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.
- Klíčová slova
- ATM, ATR, DNA damage response pathway, HBV core protein, serine phosphorylation,
- 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
- Názvy látek
- ATM protein, human MeSH Prohlížeč
- ATM protein MeSH
- checkpoint kinasa 2 MeSH
- CHEK2 protein, human MeSH Prohlížeč
- etoposid MeSH
- hepatitida B - antigeny e MeSH
- hepatitis B virus X protein MeSH Prohlížeč
- peroxid vodíku MeSH
- proteiny virového jádra MeSH
- serin MeSH
- trans-aktivátory MeSH
- virové regulační a přídatné proteiny MeSH
The dramatic progress in tumour biology and immunology in the past several years has opened new avenues for the treatment and prevention of cancer. One of the great contributions of the immunotherapeutic approaches is an increasing understanding of the immunology of cancer, which is, gradually creating conditions for the development of prophylactic anti-cancer vaccines. Efficient vaccines have been developed and employed for the prophylaxis of two frequent cancers of viral origin, namely cervical cancer and liver cancer. The new knowledge on the interactions between the immune system and the malignant tumors seems to provide means for the development of prophylactic vaccines against cancers developing due to the mutations in the proto-oncogenes converting their products into oncoproteins. According to the present estimates, these cancers form a great majority of human malignancies. Recent evidence has indicated that the immune system recognizes such mutated proteins, and that the development of cancer is due to the failure of the immune system to eliminate neoplastic cells. Followingly, it can be expected that inducing immunity against the mutated epitopes will increase the capacity of the body to deal with the initiated precancerous cells. In the present paper this hypothesis is primarily discussed in the relationship with colorectal cancer (CRC), which seems to be a well-fitting candidate for prophylactic vaccination. CRC is the third most frequent malignancy and the fourth most common cause of cancer mortality. Mutations of two proto-oncogenes, namely RAS and RAF, are involved in the majority of CRC cases and, in addition, they are shared with other human malignancies. Therefore, the strategy to be used for prophylaxis of CRC is discussed together with several other frequent human cancers, namely lung cancer, pancreatic duct cancer and melanoma. The prophylactic vaccines proposed are aimed at the reduction of the incidence of these and, to a lesser extent, some other cancers.
- Klíčová slova
- cancer, prophylactic vaccines, proto-oncogenes RAS and RAF,
- MeSH
- infekce papilomavirem * MeSH
- lidé MeSH
- melanom * MeSH
- nádory děložního čípku * MeSH
- nádory slinivky břišní * MeSH
- vakcinace MeSH
- vakcíny proti papilomavirům * MeSH
- vakcíny * MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- vakcíny proti papilomavirům * MeSH
- vakcíny * MeSH
The tumorigenic potential of mouse polyomavirus (MPyV) has been studied for decades in cell culture models and has been mainly attributed to nonstructural middle T antigen (MT), which acts as a scaffold signal adaptor, activates Src tyrosine kinases, and possesses transforming ability. We hypothesized that MPyV could also transform mouse cells independent of MT via a Toll-like receptor 4 (TLR4)-mediated inflammatory mechanism. To this end, we investigated the interaction of MPyV with TLR4 in mouse embryonic fibroblasts (MEFs) and 3T6 cells, resulting in secretion of interleukin 6 (IL-6), independent of active viral replication. TLR4 colocalized with MPyV capsid protein VP1 in MEFs. Neither TLR4 activation nor recombinant IL-6 inhibited MPyV replication in MEFs and 3T6 cells. MPyV induced STAT3 phosphorylation through both direct and MT-dependent and indirect and TLR4/IL-6-dependent mechanisms. We demonstrate that uninfected mouse fibroblasts exposed to the cytokine environment from MPyV-infected fibroblasts upregulated the expressions of MCP-1, CCL-5, and α-SMA. Moreover, the cytokine microenvironment increased the invasiveness of MEFs and CT26 carcinoma cells. Collectively, TLR4 recognition of MPyV induces a cytokine environment that promotes the cancer-associated fibroblast (CAF)-like phenotype in noninfected fibroblasts and increases cell invasiveness.
- Klíčová slova
- CAF, IL-6, MPyV, TLR4, mouse fibroblasts, mouse polyomavirus, spheroid invasiveness,
- Publikační typ
- časopisecké články 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.
