Hepatitis B virus (HBV) infection can cause liver disease and lead to hepatocellular carcinoma (HCC). To better understand the factors involved in viral infection and pathogenesis and to develop novel therapies, it is crucial to investigate virus-host interactions. HBV infection has been shown to increase the expression of the unconventional prefoldin RPB5 interactor (URI1), a cellular protein that promotes liver tumorigenesis and HCC metastasis. Our study investigated the role of URI1 in HBV infection in vitro. Although previous reports have suggested that URI1 may act as an HBV restriction factor, our results showed that URI1 silencing or overexpression did not affect HBV replication in HepG2-NTCP cells. In primary human hepatocytes, URI1 knockdown modestly reduced HBV markers but did not significantly alter acute infection. Supporting the premise that URI1 is a promising therapeutic target for HCC, our findings show that URI1 knockdown does not enhance HBV infection in an acute infection model. This suggests that URI1 may be a viable therapeutic target for patients with HBV-associated HCC without increasing HBV-related complications.

• Klíčová slova
• HBV, Hepatitis B virus, Hepatocellular carcinoma, URI1, Unconventional prefoldin RPB5 interactor,
• MeSH
• buňky Hep G2 MeSH
• genový knockdown MeSH
• hepatitida B * virologie   metabolismus   MeSH
• hepatocelulární karcinom virologie   MeSH
• hepatocyty virologie   MeSH
• interakce hostitele a patogenu * MeSH
• lidé MeSH
• nádory jater virologie   MeSH
• replikace viru MeSH
• virus hepatitidy B * fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

The Czech Republic, a part of the former Czechoslovakia, has been at the forefront of several research directions in virology, genetics and physiology [...].

• MeSH
• virologie * MeSH
• Publikační typ
• práce podpořená grantem MeSH
• úvodníky MeSH
• Geografické názvy
• Česká republika MeSH

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.

• Klíčová slova
• ER translocation, HBV precore protein, HBe, TRAP complex, hepatitis B virus,
• 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
• Názvy látek
• cystein MeSH
• hepatitida B - antigeny e * MeSH
• membránové glykoproteiny MeSH
• proteiny - lokalizační signály MeSH
• proteiny vázající vápník MeSH
• receptory cytoplazmatické a nukleární MeSH
• receptory peptidů MeSH
• signal sequence receptor MeSH Prohlížeč

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

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

Prostate-Specific Membrane Antigen (PSMA) is an established biomarker for the imaging and experimental therapy of prostate cancer (PCa), as it is strongly upregulated in high-grade primary, androgen-independent, and metastatic lesions. Here, we report on the development and functional characterization of recombinant single-chain Fv (scFv) and Fab fragments derived from the 5D3 PSMA-specific monoclonal antibody (mAb). These fragments were engineered, heterologously expressed in insect S2 cells, and purified to homogeneity with yields up to 20 mg/L. In vitro assays including ELISA, immunofluorescence and flow cytometry, revealed that the fragments retain the nanomolar affinity and single target specificity of the parent 5D3 antibody. Importantly, using a murine xenograft model of PCa, we verified the suitability of fluorescently labeled fragments for in vivo imaging of PSMA-positive tumors and compared their pharmacokinetics and tissue distribution to the parent mAb. Collectively, our data provide an experimental basis for the further development of 5D3 recombinant fragments for future clinical use.

• Klíčová slova
• NAALADase, antibody fragment, glutamate carboxypeptidase II, in vivo imaging, monoclonal antibody, prostate cancer, prostate-specific membrane antigen,
• MeSH
• antigeny povrchové imunologie   MeSH
• buněčné linie MeSH
• buňky PC-3 MeSH
• fluorescence MeSH
• glutamátkarboxypeptidasa II imunologie   MeSH
• hmyz MeSH
• jednořetězcové protilátky imunologie   MeSH
• lidé MeSH
• monoklonální protilátky imunologie   MeSH
• myši nahé MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• nádory prostaty imunologie   MeSH
• rekombinantní proteiny imunologie   MeSH
• xenogenní modely - testy protinádorové aktivity metody   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antigeny povrchové MeSH
• FOLH1 protein, human MeSH Prohlížeč
• glutamátkarboxypeptidasa II MeSH
• jednořetězcové protilátky MeSH
• monoklonální protilátky MeSH
• rekombinantní 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
• 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
• Názvy látek
• cytokiny MeSH
• interferon alfa MeSH
• kruhová DNA MeSH
• kultivační média speciální MeSH
• toll-like receptory MeSH