene which is involved in the regulation of apoptosis, cell cycle, antiviral immunity, and tumorigenesis. vIRF-3 was shown to interact with p53 and inhibit p53-mediated apoptosis. However, the molecular mechanism underlying this phenomenon has not been established. Here, we show that vIRF-3 associates with the DNA-binding domain of p53, inhibits p53 phosphorylation on serine residues S15 and S20, and antagonizes p53 oligomerization and the DNA-binding affinity. Furthermore, vIRF-3 destabilizes p53 protein by increasing the levels of p53 polyubiquitination and targeting p53 for proteasome-mediated degradation. Consequently, vIRF-3 attenuates p53-mediated transcription of the growth-regulatory p21 gene. These effects of vIRF-3 are of biological relevance since the knockdown of vIRF-3 expression in KSHV-positive BC-3 cells, derived from PEL, leads to an increase in p53 phosphorylation, enhancement of p53 stability, and activation of p21 gene transcription. Collectively, these data suggest that KSHV evolved an efficient mechanism to downregulate p53 function and thus facilitate uncontrolled cell proliferation and tumor growth.

• MeSH
• Apoptosis genetics   MeSH
• Phosphorylation MeSH
• HCT116 Cells MeSH
• HEK293 Cells MeSH
• Cyclin-Dependent Kinase Inhibitor p21 genetics   metabolism   MeSH
• Interferon Regulatory Factors genetics   metabolism   MeSH
• Humans MeSH
• Herpesvirus 8, Human genetics   metabolism   MeSH
• Protein Multimerization MeSH
• Mutation MeSH
• Cell Line, Tumor MeSH
• Tumor Suppressor Protein p53 chemistry   genetics   metabolism   MeSH
• Reverse Transcriptase Polymerase Chain Reaction MeSH
• Promoter Regions, Genetic genetics   MeSH
• bcl-2-Associated X Protein genetics   metabolism   MeSH
• Gene Expression Regulation, Neoplastic MeSH
• RNA Interference MeSH
• Serine genetics   metabolism   MeSH
• Protein Stability MeSH
• Transfection MeSH
• Ubiquitination MeSH
• Protein Binding MeSH
• Viral Proteins genetics   metabolism   MeSH
• Blotting, Western MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent associated with Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman disease (MCD). Similar to other herpesviruses, KSHV has two life cycles, latency and lytic replication. In latency, the KSHV genome persists as a circular episome in the nucleus of the host cell and only a few viral genes are expressed. In this review, we focus on oncogenic, antiapoptotic, and immunomodulating properties of KSHV-encoded homologues of cellular interferon regulatory factors (IRFs)--viral IRF1 (vIRF1) to vIRF4--and their possible role in the KSHV-mediated antiviral response, apoptosis, and oncogenicity.

• MeSH
• Apoptosis MeSH
• Models, Biological MeSH
• Interferon Regulatory Factors genetics   immunology   MeSH
• Interferons metabolism   MeSH
• Carcinogenesis MeSH
• Humans MeSH
• Herpesvirus 8, Human genetics   immunology   pathogenicity   MeSH
• Multigene Family MeSH
• Signal Transduction MeSH
• Genes, Viral MeSH
• Viral Proteins genetics   immunology   MeSH
• Inflammation etiology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH

The Kaposi sarcoma-associated herpesvirus (KSHV) has been linked to Kaposi sarcoma, body cavity-based lymphoma, and Castleman disease. vIRF-3 is a KSHV latent gene that is critical for proliferation of KSHV-positive lymphoid cells. Furthermore, vIRF-3 contributes to KSHV-associated pathogenesis by stimulating c-Myc transcription activity. Here we show that vIRF-3 can associate with Skp2, a key component of the SCF(skp2) ubiquitin ligase complex. Skp2 is a transcriptional co-factor for c-Myc that was shown to regulate the stability of c-Myc protein as well as c-Myc-dependent transcription. In this study, we show that vIRF-3 binds to the F-box of Skp2 and recruits it to c-Myc-regulated promoters to activate c-Myc-dependent transcription. Additionally, cells overexpressing vIRF-3 exhibit higher levels of c-Myc ubiquitylation, suggesting that ubiquitylation is necessary for c-Myc-mediated transcription. Moreover, vIRF-3 can stabilize the c-Myc protein by increasing its half-life. Collectively, these results indicate that vIRF-3 can effectively manipulate c-Myc stability and function and thus contribute to c-Myc-induced KSHV-associated lymphomagenesis.

• MeSH
• Transcription, Genetic genetics   MeSH
• HEK293 Cells MeSH
• HeLa Cells MeSH
• Castleman Disease genetics   metabolism   virology   MeSH
• Interferon Regulatory Factors genetics   metabolism   MeSH
• Humans MeSH
• Herpesvirus 8, Human genetics   metabolism   MeSH
• Lymphocytes metabolism   virology   MeSH
• S-Phase Kinase-Associated Proteins genetics   metabolism   MeSH
• Proto-Oncogene Proteins c-myc genetics   metabolism   MeSH
• Protein Stability MeSH
• Ubiquitination genetics   MeSH
• Protein Binding MeSH
• Viral Proteins genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

• Publication type
• Meeting Abstract MeSH