- Klíčová slova
- E3 ubiquitin-protein ligase, HBc, hepatitis B virus, post-translational modifications, ubiquitin, ubiquitination,
- 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
- Názvy látek
- arginin MeSH
- lysin MeSH
- ubikvitin MeSH
- ubikvitinligasy MeSH
- virové proteiny 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
- leukocyty mononukleární metabolismus MeSH
- lidé MeSH
- přirozená imunita účinky léků MeSH
- replikace viru účinky léků MeSH
- systémy cílené aplikace 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
- Názvy látek
- cytokiny MeSH
- interferon alfa MeSH
- kruhová DNA MeSH
- kultivační média speciální MeSH
- toll-like receptory MeSH
Chronic hepatitis B virus (HBV) infection is a major cause of chronic liver disease and cancer worldwide. The mechanisms of viral genome sensing and the evasion of innate immune responses by HBV infection are still poorly understood. Recently, the cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) was identified as a DNA sensor. In this study, we investigated the functional role of cGAS in sensing HBV infection and elucidate the mechanisms of viral evasion. We performed functional studies including loss-of-function and gain-of-function experiments combined with cGAS effector gene expression profiling in an infectious cell culture model, primary human hepatocytes, and HBV-infected human liver chimeric mice. Here, we show that cGAS is expressed in the human liver, primary human hepatocytes, and human liver chimeric mice. While naked relaxed-circular HBV DNA is sensed in a cGAS-dependent manner in hepatoma cell lines and primary human hepatocytes, host cell recognition of viral nucleic acids is abolished during HBV infection, suggesting escape from sensing, likely during packaging of the genome into the viral capsid. While the hepatocyte cGAS pathway is functionally active, as shown by reduction of viral covalently closed circular DNA levels in gain-of-function studies, HBV infection suppressed cGAS expression and function in cell culture models and humanized mice. Conclusion: HBV exploits multiple strategies to evade sensing and antiviral activity of cGAS and its effector pathways.
- MeSH
- buněčné kultury MeSH
- DNA virů imunologie MeSH
- hepatitida B imunologie patofyziologie MeSH
- hepatocyty metabolismus virologie MeSH
- hybridizace in situ fluorescenční metody MeSH
- imunitní únik imunologie fyziologie MeSH
- interakce hostitele a patogenu MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- lidé MeSH
- myši MeSH
- nukleotidy cyklické metabolismus MeSH
- stanovení celkové genové exprese metody MeSH
- virus hepatitidy B patogenita MeSH
- western blotting MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Názvy látek
- cyclic guanosine monophosphate-adenosine monophosphate MeSH Prohlížeč
- DNA virů MeSH
- nukleotidy cyklické MeSH
Depletion and functional impairment of circulating plasmacytoid dendritic cells (pDCs) are characteristic attributes of HIV-1-infection. The mechanism of dysfunction of pDCs is unclear. Here, we studied the development of phenotype of pDCs in a cohort of HIV-1-infected individuals monitored before the initiation and during a 9-month follow up with antiretroviral therapy (ART). Using polychromatic flow cytometry, we detected significantly higher pDC-surface expression of the HIV-1 receptor CD4, regulatory receptor BDCA-2, Fcγ receptor CD32, pDC dysfunction marker TIM-3, and the marker of killer pDC, TRAIL, in treatment-naïve HIV-1-infected individuals before initiation of ART when compared to healthy donors. After 9 months of ART, all of these markers approached but did not reach the expression levels observed in healthy donors. We found that the rate of decline in HIV-1 RNA level over the first 3 months of ART negatively correlated with the expression of TIM-3 on pDCs. We conclude that immunogenic phenotype of pDCs is not significantly restored after sustained suppression of HIV-1 RNA level in ART-treated patients and that the level of the TIM-3 expressed on pDCs in treatment naïve patients could be a predictive marker of the rate of decline in the HIV-1 RNA level during ART.
- Klíčová slova
- BDCA-2, HIV-1, T cell Ig and mucin-domain containing molecule 3 (TIM-3), Toll-like receptors 7 and 9 (TLR7/9), antiretroviral therapy (ART), innate and adaptive immune responses, pDC dysfunction, plasmacytoid dendritic cells (pDCs),
- MeSH
- biologické markery MeSH
- buněčný receptor 2 viru hepatitidy A genetika MeSH
- CD4-pozitivní T-lymfocyty účinky léků imunologie metabolismus MeSH
- dendritické buňky imunologie metabolismus MeSH
- dospělí MeSH
- exprese genu * MeSH
- HIV infekce farmakoterapie genetika imunologie virologie MeSH
- HIV-1 * imunologie MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladý dospělý MeSH
- počet CD4 lymfocytů MeSH
- RNA virová MeSH
- virová nálož MeSH
- vysoce aktivní antiretrovirová terapie MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladý dospělý MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- biologické markery MeSH
- buněčný receptor 2 viru hepatitidy A MeSH
- HAVCR2 protein, human MeSH Prohlížeč
- RNA virová MeSH
Recent studies have reported that the crosslinking of regulatory receptors (RRs), such as blood dendritic cell antigen 2 (BDCA-2) (CD303) or ILT7 (CD85g), of plasmacytoid dendritic cells (pDCs) efficiently suppresses the production of type I interferons (IFN-I, α/β/ω) and other cytokines in response to toll-like receptor 7 and 9 (TLR7/9) ligands. The exact mechanism of how this B cell receptor (BCR)-like signaling blocks TLR7/9-mediated IFN-I production is unknown. Here, we stimulated BCR-like signaling by ligation of RRs with BDCA-2 and ILT7 mAbs, hepatitis C virus particles, or BST2 expressing cells. We compared BCR-like signaling in proliferating pDC cell line GEN2.2 and in primary pDCs from healthy donors, and addressed the question of whether pharmacological targeting of BCR-like signaling can antagonize RR-induced pDC inhibition. To this end, we tested the TLR9-mediated production of IFN-I and proinflammatory cytokines in pDCs exposed to a panel of inhibitors of signaling molecules involved in BCR-like, MAPK, NF-ĸB, and calcium signaling pathways. We found that MEK1/2 inhibitors, PD0325901 and U0126 potentiated TLR9-mediated production of IFN-I in GEN2.2 cells. More importantly, MEK1/2 inhibitors significantly increased the TLR9-mediated IFN-I production blocked in both GEN2.2 cells and primary pDCs upon stimulation of BCR-like or phorbol 12-myristate 13-acetate-induced protein kinase C (PKC) signaling. Triggering of BCR-like and PKC signaling in pDCs resulted in an upregulation of the expression and phoshorylation of c-FOS, a downstream gene product of the MEK1/2-ERK pathway. We found that the total level of c-FOS was higher in proliferating GEN2.2 cells than in the resting primary pDCs. The PD0325901-facilitated restoration of the TLR9-mediated IFN-I production correlated with the abrogation of MEK1/2-ERK-c-FOS signaling. These results indicate that the MEK1/2-ERK pathway inhibits TLR9-mediated type I IFN production in pDCs and that pharmacological targeting of MEK1/2-ERK signaling could be a strategy to overcome immunotolerance of pDCs and re-establish their immunogenic activity.
