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.

• Keywords
• HBV, Hepatitis B virus, Hepatocellular carcinoma, URI1, Unconventional prefoldin RPB5 interactor,
• MeSH
• Hep G2 Cells MeSH
• Gene Knockdown Techniques MeSH
• Hepatitis B * virology   metabolism   MeSH
• Carcinoma, Hepatocellular virology   MeSH
• Hepatocytes virology   MeSH
• Host-Pathogen Interactions * MeSH
• Humans MeSH
• Liver Neoplasms virology   MeSH
• Virus Replication MeSH
• Hepatitis B virus * physiology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't 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
• Virology * MeSH
• Publication type
• Research Support, Non-U.S. Gov't MeSH
• Editorial MeSH
• Geographicals
• Czech Republic 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.

• Keywords
• ER translocation, HBV precore protein, HBe, TRAP complex, hepatitis B virus,
• 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
• Names of Substances
• Cysteine MeSH
• Hepatitis B e Antigens * MeSH
• Membrane Glycoproteins MeSH
• Protein Sorting Signals MeSH
• Calcium-Binding Proteins MeSH
• Receptors, Cytoplasmic and Nuclear MeSH
• Receptors, Peptide MeSH
• signal sequence receptor MeSH Browser

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.

• Keywords
• ATM, ATR, DNA damage response pathway, HBV core protein, serine phosphorylation,
• 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
• Names of Substances
• ATM protein, human MeSH Browser
• Ataxia Telangiectasia Mutated Proteins MeSH
• Checkpoint Kinase 2 MeSH
• CHEK2 protein, human MeSH Browser
• Etoposide MeSH
• Hepatitis B e Antigens MeSH
• hepatitis B virus X protein MeSH Browser
• Hydrogen Peroxide MeSH
• Viral Core Proteins MeSH
• Serine MeSH
• Trans-Activators MeSH
• Viral Regulatory and Accessory Proteins 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.

• Keywords
• E3 ubiquitin-protein ligase, HBc, hepatitis B virus, post-translational modifications, ubiquitin, ubiquitination,
• 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
• Names of Substances
• Arginine MeSH
• Lysine MeSH
• Ubiquitin MeSH
• Ubiquitin-Protein Ligases MeSH
• Viral Proteins 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.

• Keywords
• NAALADase, antibody fragment, glutamate carboxypeptidase II, in vivo imaging, monoclonal antibody, prostate cancer, prostate-specific membrane antigen,
• MeSH
• Antigens, Surface immunology   MeSH
• Cell Line MeSH
• PC-3 Cells MeSH
• Fluorescence MeSH
• Glutamate Carboxypeptidase II immunology   MeSH
• Insecta MeSH
• Single-Chain Antibodies immunology   MeSH
• Humans MeSH
• Antibodies, Monoclonal immunology   MeSH
• Mice, Nude MeSH
• Mice MeSH
• Cell Line, Tumor MeSH
• Prostatic Neoplasms immunology   MeSH
• Recombinant Proteins immunology   MeSH
• Xenograft Model Antitumor Assays methods   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Antigens, Surface MeSH
• FOLH1 protein, human MeSH Browser
• Glutamate Carboxypeptidase II MeSH
• Single-Chain Antibodies MeSH
• Antibodies, Monoclonal MeSH
• Recombinant Proteins 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
• Names of Substances
• Cytokines MeSH
• Interferon-alpha MeSH
• DNA, Circular MeSH
• Culture Media, Conditioned MeSH
• Toll-Like Receptors MeSH