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
• cystein metabolismus   MeSH
• endoplazmatické retikulum metabolismus   MeSH
• hepatitida B - antigeny e * metabolismus   MeSH
• hepatitida B * metabolismus   MeSH
• lidé MeSH
• membránové glykoproteiny MeSH
• proteiny - lokalizační signály genetika   MeSH
• proteiny vázající vápník MeSH
• receptory cytoplazmatické a nukleární MeSH
• receptory peptidů MeSH
• virus hepatitidy B metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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
• aminokyselinové motivy MeSH
• ATM protein metabolismus   MeSH
• buňky Hep G2 MeSH
• checkpoint kinasa 2 metabolismus   MeSH
• cytoplazma metabolismus   virologie   MeSH
• etoposid farmakologie   MeSH
• fosforylace MeSH
• hepatitida B - antigeny e metabolismus   MeSH
• hepatocyty virologie   MeSH
• lidé MeSH
• peroxid vodíku farmakologie   MeSH
• poškození DNA * MeSH
• proteiny virového jádra chemie   metabolismus   MeSH
• replikace viru účinky léků   MeSH
• serin metabolismus   MeSH
• trans-aktivátory genetika   metabolismus   MeSH
• virové regulační a přídatné proteiny genetika   metabolismus   MeSH
• virus hepatitidy B účinky léků   fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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
• arginin genetika   MeSH
• buněčné linie MeSH
• buňky Hep G2 MeSH
• HEK293 buňky MeSH
• hepatitida B genetika   MeSH
• hepatocelulární karcinom genetika   MeSH
• lidé MeSH
• lysin genetika   MeSH
• nádorové buněčné linie MeSH
• posttranslační úpravy proteinů genetika   MeSH
• ubikvitin genetika   MeSH
• ubikvitinace genetika   MeSH
• ubikvitinligasy genetika   MeSH
• virové proteiny genetika   MeSH
• virus hepatitidy B genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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
• fosforylace MeSH
• lidé MeSH
• posttranslační úpravy proteinů MeSH
• proteinarginin-N-methyltransferasy MeSH
• proteiny virového jádra genetika   MeSH
• ubikvitinace MeSH
• virus hepatitidy B * genetika   patogenita   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH
• přehledy 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
• buňky Hep G2 MeSH
• chronická hepatitida B virologie   MeSH
• cytokiny metabolismus   MeSH
• hepatocyty virologie   MeSH
• interferon alfa metabolismus   MeSH
• kruhová DNA metabolismus   MeSH
• kultivační média speciální farmakologie   MeSH
• lékové transportní systémy MeSH
• leukocyty mononukleární metabolismus   MeSH
• lidé MeSH
• přirozená imunita účinky léků   MeSH
• replikace viru účinky léků   MeSH
• toll-like receptory agonisté   metabolismus   MeSH
• virus hepatitidy B fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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
• fosforylace MeSH
• hmotnostní spektrometrie MeSH
• lidé MeSH
• metylace MeSH
• proteinarginin-N-methyltransferasy metabolismus   fyziologie   MeSH
• replikace viru fyziologie   MeSH
• subcelulární frakce metabolismus   MeSH
• ubikvitinace MeSH
• virus hepatitidy B enzymologie   fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články 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
• apoptóza genetika   MeSH
• fosforylace MeSH
• HCT116 buňky MeSH
• HEK293 buňky MeSH
• inhibitor p21 cyklin-dependentní kinasy genetika   metabolismus   MeSH
• interferonové regulační faktory genetika   metabolismus   MeSH
• lidé MeSH
• lidský herpesvirus 8 genetika   metabolismus   MeSH
• multimerizace proteinu MeSH
• mutace MeSH
• nádorové buněčné linie MeSH
• nádorový supresorový protein p53 chemie   genetika   metabolismus   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• promotorové oblasti (genetika) genetika   MeSH
• protein X asociovaný s bcl-2 genetika   metabolismus   MeSH
• regulace genové exprese u nádorů MeSH
• RNA interference MeSH
• serin genetika   metabolismus   MeSH
• stabilita proteinů MeSH
• transfekce MeSH
• ubikvitinace MeSH
• vazba proteinů MeSH
• virové proteiny genetika   metabolismus   MeSH
• western blotting MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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
• apoptóza MeSH
• biologické modely MeSH
• interferonové regulační faktory genetika   imunologie   MeSH
• interferony metabolismus   MeSH
• karcinogeneze MeSH
• lidé MeSH
• lidský herpesvirus 8 genetika   imunologie   patogenita   MeSH
• multigenová rodina MeSH
• signální transdukce MeSH
• virové geny MeSH
• virové proteiny genetika   imunologie   MeSH
• zánět etiologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy 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
• genetická transkripce genetika   MeSH
• HEK293 buňky MeSH
• HeLa buňky MeSH
• hyperplazie velkých lymfatických uzlin genetika   metabolismus   virologie   MeSH
• interferonové regulační faktory genetika   metabolismus   MeSH
• lidé MeSH
• lidský herpesvirus 8 genetika   metabolismus   MeSH
• lymfocyty metabolismus   virologie   MeSH
• proteiny asociované s kinázou S-fáze genetika   metabolismus   MeSH
• protoonkogenní proteiny c-myc genetika   metabolismus   MeSH
• stabilita proteinů MeSH
• ubikvitinace genetika   MeSH
• vazba proteinů MeSH
• virové proteiny genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• Publikační typ
• abstrakt z konference MeSH