- Klíčová slova
- B cell-like receptor signaling, MEK1/2, blood dendritic cell antigen 2, c-FOS, plasmacytoid dendritic cells, regulatory receptors, toll-like receptors 7 and 9 (TLR7/9), type I interferon,
- MeSH
- B-lymfocyty imunologie MeSH
- buněčné linie MeSH
- dendritické buňky fyziologie MeSH
- extracelulárním signálem regulované MAP kinasy metabolismus MeSH
- interferon typ I metabolismus MeSH
- lidé MeSH
- MAP kinasa-kinasa 1 metabolismus MeSH
- MAP kinasa-kinasa 2 metabolismus MeSH
- MAP kinasový signální systém MeSH
- NF-kappa B metabolismus MeSH
- proteinkinasa C metabolismus MeSH
- protoonkogenní proteiny c-fos metabolismus MeSH
- receptory antigenů B-buněk genetika metabolismus MeSH
- toll-like receptor 9 metabolismus MeSH
- vápníková signalizace MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- extracelulárním signálem regulované MAP kinasy MeSH
- interferon typ I MeSH
- MAP kinasa-kinasa 1 MeSH
- MAP kinasa-kinasa 2 MeSH
- NF-kappa B MeSH
- proteinkinasa C MeSH
- protoonkogenní proteiny c-fos MeSH
- receptory antigenů B-buněk MeSH
- toll-like receptor 9 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
- Názvy látek
- PRMT5 protein, human MeSH Prohlížeč
- proteinarginin-N-methyltransferasy MeSH
The innate immune cells sense microbial infection and self-ligands by pathogen recognition receptors (PRRs), such as toll-like receptors (TLRs) and regulatory receptors (RRs), associated with immunoreceptor tyrosine-based activation motif (ITAM). Rapid activation and concerted action of PRRs signaling and feedback inhibitory mechanisms must be engaged to ensure the host defense functions and to prevent cytotoxicity associated with excessive activation. ITAM-associated RRs can generate stimulatory or, paradoxically, inhibitory signals. The network of ITAM-associated RR, together with TLR-signaling pathways, are responsible for immunogenic or tolerogenic responses of macrophages and dendritic cells to their microenvironment. In macrophages, TLR4 signaling is inhibited by low-avidity ligation of ITAM-associated receptors, while high-avidity ligation of ITAM-associated receptors results in potentiation of TLR4 signaling together with resistance to extracellular cytokine microenvironment signals. In contrast to macrophages, TLR7/9 signaling in plasmacytoid DCs (pDCs) is inhibited by high-avidity ligation of ITAM-associated RR, while low-avidity ligation does not show any effect. Surprisingly, interference of ITAM-associated receptor signaling with TLR pathways has not been reported in conventional dendritic cells. Here, we present an overview of molecular mechanisms acting at the crossroads of TLR and ITAM-signaling pathways and address the question of how the high-avidity engagement of the ITAM-associated receptors in pDCs inhibits TLR7/9 signaling. Cellular context and spatiotemporal engagement of ITAM- and TLR-signaling pathways are responsible for different outcomes of macrophage versus pDC activation. While the cross-regulation of cytokine and TLR signaling, together with antigen presentation, are the principal functions of ITAM-associated RR in macrophages, the major role of these receptors in pDCs seems to be related to inhibition of cytokine production and reestablishment of a tolerogenic state following pDC activation. Pharmacologic targeting of TLR and ITAM signaling could be an attractive new therapeutic approach for treatment of chronic infections, cancer, and autoimmune and inflammatory diseases related to pDCs.
